Celebration of Native Mass Spectrometry...2 Institut Européen de Chimie et Biologie , Université...

Celebration of

Native Mass

Spectrometry 24-26th March 2019

Oxford, UK

nativemsoxford2019.wordpress.com

@nativemsoxford1

Foreword

Welcome to Oxford and a Celebration of Native Mass Spectrometry! We are delighted that you can join us to

celebrate all that has been achieved using native MS, and hear new research presented that will no doubt shape

the field in the years to come. The last decade has been a particularly exciting and momentous time for

structural biology, and for the new techniques that have enabled innumerable biological discoveries. This is

exemplified by two Nobel prizes in Chemistry - 2014 for “for the development of super-resolved fluorescence

microscopy” & 2017 ‘for developing cryo-electron microscopy for the high-resolution structure determination of

biomolecules in solution”. Such accolades rightly honour technological advances that have transformed what is

scientifically possible, but perhaps as importantly, help drive interest in methodologies and the proliferation of

cutting-edge instrumentation.

Similar to cryoEM, we in native MS have recently enjoyed a resolution revolution, driven by improvements to

classical instrumentation as well as the arrival of new commercial platforms making routinely achievable what

could only be imagined a few years ago. Thanks in large part to a growing need to precisely characterise

biomolecules in the pharma, biopharma and biotech sectors, we have also experienced an unprecedented

explosion in the native MS user base, with more and more non-specialists entering the field. These huge leaps

forward are exciting but also require us to periodically take stock and reflect.

The rapid democratisation of native MS makes defining, promoting and encouraging best practice incredibly

important. However, this can be challenging, especially when effectively capitalising on rapid technological

advances sometimes requires established dogmas to be reinforced, but at other times redefined. Responding

constructively to disruptive new ideas and critically evaluating new uses for native MS can only be done as a

community, through lively discussion, debate and co-operation. These noble ideals are particularly poignant at

present, at this uncertain time for Britain and Europe, and in the age of social media, where voicing even

considered opinions can be a perilous exercise.

It is therefore extremely encouraging that you are all here! 169 delegates from 14 European nations and 15

countries. An almost 1:2 ratio between industry and academia, and 36% student delegates suggests that the

diverse groups that make up our expanding community are well represented. In the spirit of the special interest

groups, organised by the British Mass Spectrometry Society, the format of the conference has been designed to

promote discussion and encourage participation from all. I therefore hope that this Celebration of Native Mass

Spectrometry will provide a forum not only to showcase fantastic science, but also to encourage discourse and to

respectfully challenge ideas new and old.

On behalf of the whole organising committee I would like to welcome you to Oxford and wish you an

enjoyable few days under the dreaming spires!

Dr Joe Gault, Junior Research Fellow, The Queen’s College

Sponsors Thank you to all our sponsors for supporting this event so generously. We are

particularly grateful to Waters for sponsoring the poster session & after dinner

mixer, Thermo Fisher for sponsoring the dinner, Refeyn for poster and talk prizes

and OMass Therapeutics for providing student bursaries.

Supporters Thank you to the British Mass Spectrometry Society, The Queen’s College, and

the Department of Chemistry of The University of Oxford for their support, both

logistical and financial.

Organising Committee

Dr Joseph Gault University of Oxford, UK

Prof. Michael Landreh Karolinska Instiutet, Sweden

Prof. Frank Sobott University of Leeds, UK

Prof. Justin Benesch University of Oxford, UK

Prof. Dame Carol Robinson University of Oxford, UK

Programme

Sunday 24th

1830-late Informal Mixer at The Chequers Inn, 131 High Street, Oxford OX1 4DH

0745-0845 Breakfast for Residential Delegates at The Queen's College

0730 Exhibitor Setup

0900-0915 Opening Remarks

0915-1015 OPENING PLENARY

Native MS - Past, Present, Future - Carol Robinson

1015 NATIVE MS AND ION MOBILITY IN STRUCTURAL BIOLOGY

Chair - Perdita Barran, University of Manchester

1020-1050 Can we structurally distinguish between highly similar protein complexes by native MS? - Michal Sharon, Weizmann Institute

1050-1110 Using native MS & HDX- MS to study the assembly of the ‘ring-doughnut’ structure of encapsulated ferritins - Jennifer Ross, University of Edinburgh

1110-1130 Solution and gas-phase structures of G-quadruplexes: ion mobility mass spectrometry & solution spectroscopy - Anirban Ghosh, Institut Européen de Chimie et Biologie

1130-1150 What next for native ion mobility-mass spectrometry? - Dale Cooper-Shepherd, Waters

Lunch - St John's College Hall

1300 BIOTHERAPEUTICS, GLYCOPROTEINS AND COMPLEX BIO-MOLECULES

Chair - Rebecca Burnley, UCB

1305-1335 Is high-throughput and native MS really differentiating compared to other analytical methods used in pharma? - Iain Campuzano, Amgen

1335-1355 The practical aspects and advantages of directly coupling ion exchange chromatography to high resolution native mass spectrometry - Ken Cook, Thermo Fisher

1355-1415 Intact and middle level collision induced unfolding to decipher gas-phase unfolding mechanism of hybrid & canonical mAbs - Oscar Hernandez-Alba, LSMBO/CNRS

1415-1435 Native mass spectrometry unravels highly complex glycosylation patterns in the therapeutic Fc-fusion protein Etanercept - Therese Wohlchlager, University of Salzburg

Tea/Coffee Break

1500 MEMBRANE PROTEINS

Chair - Frank Sobott,, University of Leeds

1505-1535 Structural dynamics underpinning function in membrane transporters - Argyris Politis, King's College London

1535-1555 Elucidating the effect lipids have on the function and stability of GPCRs - Idlir Liko, Omass Therapeutics

1555-1615 Structural characterization of membrane-associated amyloid-β oligomers - Eduard Puig, IRB Barcelona

1615-1635 The different effects of substrates and nucleotides on the complex formation of ABC transporters - Francesco Fiorentino, University of Oxford

POSTER SESSION

1915 Pre Dinner Mixer - The Cloisters, The Queen's College

1945 GALA DINNER - THE QUEEN'S COLLEGE HALL

hosted by Prof Paul Madden, Provost of The Queen's College & Prof Dame Carol Robinson

2145 Post Dinner Mixer - University College Bar

Monday 25th

Celebration of Native Mass Spectrometry - Conference Programme Oxford, 24-26th March 2019

0745-0845 Breakfast for Residential Delegates at The Queen's College

900 INTEGRATIVE NATIVE MS - HDX, X-LINKING, LABELLING & MODELLING IN STRUCTURAL BIOLOGY

Chair - Carla Schmidt,

0905-0935 Protein foot printing using carbenes: binding sites and bottlenecks - Neil Oldham, University of Nottingham

0935-0955 Of rabbit and men: integrative structural proteomics analysis of the 20S proteasome complex - Rosa Viner, Thermo Fisher

0955-1015 Chemical cross-linking and ion mobility for integrative modelling - Matteo Degiacomi, Durham University

1015-1035 Inter-domain dynamics mediate substrate binding by the periplasmic chaperone SurA - Antonio Calabrese, University of Leeds

Tea/Coffee Break

1100 CLOSE-UPS OF PROTEINS IN THE GAS PHASE

Chair - Erik Marklund,, Uppsala University

1105-1135 Using gas-phase methods to study solution structures: what could go wrong? - Valérie Gabelica, Université de Bordeaux

1135-1155 The effect of entropy upon electrospray charge state distribution - Rod Chalk, SGC University of Oxford

1155-1215 Exploring protein stability & dynamics with a cyclic T-Wave instrument - Charles Eldrid, UCL

1215-1235 Effect of small drug-like molecules and metal ions on the conformational ensemble of alpha-synuclein - Rani Moons, University of Antwerp

Lunch - St John's College Hall

1335 THE FUTURE OF STRUCTURAL MS - NATIVE TOP-DOWN, NEW METHODS & WORKFLOWS

Chair - Peter O'Connor, University of Warwick

1340-1410 Tackling bacterial proteins from the top (down) -Julia Chamot-Rooke, Institut Pasteur

1410-1430 Data processing tools to develop native LESA TWIMS and conformational imaging - Emma Sisley, University of Birmingham

1430-1450 Weighing molecules with light – a new way to study biomolecules - Matthias Langhorst, Refeyn

1450-1510 Native electrospray ion beam deposition: soft landing as new workflow for protein imaging - Sabine Abb, Max Planck Institute for Solid State Research

1515-1615 CLOSING PLENARY

Remain Creative with Native - Albert Heck

1615-1625 Closing Remarks

Tuesday 26th

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Go beyond with native mass

spectrometry

Queen’s to St John’s The quickest way to walk between Queen’s and St John’s is to exit Queen’s

via the late gate next to the library in back quad and to follow this route.

Chequers

St John’s

Queen’s

Late gate

Selected Talks

SELECTED TALK - Native MS and Ion Mobility in Structural Biology

Presenting Author - Jennifer Ross

Can we structurally distinguish between highly similar protein complexes by

native MS?

J. Ross1, T. Lambert1, C. Piergentili2, D. He3, J. Marles-Wright2, and D. Clarke1

1 The School of Chemistry, The University of Edinburgh, UK

2 School of Natural and Environmental Sciences, Newcastle University, UK

3 SGC, University of Oxford, UK

Title: Using Native MS and Hydrogen-Deuterium Exchange MS to Study the Assembly of the ‘Ring-Doughnut’

Structure of Encapsulated Ferritins.

Abstract:

Ferritins are ubiquitous proteins used to sequester and store iron in almost all life forms. In classical ferritins, iron

is oxidised from Fe(II) to Fe(III) and then, stored as a ferrihydrite mineral within a spherical nanocage made up of

24 ferritin subunits. The recently discovered encapsulated ferritin (EncFtn) family do not store iron in this manner,

but instead use another protein, encapsulin, for storage following iron oxidation. We have recently reported

structural information on several members of the EncFtn family, which share a conserved decameric ‘ring-

doughnut’ structure, with ten monomers arranged as a pentamer of dimers. New results elucidate the assembly

pathway of the ring-doughnut structure through the use of native ion-mobility mass spectrometry and

hydrogen-deuterium exchange mass spectrometry. Charge state distributions consistent with the EncFtn

monomer and dimers form higher order assemblies via both pH-mediated and Fe(II)-dependent pathways.

Interestingly, no odd numbered oligomerisation states are observed (except monomer), implying that oligomers

larger than dimers are caused by the association of dimer units, not monomer units. Analysis of a series of single

amino acid variants of EncFtn pinpoints a conserved His residue in the active site as an important mediator of

this oligomerisation process.


Presenting Author - Anirban Ghosh

Solution and gas-phase structures of G-quadruplexes: ion mobility mass

Spectrometry & solution spectroscopy

A. Ghosh1, F. Rosu2, V. Gabelica1*

1 Laboratoire Acides Nucléiques: Régulations Naturelle et Artificielle , Université de Bordeaux, Inserm & CNRS

(ARNA, U1212, UMR5320), IECB , 2 rue Robert Escarpit , 33607 Pessac , France

2 Institut Européen de Chimie et Biologie , Université de Bordeaux, CNRS & Inserm (IECB, UMS3033,

US001) , 2 rue Robert Escarpit , 33607 Pessac , France.

Solution and Gas-Phase Structures of G-Quadruplexes: Ion Mobility Mass Spectrometry, Solution Spectroscopy

G-quadruplexes (G4) are guanine-rich sequences found in promoters and telomeres associated with cellular

processes. Here we are using Ion mobility mass spectrometry (IM-MS) to shed light on the diverse

conformational topology of the G-quadruplexes in native MS condition. Also, we are using cosolvents

(Acetonitrile, Ethanol) to elucidate the conformational rearrangements in dehydrated conditions. CD and

1HNMR spectra in native MS buffer shows that the G4 conformation (as reported literature with high KCl

concentration) is preserved. ESI-MS illustrates different K+ bound stoichiometries (2-tetrad, 3-tetrad, & 4-tetrad)

present in solution for different G4 sequences. The collision cross-section (CCS) distributions are broad for all

hybrid (2K+ bound) and anti-parallel conformations (1K+ bound) and narrower for parallel conformations (2K+

bound). In presence of cosolvents, the K+ stoichiometry and ion mobility vary for several initially antiparallel and

hybrid sequences. We also tested whether gas-phase structure optimization with advanced quantum calculations

would generate appropriate three-dimensional models of G4 systems studied. This paves the way towards

using native ion mobility mass spectrometry based approaches to study nucleic acid conformational

polymorphism.

SELECTED TALK - Biotherapeutics, Glycoproteins and Complex Bio-Molecules

Presenting Author - Ken Cook

The practical aspects and advantages of directly coupling ion exchange

chromatography to high resolution native mass spectrometry

Ken Cook1, Kai Scheffler2, Florian Füssl3, Jonathan Bones3

1Thermo Fisher Scientific, Hemel Hempstead 2Thermo Fisher Scientific, Germering 3NIBRT, Dublin, Ireland.

Mass Spectrometry (MS) is used as a tool in the identification of protein charged variants, however the

technique of ion exchange requires high salt eluents which are incompatible with MS. Structural variants

exposed by ion exchange must be collected separately off-line, then desalted before further characterisation by

MS. Here we describe novel direct on-line coupling of ion exchange to the MS instrument in the

characterisation of Mab variants. The workflow has a fast run time and greatly reduces analysis time and sample

handling. The chromatographic resolution of MAb charged variants compares favourably with traditional salt

elution. The proteins enter the Orbitrap MS system in the native state with a reduced charge distribution and an

elevated mass to charge ratio. The mass accuracy was found to be much improved due to the chromatographic

separation of near isobaric variants which could otherwise compromise deconvolution. This can be compared to

SEC where all the charged variants elute at the same time. Confirmed identification of variant peaks found with

this direct on-line coupling in addition to the charge variant profile include fragments, N-terminal Asp loss,

succinimide Asp variants, glycosylation, deamidation, and lysine truncation. The application has applicability to

anionic and cationic proteins.


Presenting Author - O. Hernandez-Alba

Intact and middle level collision induced unfolding to decipher gas-phase

unfolding mechanism of hybrid & canonical mAbs

O. Hernandez-Alba1, T. Botzanowski1, A. Beck2, S. Cianferani1

1 Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178,

67000 Strasbourg, France

2 Centre d'Immunologie Pierre-Fabre (CIPF), Saint-Julien-en-Genevois, France

Intact- and middle-level Collision Induced Unfolding experiments to decipher gas-phase unfolding mechanism

of hybrid and canonical mAbs

Most currently approved mAbs for clinical treatment of several diseases such as cancer and cardiovascular

disorders, are selected from three human IgG isotypes (1, 2, and 4)1. Since different isotypes also differ in their

ability to support secondary immune functions, there is a real interest in developing new strategies for a better

understanding of structure-function correlation. Ion mobility spectrometry (IMS), and more particularly collision

induced unfolding (CIU), combined with native mass spectrometry (MS) are playing a crucial role in the gas-

phase structural characterization of mAb-based biotherapeutics.

Here we aim at developing new CIU-based strategies at intact and middle level to tackle structural

differences/similarities on biotherapeutic mAbs not only from different isotypes (1, 2, and 4) but also for hybrid

IgG formats. In particular, middle-level CIU analysis performed after IdeS digestion, provided specific signatures

corresponding to structural domains that exhibited different unfolding mechanism allowing the differentiation of

the IgG formats4.

Altogether, our study highlights the suitability of CIU experiments at both intact and middle levels for the

characterization of the gas-phase unfolding dynamics of different mAbs. We envision that specific signatures

characteristic of each individual mAb isotype from different IgG-subunits can be used to characterize/categorize

new IgG-based biotherapeutics.


Presenting Author - T. Wohlschlager

Native mass spectrometry unravels highly complex glycosylation patterns in the

therapeutic Fc-fusion protein Etanercept

T. Wohlschlager1,2, K. Scheffler2,3, I.C. Forstenlehner2,4, W. Esser-Skala1,2, S. Senn1,2, E. Damoc5, J. Holzmann2,4,

C.G. Huber1,2

1 Department of Biosciences, University of Salzburg, Austria

2 Christian Doppler Laboratory for Innovative Tools for Biosimilar Characterization, Salzburg, Austria

3 Thermo Fisher Scientific GmbH, Dreieich, Germany

4 Sandoz, Novartis Technical Operations Biologics Technical Development & Manufacturing, Kundl, Austria

5 Thermo Fisher Scientific GmbH, Bremen, Germany

Characterization of therapeutic glycoproteins is conventionally performed at the released glycan or glycopeptide

level using generic high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry

(MS) methods. Intact protein characterization, on the other hand, represents a powerful alternative approach in

that it may resolve co-existing glycoforms and reveal highly distinct glycosylation patterns. We implemented

native MS for the characterization of intact Etanercept (Enbrel®), a highly N- and O-glycosylated recombinant Fc-

fusion protein applied in the therapy of arthritic diseases. Taking advantage of the higher spatial resolution at

lower charge states detected under native conditions, more than 80 different isoforms of the 130 kDa protein

were distinguishable upon deconvolution of the highly complex mass spectra. Assignment of specific

glycoforms was achieved upon enzymatic digestion of the molecule using a set of glycosidases and proteases.

Information gained at lower structural levels (i.e. glycopeptides, protein subunits) was successfully integrated to

facilitate glycoform annotation at higher structural levels (i.e. whole protein upon partial deglycosylation) through

the application of advanced computational tools. Finally, we demonstrate native MS as a rapid fingerprinting tool

for the assessment of batch-to-batch variability at the intact protein level rendering our method highly attractive

for quality control as well as for comparability studies.

SELECTED TALK - Membrane Proteins

Presenting Author - Eduard Puig

Structural characterization of membrane-associated amyloid-β oligomers

E. Puig12, S. Ciudad12, T. Botzanowski3, S. Chaignepain1, S. Cianferani3, N. Carulla12

1 CBMN (UMR 5248), University of Bordeaux CNRS IPB, Institut EuropÉen de Chimie et Biologie, Pessac,

France 2 Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and

Technology, Barcelona, Spain 3 Laboratoire de SpectromÉtrie de Masse BioOrganique, UniversitÉ de

Strasbourg CNRS UMR7178, IPHC, Strasbourg, France

Structure of an Aβ42 pore-forming oligomer: Alzheimer’s disease (AD) is associated to the aggregation of the

amyloid-beta peptide (Aβ). Therefore, understanding the links between protein aggregation and neurotoxicity,

and specially obtaining 3D structures of the aggregates responsible for neurotoxicity is key to design effective

prevention and therapeutic strategies. We have been working on the hypothesis that Aβ interacts with the cell

membrane and shown that in the presence of detergent micelles Aβ is able to oligomerize and adopt a

specific and defined structure with characteristics of a β-barrel assembly that functions as a pore. We refer to

these type of oligomers as β-Barrel Pore-Forming Oligomer (βPFO). We hereby present the work carried out

to obtain the 3D structure of βPFO-type oligomers focusing on the mass spectrometry (MS) part. Specifically,

we have used size-exclusion chromatography (SEC) coupled to ion mobility-MS. This strategy has allowed us to

establish the stoichiometry of distinct oligomer species present in the sample as a function of their elution time

through a SEC column. We have also used cross-linking MS strategies to confirm the intact mass of these

oligomers and to obtain structural restraints. Altogether, both strategies have contributed to the 3D structural

characterization of βPFO-type oligomers.

SELECTED TALK - Membrane Proteins

Presenting Author - Francesco Fiorentino

The different effects of substrates and nucleotides on the complex formation of

ABC transporters

F. Fiorentino1, J. R. Bolla1, S. Mehmood1, C. V. Robinson1

1 Department of Chemistry, University of Oxford, UK

The molybdate importer (ModBC-A of A. fulgidus) and the vitamin B12 importer

(BtuCD-F of E. coli) are members of the type I and type II ABC importer families. Here we study the influence

of substrate and nucleotide binding on complex formation and stability. Using native mass spectrometry we

show that the interaction between the periplasmic substrate-binding protein (SBP) ModA and the transporter

ModBC is dependent upon binding of molybdate. By contrast, vitamin B12 disrupts interactions between the

transporter BtuCD and the SBP BtuF. Moreover, while ATP binds cooperatively to BtuCD-F, and acts

synergistically with vitamin B12 to destabilise the BtuCD-F complex, no effect is observed for ATP binding on

the stability of ModBC-A. These observations not only highlight the ability of MS to capture these importer-SBP

complexes but allow us to add molecular detail to proposed transport mechanisms.

SELECTED TALK - Integrative Native MS - HDX, X-linking, Labelling in Structrual Biology

Presenting Author - Matteo Degiacomi

Chemical cross-linking and ion mobility for integrative modelling

M. T. Degiacomi1

1 Department of Chemistry, Durham University, UK

Chemical cross-linking reports on the neighborhood relationships between amino acids in proteins. The

interpretation of cross-linking data in a structural context is typically performed by comparison against measured

interatomic distances in a reference atomic structure. However, proteins are dynamic, undergoing motions

spanning from local fluctuations of individual residues to global domain motions. We developed a method and

associated software, DynamXL, that accounts directly for molecular flexibility in the context of cross-linking

modelling. We demonstrate that our method improves significantly the rationalization of experimental cross-

linking data and enhances the performance of protein-protein docking protocols.

We sthen Coupling cross-linking and collision cross-section data we then characterize the structure of small Heat

Shock Proteins (sHSP). sHSP bind nascent unfolding proteins, preventing their potentially harmful denaturation.

Since sHSP oligomers are often polydisperse and each complex can bind multiple proteins simultaneously,

several sHSP:target stoichiometries are often observed. Mass Sectrometry (MS) is one of the few techniques

able to separate these complexes and assess them individually. Here, we produce models of sHSPs multimeric

structures and guide the docking of a sHSP complex to its target, demonstrating how a combination of MS and

integrative modelling is a powerful tool to gain insights into protein structure and function.

SELECTED TALK - Integrative Native MS - HDX, X-linking, Labelling in Structrual Biology

Presenting Author - Antonio Calabrese

Inter-domain dynamics mediate substrate binding by the periplasmic chaperone

SurA

Antonio N. Calabrese1, Bob Schiffrin1, Matthew Watson1, Jim E. Horne1, Julia R. Humes1, Theodoros K.

Karamanos1, Paul White1, Roman Tuma1, Alison E. Ashcroft1, David J. Brockwell1, Sheena E. Radford1

1 Faculty of Biological Sciences, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2

9JT, UK

The periplasmic chaperone SurA is involved in outer membrane protein (OMP) assembly in gram negative

bacteria. E. coli SurA has a three domain architecture, a core domain and two peptidyl prolyl isomerase (PPIase)

domains [P1 and P2]. However, the roles of these domains in chaperoning remain unclear. Here we have utilised

structural mass spectrometry (MS) techniques (XL-MS and HDX-MS), alongside single molecule FRET (smFRET)

and molecular dynamics (MD) simulations to study the structure of SurA, and to interrogate complexes of SurA

with model substrates. XL-MS, smFRET and MD revealed that SurA samples a broad spectrum of conformations,

with the three domains in dynamic motion relative to one another. Using XL-MS, with NHS-ester and

photoactivatable reagents, we showed that unfolded clients bind in several orientations to SurA, with binding

mediated by multiple interaction sites. Finally, HDX-MS demonstrated that conformational changes are relayed

throughout SurA upon substrate binding, whilst smFRET showed that substrate binding narrows the

conformational ensemble. Combined, the data demonstrate that inter-domain flexibility is a key structural feature

of SurA that may play a role in its chaperone function. This work demonstrates the power of an integrated

structural MS centred approach to interrogate inherently flexible multidomain proteins and their assemblies.


Presenting Author - Rod Chalk

The effect of entropy upon electrospray charge state distribution

Rod Chalk1, Ole Tietz2, Opher Gileadi1 and Nicola Burgess-Brown1

1 Structural Genomics Consortium, University of Oxford, OX3 7DQ

2 Deptartment of Oncology, University of Oxford, OX3 7DQ

Effect of Entropy upon Electrospray Charge Distribution.

We have compared the ESI charge distributions of diphenylalanine self-assembled fibrils, polylysine synthetic

oligopeptides, and synthetic tri-TAT peptides in both linear and cyclic forms against natural peptides, denatured

proteins and native protein complexes up to 1M Da. As expected, our data shows a clear relationship between

charge state and surface area, but also demonstrates a relationship between charge states and entropy.

Comparison of enzymes under physiological conditions, stabilized by pH or stabilized by chemical methylation

show alterations in both charge state and charge state distribution. We show how this can enhance our

understanding of the electrospray process. We also show how it can be used to study changes in protein

conformation resulting from ligand binding or phosphorylation, and show how to obtain time-resolved protein

structure data.


Presenting Author - C. Eldrid

Exploring protein stability & dynamics with a cyclic T-Wave instrument

C. Eldrid1, J. Ujma2, S. Kalfas1, N. Tomczyk2, K. Giles2, M. Morris2, K. Thalassinos1,3

1 Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London,

WC1E 6BT, UK 2 Waters Corporation, Stamford Road, Wilmslow, SK9 4AX, UK 3 Institute of Structural and

Molecular Biology, Birkbeck College, University of London, London, WC1E 7HX, UK

Ion mobility mass spectrometry (IM-MS) allows separation of native protein ions into “conformational families”.

Increasing the IM resolving power should allow finer structural information to be obtained, and can be achieved

by increasing the length of the IM separator. This, however, increases the time that protein ions spend in the gas

phase and previous experiments have shown that the initial conformations of small proteins can be lost within

tens of milliseconds. Here, we report on investigations of protein ion stability using a multi-pass travelling wave

(TW) cyclic IM (cIM) device. Using this device, minimal structural changes were observed for Cytochrome C after

hundreds of milliseconds, while no changes were observed for a larger multimeric complex (Concanavalin A).

The geometry of the instrument (Q-cIM-ToF) also enables complex tandem IM experiments to be performed

which were used to obtain more detailed collision induced unfolding pathways for Cytochrome C. The novel

instrument geometry provides unique capabilities with the potential to expand the field of protein analysis via

IM-MS.


Presenting Author - Rani Moons

Effect of small drug-like molecules and metal ions on the conformational

ensemble of alpha-synuclein

R. Moons1, A. Konijnenberg1, A.M. Lambeir2, F. Sobott34

1 Biomolecular and Analytical Mass Spectrometry group, University of Antwerp, Antwerp, Belgium 2 Laboratory

of Medical Biochemistry, University of Antwerp, Antwerp, Belgium 3 Astbury Centre for Structural Molecular

Biology, University of Leeds, UK 4 School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT,

UK

The intrinsically disordered protein (IDP) α-synuclein is prone to aggregation and linked with Parkinson’s

disease (PD). Major questions regarding its conformational space, oligomerisation intermediates and their link

with fibril morphology and disease etiology remain unanswered, as does the mechanism by which some small

molecules, metal ions and PTMs modulate α-synuclein monomer structure in a way which impacts aggregation

and toxicity.

The stoichiometry of binding of biologically relevant metal ions and small molecules is investigated using native

electrospray ionisation mass spectrometry (ESI-MS). Additionally, ion mobility (IM) MS approaches are optimised

and applied to study the conformational space of α-synuclein monomers in the presence of these structural

modulators.

By examining compounds with proven drug-like properties, epigallocatechin gallate (EGCG) and dopamine

(DA), our results show they bind to different conformers (more extended/compact) and have opposite

conformational effects on the α-synuclein monomer ensemble.

Recently the physiological role of some metal ions has caused considerable interest. We study the effect of a

variety of cations on α-synuclein conformation. Multivalent cations and their respective counterions show distinct

binding stoichiometries and mobility shifts.

We show how structure-modulating compounds such as small molecules and metal ions, both relevant in a

biological context, affect the conformation of α-synuclein monomers.

SELECTED TALK - The Future of Structural MS - Native Top-Down, Automation, New Methods & Workflows

Presenting Author - Emma K. Sisley

Data processing tools to develop native LESA TWIMS and conformational

imaging

E.K. Sisley1,2, R.L. Griffiths1, I.B. Styles3, H.J. Cooper1

1 School of Bioscience, University of Birmingham 2 EPSRC Centre for Doctoral Training in Physical Sciences for

Health, University of Birmingham 3 School of Computer Science, University of Birmingham

Liquid extraction surface analysis (LESA) is a sampling technique that enables direct analysis of native proteins

from biological substrates. Native LESA imaging of proteins from thaw mounted tissue sections has required

the development of in-house data processing tools. These tools deal with the summation of the spectra and the

noisy baseline. Benefits of integrating travelling wave ion mobility spectrometry (TWIMS) into native LESA MS

imaging workflows include the separation of molecular species from background noise. We have therefore

further developed these data processing tools to retain the drift times associated with the ions thus enabling the

selection of regions of interest from the .mzML files. This processing enables the generation of images of native

proteins that can only be detected by including TWIMS separation. TWIMS also allows the measurement of

collision cross sections (CCS). Using custom synthesised isomeric peptides (APLpSFLGSLPKSYVK and

APLSFLGSLPKpSYVK) with known differences in CCS, we were able to develop the code to visualise the

distributions of the different conformations.

Posters

POSTER No. 1 Native MS and Ion Mobility in Structural Biology

Presenting Author - Aneika Leney

A.C. Leney

School of Biosciences, University of Birmingham, B15 2TT, UK

Red algae live deep in the ocean where sunlight is lacking, yet can still photosynthesise using light-harvesting

systems called phycobilisomes. These colourful, 17 MDa protein complexes work with 95% efficiency making

them the most efficient “solar panels’ known. Yet, how phycobilisomes function still remains elusive. Moreover, to

add complexity, thousands of protein post-translational modifications (PTMs) are thought to exist within the

phycobilisome whereby the exact location and composition of each PTM is crucial for efficient light transmission.

The work presented will describe how a combination of MS methods can be used to gain insight into

phycobilisome function. Our work, in particular, providing an excellent example of how native MS can be used

to elucidate the heterogeneity within large protein complexes, and how recent advances in top-down mass

spectrometry can shed light on the function of PTMs embedded within them.


Presenting Author - C. Malosse

C. Malosse1, M. Rey1, E. Durand2, E. Cascales2, J. Chamot-Rooke1

1 Mass Spectrometry for Biology Unit, Institut Pasteur, USR 2000, CNRS, Paris, (75015) France.

2 Laboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée,

UMR7255, Aix-Marseille Université - CNRS, Marseille, France.

Structural analysis of the Type VI bacterial secretion system using native mass spectrometry

To support their growth in a competitive environment and cause pathogenesis, bacteria have evolved a broad

repertoire of macromolecular machineries to deliver specific effectors and toxins. Among these multiprotein

complexes, the type VI secretion system (T6SS) is a contractile nanomachine that targets both prokaryotic and

eukaryotic cells. It comprises two functional subcomplexes: a tail composed of an inner tube wrapped by a

sheath and built on the baseplate which docks the tail to the membrane complex and hence serves as an

evolutionary adaptor.

In entero-aggregative E. coli, the baseplate is composed of the TssK,F,G and E subunits that assemble into a

multimer complex. To obtain the stoichiometry of the complex, we measured its mass in native conditions. From

these measurements an unexpected stoichiometry was obtained: TssK6F2GE. We then fragmented the intact

complex also to obtain elements of topology, in particular identify the subunits localized at the periphery of the

whole complex. Our results were confirmed by cryo-EM data.

Biogenesis and structure of a type VI secretion baseplate : Y. Cherrak, C. Rapisarda, R. Pellarin, G. Bouvier, B.

Bardiaux, F. Allain, C. Malosse, M. Rey, J. Chamot-Rooke, E. Cascales, R. Fronzes and E. Durand. Nature

Microbiology, doi.org/10.1038/s41564-018-0260-1.


Presenting Author - Dai Junxiao

Dai Junxiao, Emma Norgate, Rosie Upton, Bruno Bellina, Perdita E. Barran

The Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute of Biotechnology,

University of Manchester, UK

Ion mobility/time of flight mass spectrometry (IM-MS) can be used to examine the mass and stoichiometry of

proteins and their complexes as well as their conformations. We report here two experiments. Previous studies

in our group have reported Collision Cross Section Distribution (CCSD) and Full Width at Half Maximum

(FWHM) for IgG1, IgG4, Herceptin and the NIST mAb. Here we use Variable Temperature IM-MS (VT-IM-MS) to

monitor conformational changes that occur to mAbs (both IgG1 and IgG4) as the temperature of the drift gas is

lowered from 300K to 180K. Remarkably for three mAbs studied, we observe cold denaturation at 250K but

not at 190K. Secondly, we use VT-IM-MS to observe cis-trans isomerization of a 13-residue polyproline in gas-

phase. We aim to complement the work of Clemmer et al. which has used IM-MS to probe conformation

change based on temperature changes in solutions. We focus on Pro13 [M + 2H]2+ ion, which is the dominant

ion, and report conformational change over a temperature range from 300-520K.


Presenting Author - Dominik Saman

D. Saman1, M. Collier2, C. V. Robinson1, J. L. P. Benesch1

1 Department of Chemistry, University of Oxford, UK 2 Department of Biology, Stanford University, USA

For the first time, we are able to quantitatively study the mixing between two highly polydisperse systems using

a modified Q-Exactive mass spectrometer.

Here, we show that HSPB1, HSPB5 and HSPB6 form hetero-oligomers with each other in a different fashion -

ranging from a complete mixing between HSPB1 and HSPB5 to a highly specific n:n oligomers formed

between HSPB1 and HSPB6. From these results, we hypothesize that there are additional stabilising

interactions between HSPB1 and HSPB6, compared to HSPB1 and HSPB5, that strongly favour the n:n

oligomers. To prove this hypothesis, we further investigate the effects of deletion of the N-terminal and C-

terminal domains on the fashion of co-assembly between these proteins, as these have been strongly implicated

in controlling the polydispersity of the HSPBs on their own.

The implications of studying these kinds of problems with mass spectrometry are far-reaching - so far, people

have studied these problems mostly with SAXS-coupled chromatography, which usually only give results

averaged over the whole ensemble. Here, for the first time, we are able to observe and quantify each resulting

hetero-oligomer population.


Presenting Author - Jelena Cveticanin

" J. Cveticanin1, G. Arkind1, R. Netzer1, S. Fleishman1, A. Horovitz2, M. Sharon1"

"1 Department of Biomolecular Sciences, Weizmann Institute of Science, Israel 2 Department of Structural

Biology, Weizmann Institute of Science, Israel"

Double-mutant cycle analysis provides a strategy for studying the strength of pairwise interactions within and

between proteins. We have recently shown that these pairwise interaction energies can be determined from a

single native mass spectrum by measuring the intensities of the complexes formed by the two wild-type

proteins, the complex of each wild-type protein with a mutant protein, and the complex of the two mutant

proteins. This native mass spectrometry approach, obviates the need for error-prone measurements of binding

constants, and provides information regarding multiple interactions in a single spectrum. Here, we further

advanced the MS-based approach by demonstrating that it is possible to define pairwise interaction energies by

measuring a single native MS spectrum directly from crude cell lysate of bacteria co-expressing the four proteins

forming the cycle. The applicability of this native MS-based approach was tested for three different double-

mutant cycles using the 1:1 complex between E2 colicin endonuclease (colE) and the Im2 immunity protein (Im).

This method overcomes the need for purifying the target proteins, providing an efficient and rapid mean of

determining coupling energies. The results also indicate that inter-molecular hydrogen bond strengths are not

affected by the more crowded conditions in cell lysates.


Presenting Author - Jonathan P. Williams1

Jonathan P. Williams1, Jeff Brown1, Lindsay Morriso1, Christopher Hughes1, Joe Beckman2, Valery G. Voinov2,

Frederik Lermyte3

1 Waters, 2 Oregon State University and e-MSion, 3 Warwick University

SEC-Native Ion Mobility ECD MS of Intact Proteins and Protein Complexes:

Provision of Higher Order Structure and Sequence-Specific information

Electron Capture Dissociation has been used for the structural characterization of a number of standard proteins

in both their native and denatured forms. Electron excitation-based fragmentation techniques offer more

comprehensive sequence-specific product ions following fragmentation of multiply charged protein ions

compared to the conventional vibrational excitation method of CID. Furthermore, labile post-translational

modifications are retained compared to CID.

A prototype ECD cell developed by e-MSion, which entraps low-energy electrons from a heated filament

containing two magnets and six DC electrostatic lens, was installed within the Waters Synapt G2-Si. The cell

delivers the possibility of performing ECD either before or after the Travelling-Wave Ion Mobility device. Data

have been obtained using both infusion-based ECD MS and SEC-Native ECD MS/SEC-Native Ion Mobility

ECD MS experiments. The SEC-Native MS approach can be used for routine analysis where sample amounts

are not limiting.

Proof-of-principle Native Top-Down ECD MS experiments will be described following implementation of ECD

post-Travelling Wave Ion Mobility. Data have been obtained from yeast ADH, human hemoglobin, NIST mAb,

ideS-digested NIST mAb and GroEL.


Presenting Author - Lauren Tomlinson

Lauren J. Tomlinson1, Richard Bayliss2, Patrick A. Eyers3 and Claire E. Eyers1

1 Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of

Liverpool, Crown Street, Liverpool L69 7ZB

2 School of Molecular and Cellular Biology, Astbury Centre for Structural and Molecular Biology, University of

Leeds, Leeds LS2 9JT, UK

3 Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool

L69 7ZB

Protein phosphorylation is catalysed by protein kinases, which are important drug targets in human diseases

such as cancer. A plethora of physical techniques are now available to measure inhibitor-binding propensity

amongst different conformations of protein kinases. However, kinase inhibitors often suffer from an inherent lack

of specificity, driven by off-target binding to different protein kinase conformations presented to them in the

cell. Importantly, the structural factors that influence dynamic kinase interactions with drugs are not fully

understood. Structural mass spectrometry takes advantage of the fact that conformational information of

macromolecular complexes and the effects of phosphorylation and/or, ligand binding on protein structure are

preserved in the gas phase. MS-based information pertaining to structural stability and conformation can rapidly

inform other structural evaluation (such as X-Ray crystallography), and reveal, or confirm, novel drug-binding

modes and allosteric networks in kinases. To understand the effects of different classes of small molecules on

protein kinase conformation, we have carried out ion-mobility mass spectrometry on differentially

phosphorylated variants of purified Aurora A protein kinase both in the absence and presence of chemical

inhibitors. Our analysis reveals phosphorylation- and inhibitor-mediated changes in Aurora A conformational

dynamics and stability, as evidenced by collision-induced unfolding (CIU) profiling.


Presenting Author - Margit Kaldmäe

M. Kaldmäe1, E. G. Marklund2, M. Landreh1

1 Department of Microbiology, Tumour and Cell Biology, Karolinska Institutet, Sweden 2 Department of

Chemistry BMC, Uppsala University, Sweden

Biotechnological applications of protein complexes require detailed information about their structure and

composition, which can be challenging to obtain for proteins from natural sources. Prominent examples are the

ring-shaped phycoerythrin (PE) and phycocyanin (PC) complexes isolated from the light-harvesting antennae of

red algae and cyanobacteria. Despite their wide-spread use as fluorescent probes in biotechnology and

medicine, the structures and interactions of their non-crystallisable central subunits are largely unknown. Here, we

employ ion mobility mass spectrometry to reveal varying stabilities of the PC and PE complexes and identify

their closest architectural homologues among all protein assemblies in the PDB. Our results suggest that the

central subunits of PC and PE complexes, although absent from the crystal structures, may be crucial for their

stability, and thus of unexpected importance for their biotechnological applications.


Presenting Author - Maria Mateos-Jimenez

M. Mateos-Jimenez1, L. Regan2, A. Edkins3, C. Veale4, D. Clarke5

1 School of Chemistry, The University of Edinburgh, UK 2 School of Biological Sciences, The University of

Edinburgh, UK 3 Department of Biochemistry and Microbiology, Rhodes University, SA 4 School of Chemistry

and Physics, University of KwaZulu-Natal, SA 5 School of Chemistry, The University of Edinburgh, UK

The chaperone HSP90 is essential for protein homeostasis and cell survival, particularly in cancer where high

growth rates cause major cellular perturbations. In tumoral cells, HSP90 has been found significantly

overexpressed associated with the co-chaperone HSP70-HSP90 organising protein (HOP). The HOP-

HSP90 interaction is characterised by a ‘carboxylate clamp’ present on the TPR2A domain of HOP, which

binds to the conserved C-terminal MEEVD motif of HSP90. The main challenge faced by conventional

HSP90 inhibitors is the compensatory action of other chaperones that is induced upon treatment. Recent

evidence suggests that inhibiting the HOP-HSP90 interaction does not induce this compensatory response,

constituting an attractive approach for inhibiting HSP90.

Here we present our latest results on the application of native mass spectrometry and ion mobility techniques to

screen and characterise potential small molecule inhibitors of the TPR2A-HSP90 interface. These preliminary

results may provide some insights to the design of optimised ligands in later screening assays.


Presenting Author - Olivia Hepworth

O. W. Hepworth1, J. R. Naglik2 and A. J. Borysik1

1 Department of Chemistry, King's College London, UK

2 Centre for Host-Microbiome Interactions, King's College London, UK

Candidalysin is a cytolytic peptide toxin, released and produced by the fungus Candida albicans. It is the first

described fungal toxin established as the hyphal moiety responsible for mucosal damage and epithelial cell

activation in response to infection. Many research groups have provided a solid biological understanding of

Candidalysin. However, the molecular mechanism of toxicity is currently unknown, and the peptide’s structure

and dynamics are still poorly understood. In this study we use a range of mass spectrometry and biophysical

techniques to characterize the membrane embedded peptide assemblies with a view to understanding the

molecular basis of their toxicity. Using native mass spectrometry, we have identified that the peptide forms

oligomers, and we aim to model the peptide’s binding affinity in order to characterize the kinetics of association.

Analysis of Candidalysin’s interaction with phospholipid bilayers will also be investigated to identify a specific

membrane target of peptide binding. To determine the thermodynamics of formation, we examined the sub-

unit exchange between oligomers, and observed that the peptide forms stable structures in solution. The

combination of these findings will hopefully provide significant insights into Candidalysin’s structure and its

biological function.


Presenting Author - Rebecca Miller

R. Miller1,2, A. Dykstra2, W. Wei2, C. Holsclaw2, R. Schworer3, O. Zubkova3, P. Tyler, J3. Turnbull, J4. Leary2

1 Copenhagen Centre for Glycomics, University Of Copenhagen, Denmark.

2 Department of Molecular and Cellular Biology and Chemistry, University of California, US

3 Ferrier Research Institute, Victoria University of Wellington, New Zealand

4 Department of Biochemistry, University of Liverpool, UK

Heparin and heparan sulfate (HS) are poly-anionic molecules whose biological properties are determined by

their charge, sequence and conformation. They bind to and regulate many proteins including MCP-1. MCP-1 is

predominantly upregulated in rheumatoid arthritis, resulting in the upregulation of cytokines and an

inflammatory response that causes the enzymatic digestion of connective tissues. Small anionic molecules,

nucleotides, and peptides have shown promise for immune suppression; nevertheless logically HS is optimal as

it is a natural regulator of MCP-1 activation and inhibition.

The highly anionic charge of heparin/HS leads to a high proportion of attached sodium ions, resulting in

multiple sodiated complexes, salt clustering, increased complexity and reduced signal-to-noise quality. Studying

HS and HS:protein complexes requires improved desalting methods. The Solvay method (producing sodium

carbonate) was used to remove sodium from samples using carbon dioxide. This permitted ion mobility mass

spectrometry of previously inaccessible MCP-1: HS dimeric and tetrameric complexes. Peak broadening was

reduced by 32 Da to 5 Da and 24 Da to 4 Da respectively. This method of desalting was effective in reducing

sodium ion adductions and salt clusters, improving mass measuring accuracy and signal-to-noise quality from

oligosaccharides and protein:oligosaccharide complexes, thus relieving a critical bottleneck in the field.


Presenting Author - Shay Vimer

S. Vimer1, G. Ben-Nissan1, D. Morgenstern2, R.S. Quintyn3, V.H. Wysocki3 M. Sharon1*

1Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel

2Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot, Israel

3Department of Chemistry and Biochemistry, The Ohio State University, Columbus, USA

Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during

the course of evolution, each organism adapts to meet its physiological needs and the environmental challenges

imposed by its niche. This selection pressure leads to structural divergence of protein complexes, which are

often difficult to specify, especially in the lack of high-resolution structures. Here, we describe a multi-level

experimental approach that is based on native mass spectrometry tools for elucidating the structural variation

and preservation among highly related protein complexes. The 20S proteasome, an essential protein

degradation machinery, served as a model system, wherein we examined five complexes isolated from

different organisms. We show that along evolution, from the archaeal prokaryotic complex to the eukaryotic 20S

proteasomes from yeast and mammals, the proteasome increased both in size and stability. However,

unexpectedly, the yeast complex, and not the mammalians ones, exhibited the largest size and degree of

stability. Structural signatures of the rat and rabbit 20S proteasomes, which are lacking high-resolution three-

dimensional structures, indicated their high resemblance to the human complex. Moreover, we also identified a

new isoform of the PSMA7 subunit that resides within the two rodent complexes, which has not been

described before. Altogether, our strategy allows elucidating the unique structural properties of ortholog protein

complexes, an approach that can significantly deepen our understanding of the evolution of these complexes.


Presenting Author - Yuko P. Y. Lam

Yuko P. Y. Lam1, Cookson K. C. Chiu1, Christopher A. Wootton1, Meng Li1, Ian Hands-Portman2, Mark P. Barrow1,

Peter B. O’Connor1

1 Department of Chemistry, University of Warwick, Coventry, UK

2 Department of Life Sciences, University of Warwick, Coventry, UK

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic protein that aggregates rapidly in humans.

Classic structure-based design for therapeutics drug development against amyloid diseases is challenging as

most amyloid proteins are inherently disordered with high conformational flexibility. Herein, the measurements

involving mass spectrometry, fluorescence spectroscopy, and transmission electron microscopy have been

shown to be critical to precisely identify target regions of action for different classes of inhibitors. From our

results, two mechanisms are shown to effectively inhibit the aggregation of wild-type hIAPP either by site-

specific interaction with the amyloid critical aggregation region near the c-terminus of hIAPP or by accelerating

in the formation of non-toxic amorphous aggregates. Our data further shows that, upon deamidation of hIAPP,

aggregation could no longer be effectively prevented by some site-specific inhibition compounds, instead an

amorphous aggregates were rapidly formed. Insulin and (-)-Epigallocatechin 3-gallate (EGCG) prevent fibril

formation via different mechanisms. These methods not only help to explore the potential inhibition

mechanisms of hIAPP, but also apply to other amyloid protein-inhibitor studies, which aims to accelerate the

development of therapeutic drugs in amyloidogenesis.


Presenting Author - Zainab Ahdash

Z.Ahdash1, A.M.Lau1, R.T.Byrne2, K.Lammens2, A.Stüetzer3, H.Urlaub3,4, P.J.Booth1, E.Reading1, K.Hopfner2,

A.Politis1*

1 Department of Chemistry, King’s College London, 7 Trinity Street, London SE1 1DB, UK,

2 Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universitat Munchen, Feodor-Lynen-

Strasse 25, 81377 Munchen, Germany

3 Bioanalytical Mass Spectrometry Group, MPI for Biophysical Chemistry, D-37077 Gottingen, Germany

4 Bioanalytics Group, Institute for Clinical Chemistry, University Medical Center Gottingen, D-37075 Gottingen,

Germany

Mechanistic insight into the assembly of the HerA-NurA helicase-nuclease DNA end resection complex

The HerA-NurA helicase-nuclease complex cooperates with Mre11 and Rad50 to coordinate the repair of

double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the

HerA-NurA. By combining hybrid mass spectrometry with cryo-EM, computational and biochemical data, we

investigate the oligomeric formation of HerA and detail the mechanism of nucleotide binding to the HerA-

NurA complex from thermophilic archaea. We reveal that ATP-free HerA and HerA-DNA complexes

predominantly exist in solution as a heptamer and act as a DNA loading intermediate. The binding of either

NurA or ATP stabilizes the hexameric HerA, indicating that HerA-NurA is activated by substrates and complex

assembly. To examine the role of ATP in DNA translocation and processing, we investigated how nucleotides

interact with the HerA-NurA. We show that while the hexameric HerA binds six nucleotides in an “all-or-none’

fashion, HerA-NurA harbors a highly coordinated pairwise binding mechanism and enables the translocation and

processing of double-stranded DNA. Using molecular dynamics simulations, we reveal novel inter-residue

interactions between the external ATP and the internal DNA binding sites. Overall, here we propose a stepwise

assembly mechanism detailing the synergistic activation of HerA-NurA by ATP, which allows efficient processing

of double-stranded DNA.

POSTER No. 16 Biotherapeutics, Glycoproteins and Complex Bio-Molecules

Presenting Author - Anthony Ehkirch

A. Ehkirch1, O. Hernandez-Alba1, A. Goyon2, V. D’Atri2, F. Rouvière3, A. Beck4, S. Heinisch3, D. Guillarme2*, S.

Cianférani1

1 Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, France

2 School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Switzerland

3 Université de Lyon, Institut des Sciences Analytiques, CNRS UMR5280, Université de Lyon, France

4 IRPF - Centre d’Immunologie Pierre-Fabre (CIPF), France

Hyphenation of non-denaturing chromatographic methods to native mass spectrometry and ion mobility for

therapeutic protein characterization.

The development of therapeutic monoclonal antibodies (mAbs) progresses exponentially, with more than 75

therapeutic mAbs already approved by the FDA. Native mass spectrometry and its hyphenation to ion mobillity

(IM-MS) have gained interest for qualitative and quantitative characterization of biotherapeutics. However, online

hyphenation of non-denaturing liquid chromatography (ndLC) to native MS/IM-MS is still not straightforward,

which hampers its routine use in high throughput automated environments.

Native MS is mostly performed in ammonium acetate (AcONH4) while ndLC (e.g. size exclusion SEC or

hydrophobic interaction chromatography HIC) require large amounts of non-volatile salts. We first highlight the

benefits of 1D SEC-native MS in AcONH4 for automated online buffer exchange and aggregation studies. As

AcONH4 does not provide optimal chromatographic performances for HIC or SEC, bi-dimensional LCxLC

approaches combining HIC/SEC in first dimension and SEC in second one (HICxSEC for cysteine-linked ADC

or SECxSEC for forced degraded mAb studies) were next developed. The first dimension provides optimal

chromatographic separation, while second dimension serves for fast desalting. Our results demonstrate benefits

of ndLCxLC-nativeMS/IM-MS setups for comprehensive and streamlined characterization of mAb-based

products. The synergic online HIC/SEC coupling to native MS/IM-MS is envisioned to definitely push native MS

approaches at the forefront in biopharma companies.


Presenting Author - Ashley Bell

A. Bell1, E. Redman1, J. Mellors1

1 908 Devices Inc., Boston MA, USA

The field of native mass spectrometry has advanced significantly over the past several years. We believe that the

integration of a powerful online separation can advance the field of native mass spec even further. Capillary

Electrophoresis (CE), coupled via ESI, is a natural fit for native mass spec analysis because it can be performed in

native solvent conditions without concern for interactions with a chromatographic stationary phase. Additionally,

the slow diffusion of large molecules is a benefit to electrophoretic separation efficiency, often enabling

resolution between very minor structural differences between proteoforms. Successfully exploiting CE-ESI-MS

for native analysis requires a level of optimization (of surface chemistry, channel geometry, solvent conditions,

etc.) that has been beyond the reach of traditional CE-MS platforms. The continued advancement of our fully

integrated microfluidic CE-ESI platform has enabled us to start tackling these extremely challenging

applications. The work presented here demonstrates the separation and mass spec analysis of native proteins

and protein complexes for applications in biopharmaceutical characterization.


Presenting Author - Elizabeth Hecht

E.S. Hecht1, N. Swanson2. D. Tesar2, W. Sandoval

1 Microchemistry, Lipidomics, and Proteomics, Genentech, Inc., South San Francisco, CA USA

2 Pharmaceutical Development, Genentech, Inc. South San Francisco, CA USA

Understanding the interactions of host cell proteins and antibodies is critical to efforts to improve the drug

product purification process and prevent impurities. Polysorbase degradation has plagued the biotherapeutic

industry in recent years, with multiple reports of degradative products found, resulting in a shortened product

shelf life. Recent work has demonstrated the presence of Chinese hamster ovary phospholipase B-like 2 protein

(PLBL2) and lysosomal phospholipase A2 (LPLA2) in formulations of clinical antibody products. Ongoing efforts

to discover the mechanism by which lipases survive the purification process has led to the hypothesis that

LPLA2 may bind to an antibody when it is bound to the protein purification column, preventing dissociation.

This interaction has been supported and disproven using a variety of in vitro assays. The gold standard to assess

the protein antibody interaction would be native MS. Initially SEC-MS was attempted on an Exactive EMR but

the extensive and heterogeneous glycosylation on the lipase was problematic. Through transitioning

experiments to the UHMR and incorporating charge stripping, it was demonstrated that the protein complex

may be resolved. Furthermore, confirmation of the complex was achieved by top-down studies, leading to new

insights into the top-down behavior of antibody complexes.


Presenting Author - Florian Füssl

F. Füssl1, A. Criscuolo2&3, K. Cook4, J. Bones1

1 NIBRT - The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion,

Blackrock, Co. Dublin, A94 X099, Ireland

2 Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Germany

3 Thermo Fisher Scientific, Hanna-Kunath Strasse 11, 28199 Bremen, Germany

4 Thermo Fisher Scientific, Stafford House, 1 Boundary Park, Hemel Hempstead, HP2 7GE, United Kingdom

Unravelling the heterogeneity of chicken ovalbumin by novel MS friendly anion exchange chromatography on-

line hyphenated to native intact Orbitrap mass spectrometry

Proteins can be modified by a variety of different posttranslational modifications (PTMs), many of which are

directly related to protein function. These PTMs are responsible for the maintenance of fundamental cellular

processes and play essential roles in health and disease. In consequence PTMs need to be analysed by powerful

and reliable methods which can provide comprehensive qualitative and quantitative information. Since most

cellular proteins are anionic in nature, we developed an anion exchange chromatography - mass spectrometry

method for the on-line analysis of intact native anionic proteins. Elution is performed via a pH-gradient

maintained by low ionic strength volatile buffers that provide excellent chromatographic selectivity while

facilitating the acquisition of high quality MS data. Using ovalbumin as a model system, LC-MS analysis resulted

in the identification and relative quantification of fifteen different ovalbumin charge variants together with their

glycoform distributions. Furthermore we identified numerous species corresponding to ovalbumin fragments

and dimers, in total culminating in more than 100 different proteoforms. This novel method is incredibly

powerful and can provide new insight into protein microheterogeneity of anionic proteins.


Presenting Author - Johanna Paris

J. Paris 1, T. Morgan1, Y. Lam1, C. Wootton1, J. O'hara2, P. O'connor1

1 Departement of Chemistry, University of Warwick, UK 2 UCB, Slough, UK

Recombinant monoclonal antibodies and derivatives are widely used as therapeutic drugs. They are susceptible

to posttranslational modifications (PTMs) that could occur during the manufacturing process and storage,

resulting in product-related impurities. PTMs can change the efficacy, toxicity and the clearance of the antibody;

therefore, they need to be well monitored and characterised.

The poster will investigate the potential of 2D MS for proteomics, and especially for the analysis of antibodies.

2D MS is a powerful technique, which enables the correlation between the precursor and its fragments in a

single experiment without prior isolation. The trypsin digest of an antibody was analysed by 2DMS using

IRMPD on the 12T FT-ICR mass spectrometer. The analysis of posttranslational modifications was investigated for

the analysis of phosphorylation on proteomics samples. Finally, the use of 2D MS in a top down procedure for

the analysis of the antibody will be discussed around the MS/2DMS analysis of the light chain.


Presenting Author - Jonathan Bones

F. Fuessl1, C. Jakes1, S. Carillo1, A. Bell2, E. Redman2, K. Cook3, J. Bones1,3

1 National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Co. Dublin, A94

X099, Ireland

2 908 Devices, 645 Summer Street, Boston, Massachusetts, 02210, USA

3 Thermo Fisher Scientific, Stafford House, 1 Boundary Park, Hemel Hempstead HP2 7GE, United Kingdom

4 School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4, D04 V1W8,

Ireland

Cetuximab is a chimeric human-mouse monoclonal antibody (mAb) targeting epidermal growth factor receptor.

Cetuximab is a complex mAb, with two N-glycosylation sites present at asparagine 88 and 299 of the heavy

chain. C-terminal lysine variants are also present. These posttranslational modifications create a range of charge

variant isoforms of Cetuximab. Cation exchange chromatography and capillary electrophoresis are routinely used

for charge variant analysis of mAbs. Hyphenation to mass spectrometry has proved difficult due to

incompatibility of mobile phases or the background electrolyte. Characterization of Cetuximab using pH

gradient cation exchange chromatography using volatile buffers and microchip electrophoresis coupled to

native mass spectrometry is described, demonstrating comparable performance for unravelling the complexity

of proteoforms present within the separated charge variants of Cetuximab. Excellent comparability between the

CVA-MS and the ZipChip CE-MS platforms with similar speed of analysis revealing complementarity between

both methods. A key feature of the ZipChip CE-MS was lower sample requirements, however, CVA-MS was

more tolerant of buffer additives due to sample focusing effects on column and the higher analytical flow rate.

Both methods are ideally suited for rapid characterization of complex biopharmaceuticals, facilitating an insight

into the natural dynamic range of proteoforms of the molecule.


Presenting Author - Meng Li

M. Li1, Y. P. Y. Lam1, P. Chen2, R. Gavard3, C. K. C. Chiu1, C. A. Wootton1, Q. Wu2, M. P. Barrow1, H. Fu2, P. B.

O’Connor1

1 University of Warwick, Department of Chemistry, Coventry, UK 2 Peking University, School of Pharmaceutical

Science, Beijing, China 3 University of Warwick, MAS CDT, Coventry, UK

Of the 1400 species of scorpions worldwide, the Chinese scorpion is used in Chinese medicines for

cardiovascular problems, antimicrobial, and tumors. The venom is a complex mixture of proteins containing

bioactive components, some showing inhibition of enzyme factor Xa, which prevents blood coagulation. Crude

scorpion venom was fractionated using step elution on C18 cartridge, and in-vitro inhibition of factor Xa was

tested. Nano-LC-FTICR MS/MS analysis of each fraction was acquired using FT-ICR MS.

Using direct infusion, more than hundred unique molecular species were observed in the crude venom, but

with bottom-up proteomics, only a small number of proteins can be identified, even though many of these

unidentified peptides have good fragmentation spectra. To accurately identify potential bioactive compounds,

methodologies were developed to correlate the spectra with the bioactivity results. A list of compounds that

strongly correlate to the bioactivity could be potential pharmaceutical targets. These potential targets are then

sequenced using ECD, IRMPD and CAD. The potential factor Xa inhibitors were de novo sequenced using

both top-down and bottom-up approaches. The high mass accuracy and resolving power of the FTICR-MS aids

the sequencing study of these novel scorpion venom proteins. The method is applicable to identifying potential

pharmaceutical leads from other natural products.


Presenting Author - Sara Carillo

S. Carillo1, F. Füssl1, N. Dorival-Garcia1, C. Ta1, P.S. Kelly2, N. Barron2, J. Bones2,3

1 Characterisation and Comparability Laboratory, NIBRT. Foster Avenue, Mount Merrion, Blackrock, co. Dublin.

Ireland

2 National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland

3 School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4

CVA-MS To Investigate The Effects Of Leachables From Single-Use Bioreactors On MAbs

The implementation of single-use disposable bioreactors in bioprocess introduces a variety of new materials into

the pipeline. These plastics, when used under normal conditions, can degrade and leach breakdown products

into the culture media.

In this work, media was incubated in two types of single-use bioreactors to obtain a preconditioned media

containing leachables arising from operation under normal operational conditions (37ÂºC/7 days in rocking

incubator). The media was used to evaluate the growth curve of CHO-DP12 cell line showing a detrimental

effect on cell growth. The anti-IL8 IgG1 produced by CHO DP12 cell line was analyzed as well.

Charge variant analysis (CVA) is a powerful tool for mAbs characterisation and comparability, especially when

hyphenated with high resolution mass spectrometry (CVA-MS) for the identification of proteoforms under

native conditions. We were able to highlight significant differences in the CVA profiles between the product

obtained from the media preconditioned in the single-use bioreactor and the control, including monitoring of

N-glycans variation, increased levels of C-terminal lysine loss and proline amidation, and formation of dimeric

species. These results outline a picture of the potential effects of leachables exposure on mAb bioproduction.


Presenting Author - Victor Mikhailov

V. Mikhailov, S. Liu, C. Schofield, J. McCullagh

Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK

Native Mass Spectrometry for an Enzyme/Substrate/Inhibitor System: Competitive Binding and Binding

Affinities.

We have developed a native MS method for quantification of interactions between substrates and inhibitors with

a target enzyme. Our model enzyme is human isocitrate dehydrogenase 1 (IDH1) is a cytoplasmic enzyme

which catalyses the reversible conversion of isocitrate to 2-oxoglutarate (2OG) and CO2. A single point R132H

mutation of IDH1 turns 2OG to 2-hydroxyglutarate (2HG) using NADPH as the cofactor. This inhibits some

epigenetic regulators, and is associated with a variety of cancers including over 70% of gliomas and 30% of

myeloid leukemias. With isocitrate and 2OG being our model substrates for the wild type and mutant IDH1,

respectively, our model inhibitors are the ones that are currently in preclinical and clinical trials by several pharma

companies.

In this work we use “native’ (non-denaturing) MS to investigate the following: (1) binding of the substrates to wild

type (wt) and R132H mutant of IDH1, (2)effect of Mg2+ ions on the substrate binding, (3) binding of several of

IDH1 inhibitors to the WT and R132H variants, (4) competitiveness of the binding between the inhibitors and the

substrates.

Our results demonstrate the versatility of the native MS approach to study enzyme/substrate/inhibitor systems

and its consistency with the data from other methods.


Presenting Author - Wendy Sandoval

W Sandoval1, W Phung1, S Polderdijk2, AO Bailey3, G Han3, PJ Carter2, J Lill1

1Department of Microchemistry, Proteomics & Lipidomics, Genentech, Inc. South San Francisco, CA, USA.

2Department of Antibody Engineering, Genentech, Inc. South San Francisco, CA, USA.

3Department of Proteomics, BGI Americas, San Jose, CA, USA.

Bispecific antibodies (BsAb) combine the specificities of two antibodies to target different antigens. While

production and in vivo assembly of BsAb using a single-cell host is less resource intensive compared to two-cell

production, unwanted mis-paired species are produced. Different pairing strategies can be employed to

increase correct BsAb content, in which species must be both identified and quantified. Quantitative analysis by

mass spectrometry is limited to chromatographic separation of the antibody assemblies due to their similar

properties. This is particularly challenging for distinguishing correct and double light chain mis-paired antibodies

which have the same molecular weight. Traditionally, iCIEF is used for analyzing charge-based heterogeneities,

but lacks resolution. Here we describe a powerful analytical platform using native charge-variant MS to

characterize bispecific and mis-paired antibody species. We investigate elution order through analytical methods,

imaging, and molecular modeling in an effort to understand the intrinsic charge, size and shape differences of

these molecules. Although isoelectric points of the variants are similar, localized charge patches are

hypothesized to offer sufficient charge difference to be able to resolve then identify isobars using CV-MS.


Presenting Author - Y. Xu1,2

Y. Xu1,2, R. J. Foster1,3, F. Sobott1,2, S. E. Radford1,2

1 Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK 2 School of Molecular

and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK 3 Department of Chemistry, University of Leeds,

Leeds LS2 9JT, UK

There are more than 50 human diseases which are characterised by the aberrant aggregation of proteins and

peptides into amyloid fibrils. Despite the numerous efforts to develop inhibitors targeting protein aggregation,

only one small-molecular inhibitor has been successfully discovered. This is largely due to the lack of structures of

the intrinsically disordered target proteins/peptides and the poor understanding of the mechanism of the

amyloid formation. Therefore, the development of small-molecule modulators which can specifically bind to the

amyloidogenic proteins/peptides and inhibit amyloid assembly is of great importance for the investigation of

the fundamental molecular mechanism of amyloid-induced cytotoxicity and for the treatment amyloid diseases.

Recently, our group has applied a rapid method, native electrospray ionisation-ion mobility -mass spectrometry

(ESI-IM-MS), for the analysis of protein aggregation and its inhibition by small molecules. The results show that

this method can effectively differentiate the mode of inhibition of the inhibitors and identify individual species

which the inhibitors bind to. Here we apply the technique for the screening of small molecules from different

compound libraries against human islet amyloid polypeptide (hIAPP or amylin), a peptide closely-related to

type-2 diabetes mellitus. Preliminary results demonstrate the ability of the ESI-IM-MS technique for the high-

throughput screening of new small-molecular binders towards hIAPP. Base on the MS profiles, a classification

system has been developed to define the mode of inhibition and to distinguish the molecules from the

screened compound library. Hit compounds found in the MS screening will be further validated by different

biophysical assays, such as NMR, thioflavin-T bioassay, and transmission electron microscopy (TEM).

POSTER No. 27 Membrane Proteins

Presenting Author - A. Moysa

A. Moysa1, D. Hammerschmid2, R. Szczepanowski3, F. Sobott2,M. Dadlez1

1 Institute of Biochemistry and Biophysics, PAN, 2 Biomolecular & Analytical Mass Spectrometry, University of

Antwerp, 3 International Institute of Molecular and Cell Biology

The pattern recognition receptor RAGE (receptor for advanced glycation end-products) transmits

proinflammatory signals in several inflammation-related pathological states, including vascular diseases, cancer,

neurodegeneration and diabetes. Its oligomerization is believed to be important in signal transduction, but

RAGE oligomeric structures and stoichiometries remain unclear. Different oligomerization modes have been

proposed in studies involving different truncated versions of the extracellular parts of RAGE. Here, we provide

basic characterization of the oligomerization patterns of full-length RAGE (including the transmembrane (TM)

and cytosolic regions) and compare the results with oligomerization modes of its four truncated fragments. For

this purpose, we used native mass spectrometry, analytical ultracentrifugation, and size-exclusion

chromatography coupled with multi-angle light scattering. Our results confirm known oligomerization

tendencies of separate domains and highlight the enhanced oligomerization properties of full-length RAGE.

Mutational analyses within the GxxxG motif of the TM region show sensitivity of oligomeric distributions to the

TM sequence. Using hydrogen-deuterium exchange, we mapped regions involved in TM-dependent RAGE

oligomerization. Our data provide experimental evidence for the major role of the C2 and TM domains in

oligomerization, underscoring synergy among different oligomerization contact regions along the RAGE

sequence. These results also explain the variability of obtained oligomerization modes in RAGE fragments.


Presenting Author - Dietmar Hammerschmid

D. Hammerschmid1,2, C. Venien-Bryan3,,S. Dewilde1,, F. Sobott2,4,5

1 Department of Biomedical Sciences, University of Antwerp, Belgium

2 Chemistry Department, University of Antwerp, Belgium

3 Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, Sorbonne University, France

4 Astbury Centre for Structural Molecular Biology, University of Leeds, UK

5 School of Molecular and Cellular Biology, University of Leeds, UK

Native MS has become impressively powerful in characterizing membrane proteins ranging from detergent

screen experiments, lipid binding studies, top-down fragmentation, and collision-induced unfolding (CIU)

experiments. Here we present an example for the application of this comprehensive toolbox for characterization

of a membrane-bound globin coupled sensor from Geobacter sulfurreducens (GsGCS).

If extracted and purified in OG detergent micelles, GsGCS assembles to an equilibrium between trimeric and

tetrameric species. Other detergents, e.g. DDM, lead to a disruption of these higher-order oligomers, leaving

only monomers behind. However, applying “mixed” micelles (OG + DDM) could maintain the oligomeric

structure highlighting that perhaps only a few surrounding molecules are crucial for the protein’s assembly state.

CIU experiments emphasize the important role of a few bound detergent molecules. Additionally, CIU indicates

a higher stability of the ferrous form of the protein (central Fe2+ of the prosthetic heme group), compared to the

Fe3+ state. Interestingly, top-down fragmentation with precursor selection (UHMR Orbitrap) and without (Synapt

G2 HDMS) result in the same y-ion fragment series which corresponds to a membrane helix that might play a

role in the (in)activation of the protein. Bringing these data together enables more target-oriented planning of

further high-resolution and/or patch-clamp electrophysiology experiments.


Presenting Author - Euan Pyle

E. Pyle1,2, A. C. Kalli3, S. Amillis4, Z. Hall5, A. M. Lau2, A. C. Hanyaloglu6, G. Diallinas4, B. Byrne1, A. Politis2

1 Department of Life Sciences, Imperial College London, London SW7 2AZ, UK 2 Department of Chemistry,

King's College London, London SE1 1DB, UK 3 Leeds Institute of Cancer & Pathology and Astbury Centre for

Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK 4 Department of Biology, National and

Kapodistrian University of Athens, Panepistimioupolis, 15781 Athens, Greece 5 Department of Biochemistry,

University of Cambridge, Cambridge CB2 1GA, UK 6 Institute of Reproductive and Developmental Biology,

Department of Surgery and Cancer, Imperial College London, London W12 0NN, UK

The role of membrane lipids in modulating eukaryotic transporter assembly and function remains unclear. We

investigated the effect of membrane lipids in the structure and transport activity of the purine transporter UapA

from Aspergillus nidulans. We found that UapA exists mainly as a dimer and that two lipid molecules bind per

UapA dimer. We identified three phospholipid classes that co-purified with UapA: phosphatidylcholine,

phosphatidylethanolamine (PE), and phosphatidylinositol (PI). UapA delipidation caused dissociation of the

dimer into monomers. Subsequent addition of PI or PE rescued the UapA dimer and allowed recovery of

bound lipids, suggesting a central role of these lipids in stabilizing the dimer. Molecular dynamics simulations

predicted a lipid binding site near the UapA dimer interface. Mutational analyses established that lipid binding

at this site is essential for formation of functional UapA dimers. We propose that structural lipids have a central

role in the formation of functional, dimeric UapA.


Presenting Author - Jani Reddy Bolla

J. Bolla1, A.Howes1, F.Fiorentino1, C.Robinson1

1. Department of Chemistry, University of Oxford, OX1 3QZ

Translocation of lipid II across the cytoplasmic membrane is essential in peptidoglycan biogenesis. Although

most steps are understood, identifying the lipid II flippase has yielded conflicting results, and the lipid II binding

properties of two candidate flippases—MurJ and FtsW—remain largely unknown. Here we apply native mass

spectrometry to both proteins and characterize lipid II binding. We observed lower levels of lipid II binding to

FtsW compared to MurJ, consistent with MurJ having a higher affinity. Site-directed mutagenesis of MurJ

suggests that mutations at A29 and D269 attenuate lipid II binding to MurJ, whereas chemical modification of

A29 eliminates binding. The antibiotic ramoplanin dissociates lipid II from MurJ, whereas vancomycin binds to

form a stable complex with MurJ:lipid II. Furthermore, we reveal cardiolipins associate with MurJ but not FtsW,

and exogenous cardiolipins reduce lipid II binding to MurJ. These observations provide insights into

determinants of lipid II binding to MurJ and suggest roles for endogenous lipids in regulating substrate binding.


Presenting Author - Julian Bender

J. Bender1, C. Schmidt1

1 Interdisciplinary research center HALOmem, Charles Tanford Protein Centre, Martin Luther University Halle-

Wittenberg, Halle (Saale), Germany.

Fusion of synaptic vesicles with the presynaptic membrane is regulated by the calcium-sensor Synaptotagmin-1

(Syt-1). Syt-1 consists of two soluble calcium-binding C2-domains anchored to the vesicle membrane by a flexible

linker. Its functional role requires Syt-1 interacting with the presynaptic membrane.

We combine biochemical and biophysical techniques with structural mass spectrometry (MS) to characterise lipid

interactions of Syt-1. For this, we recombinantly expressed Syt-1, purified the protein via a cleavable His-tag, and

confirmed its identity by mass spectrometry. We generated three variants: the full-length protein (FL), the

cytoplasmic domain (Cyt), and the C2-domains lacking the flexible linker (C2AB).

Native MS and cross-linking (XL-MS) of Cyt and C2AB revealed oligomerisation in the absence of a lipid

membrane, while native and ion-mobility MS helped investigating binding of negatively charged lipid

headgroups to positively charged polybasic stretches involved in membrane-binding of Syt-1. We further

assessed binding of soluble Syt-1 variants to lipid membranes using flotation assays of liposomes with different

lipid compositions.

Finally, FL was incorporated into liposomes and analysed by XL-MS. In contrast to the soluble variants, Syt-1 did

not oligomerise in a native-like environment.

We applied, for the first time, mass spectrometry to explore interactions of Syt-1 with a lipid membrane.


Presenting Author - Mark T. Agasid

Mark T. Agasid, Alexandra Wilson, Joseph Gault, Idlir Liko, Carol V. Robinson

University of Oxford

The physiological composition of salts may have profound impacts on the functional properties of proteins. We

present work on nano-electrospray ionisation tips to achieve high-resolution mass spectra of membrane proteins

introduced into the gas-phase from high salt buffers.


Presenting Author - Tiago Moreira

Tiago Moreira1, Rod Chalk1, Emma Mead2, Katharina Duerr1, and Nicola Burgess-Brown1

1 Structural Genomics Consortium, University of Oxford, OX3 7DQ

2 Oxford Drug Discovery Institute, University of Oxford, OX3 7DQ

Development of a novel MS platform for native structure determination and ligand identification for detergent

solubilised membrane proteins.

We have previously developed methods for top-down, intact mass analysis of membrane proteins in a range of

detergents under denaturing LC-MS conditions, but they reveal no information regarding tertiary or quarternary

structure, nor do they reveal functional information such as ligand binding and conformational changes

associated with it. Application of collisional activation to cause gas-phase detergent stripping has been successful

with, for example KCNK10, but detergent interference in the spectrum remains a problem. In collaboration with

Agilent we established that cooling in the source is not possible on the 6500 series without total redesign of

the ion optics. However, it can be introduced in the collision cell and we found that introduction of N2 at 40 psi

dramatically improves the ion signal. Upgrading the collision gas from N2 to Ar also improves ion transmission

and makes detergent stripping by collisional activation more efficient. We show how this system performs in

terms of resolution and ligand identification and show how it may be used analysis of membrane proteins and

amyloid proteins.

POSTER No. 35 Integrative Native MS - HDX, X-linking, Labelling in Structrual Biology

Presenting Author - Friedel Drepper

F. Drepper, D. Wendscheck, S. Fischer, J. Bender, A. Schummer, S. Oeljeklaus, B. Warscheid

Institute of Biology II, University of Freiburg, Germany

Biogenesis of peroxisomes requires the import of nuclear-encoded matrix proteins into the organelle facilitated

by the peroxisomal matrix protein import machinery. The receptor Pex5p recognizes matrix proteins in the

cytosol and recruits them to a docking complex consisting of peroxisomal membrane proteins Pex14p and

Pex13p. In yeast, it additionally contains Pex17p and the soluble dynein light chain protein Dyn2p. Subsequently,

a dynamic import pore is formed by which the cargo is translocated across the membrane and released into the

lumen of the organelle by an unknown mechanism. We have analysed dynamic protein interactions of yeast

Pex14p and Pex5p using affinity-purified native Pex14p complexes and recombinantly expressed proteins.

Chemical cross-linking combined with mass spectrometry revealed homo- and hetero-oligomeric interactions of

Pex14p, Pex17p and Dyn2p. Native MS studies showed a predominant trimeric association of Pex14p

dependent on the presence of its predicted coiled-coil domains. For Dyn2p, solely dimeric species were

observed. Using gas phase collisional activation, we analysed the stability of reconstituted Pex5p receptor-cargo

complexes. To reveal regulatory mechanisms, we mapped in vivo phosphorylation sites in Pex14p and Pex5p

and generated phosphomimicking site mutants. Native MS analyses suggest that phosphorylation of specific

serine residues modulates their dynamic protein interactions.


Presenting Author - J. Bellamy-Carter

J. Bellamy-Carter, L. Manzi, N. Oldham

School of Chemistry, University of Nottingham, UK

Identifying Interactions between proteins, or between proteins and small molecules is key to understanding

biology at a molecular level, and can greatly assist in the drug discovery process. Footprinting techniques utilise a

covalent label, such as hydroxy-radicals or carbenes, to map solvent accessible residues and sample native

structure with bottom-up approaches. A differential experimental design using the presence or absence of a

binding partner then allows identification of interaction sites through site-specific masking of protein labelling.

LC-MS analysis of digested protein permits high resolution location and quantitation of modified sites. While

data generation is rapid, the data are complex, making data processing laborious and potentially prone to error.

An open-source graphical user interface software - PepFoot - has been developed to enable semi-automated

processing of footprinting data, allowing for rapid characterisation of protein interaction sites. The platforms

removes the bottleneck for high-throughput screening of binding site mapping. It inputs data from footprinting

experiments, identifies peptides of interest, quantifies labelling, performs batch extraction from all experiment

files and provides analysis features for structural interpretation of the data.


Presenting Author - Jake Busuttil-Goodfellow

J.A. Busuttil-Goodfellow, A.E. Ashcroft, J.R. Ault, F. Sobott

Astbury Centre for Structural Molecular Biology, University of Leeds, UK

This project involves perturbing native structures of proteins and analysing changes in the structure via fast

photochemical oxidation of proteins (FPOP). The structures will be perturbed by excipients, crowding agents

and a high concentration of alternative proteins. The aim of the project is to gain more knowledge into the

accessibility of hydroxyl radicals from this method, and therefore attempt to discover the relationship between

solvent accessibility and amino acid reactivity in relation to hydroxyl labelling.

FPOP has the advantage of being significantly faster to label than alternative techniques such as HDX, in

addition to creating permanent covalently bonded labels. Analysis can be performed on both intact proteins and

via MS/MS of peptides, offering residue level information. Further, labels are added to side chains as opposed

to the amide backbone, giving information on the location of specific amino acid side chains.

CaM and Myo will be used as test systems, with both holo and apo forms of Myo interrogated. Given these

structures are well characterised in literature, utilising FPOP on their perturbed structure will give us a strong

indication how labelling changes in these test systems.


Presenting Author - Julien Marcoux

J. Lesne1, J. Parra1, D. Zivkovic1, T. Menneteau1, M. Chavent1, M. Locard-Paulet1, M.P. Bousquet-Dubouch1, O.

Burlet-Schiltz1, J. Marcoux1

1 Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, Université Paul Sabatier,

31000 Toulouse, France

Study of the largest and most heterogeneous macromolecular complex by HDX-MS brings new important

mechanistic insights in proteasome regulation.

The 20S proteasome is composed of four heptameric-stacked rings. It degrades proteins in a tightly controlled

fashion, thereby directly regulating intracellular concentration of cytokines and hub proteins, and may generate

immunogenic peptides. Alteration of its activity can lead to cancers, heart and auto-inflammatory diseases. It can

be regulated by replacing its constitutive catalytic subunits and/or by interacting with different activators.

However, whether its catalytic subunits composition favors the interaction with a particular regulator is still

unclear.

We utilized HDX-MS to investigate the impact of the catalytic subunit composition of the 20S on its structure

and association to specific activators.

Human standard and immuno proteasomes were deuterated alone or bound to the PA28αβ²/PA28γ ³

activators. We successfully optimized the classical HDX-MS workflow in terms of sample preparation,

chromatography and MS acquisition to work on both poorly concentrated and very heterogeneous protein

complexes. The average sequence coverage was excellent: 82% for twenty ~30kDa monomers. Our dataset

suggests a reciprocal crosstalk between the inner and outer rings that not only represents a methodological

breakthrough but also brings invaluable insights into the proteasome dynamics and regulation.


Presenting Author - R. Parakra

R. Parakra1, J. Phillips2

Living Systems Institute, University of Exeter

Hydrogen-deuterium mass spectrometry (HDX-MS) is a widely used technique for probing protein-ligand

binding. The existing notion is that ligand binding should reduce deuteration rates, and it is often logical that this

reduction will be most pronounced in the vicinity of the interaction site. Protein biosensors are ubiquitous in

nature and are at the forefront of synthetic biology. It is of utmost importance to understand how they transduce

a signal: binding a target ligand with subsequent correlated conformational and functional changes. We have

developed a statistical method to inform on the structural mechanism of signal transduction, using HDX-MS

data. This method uses a clustering algorithm to define subpopulations of protein biosensor conformers that

contribute to ligand binding and allostery. This method makes a step forward using HDX-MS data to build

mechanistic models of protein biosensing/functional switching.


Presenting Author - Sabine Wittig

S. Wittig1, M. Ganzella2, S. Kostmann1, A. Pérez-Lara2, R. Jahn2, C. Schmidt1

1 Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Martin Luther University Halle-

Wittenberg, Germany 2 Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen,

Germany

Synaptic vesicles (SVs) are small storage organelles for neurotransmitters. They are densely packed with proteins

and pass through a trafficking cycle in the nerve terminal. Available models assume random distribution of the

proteins in the membrane, however, there is evidence that they form functionally active assemblies. We set out

to unravel these assemblies by employing chemical cross-linking experiments combined with native mass

spectrometry.

In initial experiments proteins within intact SVs were chemically cross-linked using BS3 cross-linker to identify

first interaction networks in synaptic vesicles. These networks revealed many protein interactions with

Synaptobrevin-2 which plays a central role in these assemblies. To study Synaptobrevin-2 in detail we purified

three variants, the full-length protein, the cytosolic domain as well as a shorter version. Combining chemical

cross-linking and native mass spectrometry we found that Synaptobrevin-2 oligomerises in solution presumably

due to its disordered structure. Using different approaches, we targeted Synaptobrevin-2 in its natural

environment, i.e. the SV membrane. We found that many protein interactions between Synaptobrevin-2 and

other SV proteins are formed due to its unstructured and dynamic nature. We further identified local networks

independent of Synaptobrevin-2 suggesting their importance for the SV trafficking cycle.


Presenting Author - W. Sadowska1,2

K. Przygoñska1, M. Dadlez1,3

1 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland, 2 Faculty of Chemistry,

University of Warsaw, Warsaw, Poland, 3 Institute of Genetics and Biotechnology, Department of Biology,

University of Warsaw, Warsaw, Poland

Native Mass Spectrometry in analysis of coexisting oligomeric forms of Amyloid βpeptides

Recent data shows that the soluble oligomeric forms of Amyloid β (Aβ) peptide are the main neurotoxic agent

in Alzheimer’s disease. Native MS supported by Ion Mobility (IM) separation and gas-phase Hydrogen-

Deuterium exchange (HDX) enables structural analysis of Aβpeptide oligomers, which brings more information

about the mechanism of Aβpeptide oligomerization.

In this research, the analysis of Aβpeptides with point-mutations in the sequence provide us with information

about which amino acids are crucial in the process of oligomerization. In MS experiments, native Aβpeptides

milliseconds after ESI are exposed to ND3 gas, which cause exchange of the heteroatom-bound non-amide

side-chains hydrogens. Labeling is performed by the cone-exit methodology. After that labeled species are

separated by IM. Combination of these techniques is very unique since it allows to analyze oligomeric forms

coexisting in the gas-phase, also it gives insight into higher-ordered structures of intact proteins, which are

preserved by using soft ionization.

POSTER No. 42 Close-Up's of Proteins in the Gas Phase (Structrue and Modelling)

Presenting Author - Anna L. Simmonds

A.L. Simmonds1,2, P.J. Winn1, J.K. Heath1, D.H. Russell3, I.B. Styles2,4 and H.J. Cooper1

1School of Biosciences, University of Birmingham, U.K., 2Physical Sciences for Health CDT, University of

Birmingham, U.K., 3Texas A&M University, College Station, U.S.A., 4School of Computer Science, University of

Birmingham, U.K.

In order to understand protein structure in the gas phase it is important to understand the way that mass

spectrometry techniques such as fragmentation and ion mobility spectrometry (IMS) are affected by their

structure. We have been working on a number of peptides to study how the presence of non-covalent

interactions affects electron capture dissociation (ECD), electron transfer dissociation (ETD) and travelling wave

ion mobility spectrometry (TWIMS). We show that ECD can be used to refine the results of an IMS/molecular

modelling (MM) workflow on an intact peptide derived from the sequence of GSK3β and that ETD-TWIMS can

be used to allow an IMS/MM workflow to be applied to fragments of that peptide. Modelling of ETD c+ and z•

fragments has been facilitated by parameterisation of the residues adjacent to the cleavage site and assuming

that the charge/radical imparted by the cleavage process is localised to the residue adjacent to the cleavage site.

POSTER No. 43 The Future of Structural MS - Native Top-Down, Automation, New Methods & Workflows

Presenting Author - Anisha Haris

Anisha Haris, Yuko P. Y. Lam, Christopher A. Wootton, Alina Theisen, Cookson K. C. Chiu, Tomos E. Morgan, Mark

P. Barrow, and Peter B. O’Connor

Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK

Comparison of UVPD and ECD to distinguish between the isomeric products of deamidation using ultra high

resolution FT-ICR MS.

Deamidation is a spontaneous non-enzymatic post translational modification (PTM), whereby asparagine (Asn) is

converted to aspartic acid (Asp) and isoaspartic acid (isoAsp). The isoforms of Asp (L-Asp, D-Asp, L-isoAsp and

D-isoAsp) can inactivate or contribute to unwanted isomers. Understanding the balance between these forms as

well as being able to distinguish between the isomeric pairs is important. For this research, only Asp and isoAsp

were compared, but D- and L-stereoisomer studies are planned.

The ability to distinguish between Asp and isoAsp will help us to gain a deeper understanding of the effect that

they have on protein structure, which is known to affect functionality. For example, in monoclonal antibodies, the

deamidation of Asn55 in the heavy chain complementarity determining region (CDR) to Asp and isoAsp

reduces the antigen binding affinity and thereby reduces the overall antibody activity.

Electron capture dissociation (ECD) is beneficial in distinguishing between Asp and isoAsp as the diagnostic

fragment ions (c+57/z-57) are generated for isoAsp. Another activation method is ultraviolet photodissociation

(UVPD), which can be used to activate and fragment ions using photons with the product ions detected by FT-

ICR MS. UVPD produces a wide array of fragments and at 213 nm, UVPD is employed to distinguish between

the isomers Asp and isoAsp in bovine serum albumin peptides.


Presenting Author - Eva Illes-Toth

E. Illes-Toth1, H. J. Cooper1

1 University of Birmingham, School of Biosciences, Edgbaston, Birmingham, B15 2TT

Probing protein-ligand interactions of bovine carbonic anhydrase by native LESA mass spectrometry

Native LESA (Liquid Extraction Surface Analysis) mass spectrometry is a technique that enables direct sampling

of proteins from a variety of substrates under near native conditions. Our aim is to utilise this tool to characterise

protein-ligand interactions with respect to stoichiometry and binding affinities directly from their physiological

environment.

Here, we explored the binding of bovine carbonic anhydrase (CAH) with chlorothiazide, dansylamide and

sulfanilamide using both LESA and direct infusion electrospray in combination with high resolution- and ion

mobility mass spectrometry. For LESA, solutions containing CAH in 25 mM NH4OAc mixed with each ligand

at a series of concentrations were deposited and dried onto glass covered with Al foil. Subsequently, the dried

sample spots were extracted using 25 mM NH4OAc. Relative intensities of bound ligands showed good

agreement between LESA and direct infusion. We illustrate that chlorothiazide binds most tightly, followed by

dansylamide and then by sulfanilamide. Ligand binding was first observed with chlorothiazide at a protein:ligand

ratio of 1:0.01, with dansylamide at 1:0.1 and with sulfanilamide at 1:0.5. Respective collision cross sections of

the protein-ligand complexes have been determined.

Our data demonstrate the suitability of LESA for studying protein-ligand interactions under native conditions.

Future efforts will focus on determination of binding affinities (Kds).


Presenting Author - Hannah Ochner

Hannah Ochner1, Sven Szilagyi1, Sabine Abb1, Stephan Rauschenbach1,2, and Klaus Kern1,3

1 Max-Planck-Institut für Festkörperforschung, Heisenbergstr. 1, 70569 Stuttgart

2 Chemistry Research Laboratory, Department of Chemistry, University of Oxford

3 École Polytechnique Fédérale de Lausanne

Protein functionality is intimately linked to the protein’s native 3D folding structure. Determining these structures

is of tremendous importance for understanding such functionality as well as related biological processes.

Advances in structure determination are strongly coupled to imaging method developments. The imaging

method to be presented here, Low Energy Electron Holography (LEEH) works very well in conjunction with

native Electrospray Ion Beam Deposition as sample preparation method. Recently, it has been shown that this

approach enables imaging of proteins at the single molecule level (without averaging), while avoiding radiation

damage [1]. LEEH [2] is a lens-free imaging method in which the sample is radiated by coherent low energy

electrons (50-200eV) [3] to form holograms that in principle contain full 3D information of the object.

Thus, this technique can serve as a complementary method for protein structure determination, especially for

types of proteins that are hard to access using other methods such as Cryo-EM or X-ray crystallography.

We present the current state of the experiment and the reconstruction process along with future plans to

enhance resolution and to improve the reconstruction towards including 3D information.

References

[1] PNAS 114, 1474-1479 (2017)

[2] Phy. Rev. Lett, 1990, 65(10), 1204-1206.

[3] Phys. Scr., 1988, 38, 260


Presenting Author - Moritz Heusel

Moritz Heusel

Lund University

Quantitative mapping of virulence factor proteoform landscapes and the adaptive immune response.

Infectious diseases pose a major burden on contemporary human health, especially with the rapid emergence

of antibiotic resistances and strains of high virulence. The coevolutionary race between host defense and

pathogen virulence factors occurs at the biomolecular and proteoform level. On the one hand the host adaptive

response system generates panels of neutralizing antibodies that, if identified, bear the potential for therapeutic

application. On the other hand, pathogen virulence factor structure and function is modulated through a plethora

of chemical modifications. We develop multi-layer including top-down mass spectrometric strategies to shed

light both on the adaptive host immune response as well as the quantitative proteoform pattern observed across

bacterial strains of varying virulence.


Presenting Author - Paul Fremdling

P. Fremdling1, J. Gault1, I. Pokun1, S. Rauschenbach1

1 Department of Chemistry, University of Oxford, UK

Establishing the exact molecular architecture of biological macromolecules like proteins is crucial for the

understanding of their interactions within a living cell1. However, traditional structure determination techniques

exhibit severe limitations: X-ray crystallography fails on any molecule refractory to crystallization2, whereas Cryo-

Transmission-Electron-Microscopy (Cryo-TEM) critically relies on homogeneous samples3.

Chemical selection prior to the imaging process has significant potential to control the sample composition and

reduce heterogeneity. We achieve this with preparative Electrospray Ion Beam Deposition (ES-IBD), allowing us

to deposit mass-filtered macromolecules for further characterization with TEM or Scanning Probe Microscopy

(SPM)4.

To facilitate deposition for analytical imaging, a commercially available mass-spectrometer has been modified

accordingly. We demonstrate the performance of this setup through ion current and beam energy

characterization and, successfully deposited BSA on a graphite surface and confirmed its presence with SPM.

1. Robinson, C. V., Sali, A. & Baumeister, W. Nature 450, 973-982 (2007).

2. Drenth, J. & Mesters, J. Principles of Protein X-Ray Crystallography. (Springer, 2007).

3. Renaud, J.-P. et al.. Nat. Rev. Drug Discov. 17, 471-492 (2018).

4. Rauschenbach, S., Ternes, M., Harnau, L. & Kern, K. Annu. Rev. Anal. Chem. 9, 473-498 (2016).


Presenting Author - Sven Szilagyi

S. Szilagyi1, H. Ochner1, L. Krumbein1, J. Gault2, A. Konijnenberg3, E. Martin3, J. Benesch2, F. Sobott3, C.

Robinson2, S. Abb1, S. Rauschenbach1,2, K. Kern1,4

1 Department of Nanoscale Science, Max Planck Institut for Solid State Research Stuttgart, DE 2 Department of

Chemistry, University of Oxford, UK 3 Department of Chemistry, University of Antwerp, NL 4 Laboratory of

Nanosclae Science, École polytechnique fédérale de Lausanne, CH

Native electrospray ionization has been shown to successfully bring proteins and protein complexes in their

natively folded state into the gas phase, where further analysis by mass spectrometry and ion mobility

spectrometry can be performed [1]. However, these methods are not sufficient for determining structural details

at the level of imaging techniques such as TEM, AFM, STM or low energy electron holography (LEEH), which

require a very clean sample preparation process. Here, we demonstrate the usage of electrospray ion beam

deposition (ES-IBD) as a tool for sample preparation of folded proteins for single

molecule microscopy [2]. We present examples of successfully deposited molecules imaged using the above

techniques and explore the infuence of different substrates and environmental conditions.

[1] Nat. Meth., 5(11), 2008, 927-933.

[2] Annu. Rev. Anal. Chem. 2016, 9:16.1-16.26.

Delegates

Dr Sabine Abb MPI for Solid State Research, Germany [email protected]

Dr Mark Agasid University of Oxford, UK [email protected]

Zainab Ahdash King's College London, UK [email protected]

Prof Perdita Barran University of Manchester, UK [email protected]

Dr Cherine Bechara IGF, France [email protected]

Ashley Bell 908 Devices, USA [email protected]

Jedd Bellamy-Carter University of Nottingham, UK [email protected]

Julian Bender MLU Halle-Wittenberg, Germany [email protected]

Prof Justin Benesch University of Oxford, UK [email protected]

Dr Jani Bolla University of Oxford, UK [email protected]

Dr Jonathan Bones NIBRT, Ireland [email protected]

Dr Hannah Britt University College London, UK [email protected]

Maxim Brodmerkel Uppsala Universitet, Sweden [email protected]

Dr Kelly Broster Thermo Fisher Scientific, UK [email protected]

Dr Rebecca Burnley UCB, UK [email protected]

Jake Busuttil-Goodfellow University of Leeds, UK [email protected]

Dr Antonio Calabrese University of Leeds, UK [email protected]

Dr Iain Campuzano Amgen, USA [email protected]

Dr Sara Carillo NIBRT, Ireland [email protected]

Dr Rod Chalk SGC University of Oxford, UK [email protected]

Dr Julia Chamot-Rooke Institut Pasteur, France [email protected]

Shane Chandler University of Oxford, UK [email protected]

Siyun Chen University of Oxford, UK [email protected]

Dr Dror Chorev University of Oxford, UK [email protected]

Dr Sarah Cianferani CNRS University of Strasbourg, France [email protected]

Jan Commandeur MS Vision, The Netherlands [email protected]

Dr Ken Cook Thermo Fisher Scientific, UK [email protected]

Prof Helen Cooper University of Birmingham, UK [email protected]

Dr Dale Cooper-Shepherd Waters, UK [email protected]

Joana Costeira-Paulo Uppsala University, Sweden [email protected]

Dr Jelena Cveticanin Weizmann Institute of Science, Israel [email protected]

Junxiao Dai Unviersity of Manchester, UK [email protected]

Eugen Damoc Thermo Fisher Scientific, Germany [email protected]

Dr Rachel Davis UCB, UK [email protected]

Dr Matteo Degiacomi Durham University, UK [email protected]

Evolène Desligniere CNRS University of Strasbourg, France [email protected]

Dr Andrew Dolan University of Oxford, UK [email protected]

Marek Domin Boston College, USA [email protected]

Dr Katharina Dornblut Abcam, UK [email protected]

Dr Adam Dowle University of York, UK [email protected]

Dr Friedel Drepper University of Freiburg, Germany [email protected]

Dr Kyle D'Silva Thermo Fisher Scientific, UK [email protected]

Dr Tom Durrant University of Oxford, UK [email protected]

Anthony Ehkirch LSMBO CNRS, France [email protected]

Charles Eldrid University College London, UK [email protected]

Dr Victoria Ellis UCB, UK [email protected]

mailto:[email protected]










Prof Claire Eyers University of Liverpool, UK [email protected]

Simon Fairless Thermo Fisher Scientific, UK [email protected]

Francesco Fiorentino University of Oxford, UK [email protected]

Dr Kyle Fort Thermo Fisher Scientific, Germany [email protected]

Tristan Free Future Science, UK [email protected]

Paul Fremdling University of Oxford, UK [email protected]

Dr Florian Fuessl NIBRT, Ireland [email protected]

Dr Valérie Gabelica INSERM-CNRS University of

Bordeaux, France

[email protected]

Dr Joseph Gault University of Oxford, UK [email protected]

Dr Agni Gavriilidou OMass Therapeutics, UK [email protected]

Dr Anirban Ghosh IECB University of Bordeaux, France [email protected]

Dietmar Hammerschmid University of Antwerp, Belgium [email protected]

Anisha Haris University of Warwick, UK [email protected]

Dr Katharina Häussermann Refeyn Ltd, UK [email protected]

Dr Elizabeth Hecht Genentech Inc, USA [email protected]

Prof Albert Heck Utrecht University, The Netherlands [email protected]

Olivia Hepworth King's College London, UK [email protected]

Dr Oscar Hernandez-Alba LSMBO CNRS, France [email protected]

Dr Moritz Heusel Lund Univeristy, Germany [email protected]

Dr Anna Higgins University of Leeds, UK [email protected]

Dr Kin Kuan Hoi University of Oxford, UK [email protected]

Adam Hold LC/MS Consulting Ltd, UK [email protected]

Dr Jonathan Hopper OMass Therapeutics, UK [email protected]

Dr Steven Howell The Francis Crick Institute, UK [email protected]

Anna Howes MRC-LMB Cambridge, UK [email protected]

Dr Timea Eva Illes-Toth University of Birmingham, UK [email protected]

Dr Ali Jazayeri OMass Therapeutics, UK [email protected]

Kiani Jeacock University of Edinburgh, Scotland [email protected]

Prof Keith Jennings The Queen's College, Oxford, UK [email protected]

Dr Margit Kaldmäe Karolinska Institutet, Sweden [email protected]

Dr Gabriella Kiss Refeyn Ltd, UK [email protected]

Dr Albert Konijnenberg Thermo Fisher Scientific, The

Netherlands

[email protected]

Dr Pui Yiu Lam University of Warwick, UK [email protected]

Thomas Lambert Univeristy of Edinburgh, UK [email protected]

Dr Michael Landreh Karolinska Institutet, Sweden [email protected]

Dr Matthias Langhorst Refeyn Ltd, UK [email protected]

Dr Aneika Leney University of Birmingham, UK [email protected]

Meng Li University of Warwick, UK [email protected]

Dr Idlir Liko OMass Therapeutics, UK [email protected]

Justin Lock Waters, UK [email protected]

Prof Paul Madden The Queen's College, Oxford, UK [email protected]

Christian Malosse Institut Pasteur, France [email protected]

Dr Julien Marcoux IPBS, France [email protected]

Dr Erik Marklund Uppsala University, Sweden [email protected]

Dr Emma Marsden-Edwards Waters, UK [email protected]












Sarah Maslen MRC-LMB Cambridge, UK [email protected]

Maria Mateos Jimenez The University of Edinburgh, UK [email protected]

Dr Shahid Mehmood The Francis Crick Institute, UK [email protected]

Dr Thomas Menneteau UCL, UK [email protected]

Dr Victor Mikhailov University of Oxford, UK [email protected]

Prof Rebecca Miller University of Copenhagen, Denmark [email protected]

Rani Moons University of Antwerp, Belgium [email protected]

Dr Tiago Moreira SGC University of Oxford, UK [email protected]

Tomos Morgan University of Warwick, UK [email protected]

Alexander Moysa IBB PAN, Poland [email protected]

Dr Chris Nortcliffe SCIEX, UK [email protected]

Hannah Ochner MPI for Solid State Research, Germany [email protected]

Prof Peter O'Connor University of Warwick, UK [email protected]

Dr Krzysztof Okrasa Sosei Heptares, UK [email protected]

Prof Neil Oldham University of Nottingham, UK [email protected]

Anna Olerinyova University of Oxford, UK [email protected]

Dr Abraham Oluwole University of Oxford, UK [email protected]

Dr Madalina Oppermann Thermo Fisher Scientific, Sweden [email protected]

Nicklas Österlund Stockholm University, Sweden [email protected]

Dr Martin Palmer Waters, UK [email protected]

Rinky Parakra University of Warwick, UK [email protected]

Johanna Paris University of Warwick, UK [email protected]

Dr Nisha Patel UCB Celltech, UK [email protected]

Dr Gary Paul 908 Devices, UK [email protected]

Dr Jonathan Phillips University of Exeter, UK [email protected]

Ikhlaas Pokun University of Oxford, UK [email protected]

Dr Argyris Politis King's College London, UK [email protected]

Dr Tanya Prentice Oxford Nanopore Technologies, UK [email protected]

Eduard Puig IRB Barcelona, Spain [email protected]

Euan Pyle Imperial College London, UK [email protected]

Xingyu Qiu University of Oxford, UK [email protected]

Daniel Quetschlich University of Oxford, UK [email protected]

Dr Ritu Raj University of Oxford, UK [email protected]

Oliver Rather Bruker Daltonik GmbH, Germany [email protected]

Prof Stephan Rauschenbach University of Oxford, UK [email protected]

Prof Dame Carol Robinson University of Oxford, UK [email protected]

Jennifer Ross University of Edinburgh, UK [email protected]

Wiktoria Sadowska University of Warsaw, Poland [email protected]

Dr Bodhisattwa Saha University of Oxford, UK [email protected]

Dominik Saman University of Oxford, UK [email protected]

Wendy Sandoval Genentech Inc, USA [email protected]

Joshua Sauer University of Oxford, UK [email protected]

Dr Charlotte Scarff University of Leeds, UK [email protected]

Prof Carla Schmidt MLU Halle-Wittenberg, Germany [email protected]

Dr Kundan Sharmaudsc University of Oxford, UK [email protected]

Prof Michal Sharon Weizmann Institute of Science, Israel [email protected]









Denis Shutin University of Oxford, UK [email protected]

Anna Simmonds University of Birmingham, UK [email protected]

Emma Sisley University of Birmingham, UK [email protected]

Dr Mark Skehel MRC Cambridge, UK [email protected]

Prof Frank Sobott University of Leeds, UK [email protected]

Dr Zoja Soloviev GlaxoSmithKline, UK [email protected]

Fabian Soltermann University of Oxford, UK [email protected]

Dr Lars Sørensen Novo Nordisk, Denmark [email protected]

Hamish Stewart Thermo Fisher Scientific, Germany [email protected]

Sven Szilagyi MPI for Solid State Research, Germany [email protected]

Dr Haiping Tang University of Oxford, UK [email protected]

Dr Christopher Taylor University of York, UK [email protected]

Dr Rohan Thakur Bruker Daltonik GmbH, Germany [email protected]

Dr Konstantinos Thalassinos University College London, UK [email protected]

Dr Daniel Tome Bruker UK Ltd, UK [email protected]

Lauren Tomlinson University of Liverpool, UK [email protected]

Andreas Topp F.Hoffmann - La Roche Ltd.,

Switzerland

[email protected]

James Town University of Warwick, UK [email protected]

Dr Leonhard Urner University of Oxford, UK [email protected]

Gerard van der Laan MS Vision, The Netherlands [email protected]

Shay Vimer Weizmann Institute of Science, Israel [email protected]

Dr Rosa Viner Thermo Fisher Scientific, USA [email protected]

Zihao Wang University of Oxford, UK [email protected]

Dr Jonathan Williams Waters, UK [email protected]

Sabine Wittig MLU Halle Wittenberg, Germany [email protected]

Dr Therese Wohlschlager University of Salzburg, Austria [email protected]

Dr Lucy Woods Bruker Daltonik GmbH, Germany [email protected]

George Wright University of Oxford, UK [email protected]

Dr Di Wu University of Oxford, UK [email protected]

Dr Yong Xu University of Leeds, UK [email protected]

Dr Hsin-Yung Yen OMass Therapeutics, UK [email protected]

Oliver Yu University of Oxford, UK [email protected]

Michal Zawadzki Syngenta, UK [email protected]





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Celebration of Native Mass Spectrometry...2 Institut Européen de Chimie et Biologie , Université...

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