DAH3.1 Mass Spectrometry

Kathryn Lilley

Cambridge Centre for Proteomics

Department of Biochemistry

University of Cambridge

k.s.lilley@bioc.cam.ac.uk

www.bio.cam.ac.uk/proteomics/

Part III Systems Biology

Definition of the Proteome

The analysis of the entire PROTEin complement expressed by a genOME.

Wasinger et al Electrophoresis 16 (1995)

Could be:Cellular extractSecreted fluidTissueWhole organism

Why bother studying it????

CCaammbbrriiddggee CCeennttrree ffoorr PPrrootteeoommiiccss

A Proteomicist’s Tools

• Mass spectrometry• Protein and peptide separation methods• Databases and software

• Validation tools – Western blotting– GFP tagging and confocal microscopy

Instruments for mass analysis

Mass Spectrometers measure m/z of gaseous ion

Mass spectrometers comprise:

A sourcewhich is responsible for ionising the sample, e.g. electrospray, laser desorption

An analyserwhich separates and carries the ions to the detectore.g. Quadrupole, Ion-trap (mass range 2000-4000 m/z)TOF (time of flight) (mass range 0-200,000+ m/z)

A detectore.g. electron multiplier

Outline

• Proteomics workflows

• Protein identification

• Post translational modification

Protein analysis on pure proteins/complexes

Mass of protein

Modification statuscan be difficult to deconvolute with many isoforms

Higher order structuresability to spray whole complexes and look at components and

stoichiometries

Low through put methods

Usually carried out on pure proteinsor complexes

Already know what the protein is

Protein analysis on complex mixtures

For more complex samples you cannot purify each one and then analyse it.

Methods need to be applied where proteins can be analysed simultaneously

Proteins can be separated then analysed or converted to peptides which are then analysed

The peptides act as surrogates for the protein

Types of protein analysis

Proteins present PPI SCL Function

Mass Spectrometry

Western blotting

GFP tagging

Immuno- histochemistry

Enzyme assay

Arrays

Abundance

Quantitative Mass Spec

Western blotting

GFP tagging

Immuno- histochemistry

Enzyme assay

Arrays

Isoform status

Mass Spectrometry

Western blotting

Functional arrays

Y2H

Tagging + Mass Spectrometry

Western blotting

Structural studies

Protein Arrays

Biophysical assays (e.g.ITC,

AUC)

Mass Spectrometry

GFP tagging

Immuno- histochemistry

Enzyme assay

Functional arrays

Enzyme assay

Genetic approaches

Which proteins are present

Trypsin

Peptides

Mass Spectrometry

Western blotting

GFP tagging

Immuno- histochemistry

Enzyme assay

1D gel 2D gel Solution Digest

Workflow 1

• MALDI/MS

• Peptide mass fingerprinting

Mass Spectrometry

Western blotting

GFP tagging

Immuno- histochemistry

Enzyme assay

ExciseDigest Apply to MALDI ToF

Matrix Assisted Laser Desorption Ionisation (MALDI)

-cyano-4-hydroxycinnamic acid

Linear Detector

Sample target

N2 Laser

Ion Beam

Matrix Suppression

Lens

Reflectron Detector

Reflectron Assembly

Gas Cell

The chemical matrix absorbs energy from the laser pulse which is transferred to the proteinThe sample ions are then accelerated towards the detectorPrincipally produces M+H+ ions (sometimes M+2H+ )

MALDI Tof MS

Trypsin

K

RR

K

1457.351765.331975.722055/782589.31

Matrix assisted desorption time of flight mass spectrometry

Mass list

Database search of virtual trypsin digested translated genome

Identification !!!!

Peptides

Peptide Mass Fingerprinting

Score = 110

Limitations

Only works well for purified proteins

Require well annotated genome

Strengths

QuickCheap

Workflow 2

• HPLC peptide separation

• Electrospray ionisation

• LC MS/MS

Mass Spectrometry

Western blotting

GFP tagging

Immuno- histochemistry

Enzyme assay

Digest

MS CID MS

Chromatography separations

High Performance Liquid Chromatography (HPLC)

Strong cation exchange (SCX)Separation based on net charge of peptide

Weak anion exchange (WAX)Separation based on net charge of peptide

Reverse phase (RP)Separation based on hydrophobicity

Hydrophobic interaction chromatography (HILIC)Separation based on hydrophobicity

Bind peptides

Elute with gradient

e.g. acetonitrile for reverse phaseIncreasing salt for SCX

Mass Spectrometry

Western blotting

GFP tagging

Immuno- histochemistry

Enzyme assay

LC-MS/MSMolecular ion (precursor) is accelerated into collision cell where it collides with an inert gasSome of the kinetic energy is converted to internal (vibrational) energyPeptide cleavage takes place largely at the peptide bond nearest a mobile proton

Net result is:

Detect positively charged fragments which contain either the original N-terminus or C-terminus of the peptide

Tandem Mass Spectrometry LC-MS/MS:

Data Dependent Acquisition in MS

Q CID ToF

Precursor ionselection basedon intensity

Precursors scannedout of first quad.

Collision induced dissociation

All fragment ions analysed

Typical output

• List of peptide masses

– Precursor mass (parent ion mass)

• Fragment ion masses

– y-ions– b-ions

Protein identification

• Search engines

MASCOT - http://www.matrixscience.com

SEQUEST - fields.scripps.edu/sequest/

X ! Tandem -www.thegpm.org/tandem/index.html

Phenyx- www.genebio.com/products/phenyx/

Score = 960

MUDPIT

• Data dependent acquisition means that only the most intense ions at any given time are taken for MS/MS

• To improve coverage, peptide simplification is required

MUDPIT

Multi dimensional proteinIdentification technology

Washburn et al (2001)Nat. Biotech 19:242

Strengths and weaknesses

• Can be used with very complex mixtures of proteins

• If the genome is not sequenced then sequence returned may show similarity or identity to related organisms

• De novo sequencing

• More time consuming• Equipment more expensive

Can you be sure?Validation??

• GFPUsing molecular biology techniques fuse gene encoding a

fluorescent protein to your protein of interest.

• Western blotting

Proteins from gel blotted onto PVDF membrane

Primary antibody – raised against your protein of interest

Secondary antibody – raised against the first antibody constant regions

Enzyme, fluorescent tag

Quantitative Western Blotting on a System-wide Scale

Quantitative western blotting of 75% of yeast proteome

Ghaemmaghami et al, 2003

A massive amount of work

Not transferable to many organisms

GFP tagging of yeast proteome

Huh et al, 2003

GFP tagged proteins

75% of the yeast proteome classified to 22 distinct location

Systems wide immuno-

histochemistry

Barbe et al, 2008

Antibodies to 488 proteins applied to 3 different human cell lines and images stored and publically accessible

Blue = DAPI staining of nucleus

Types of protein analysis

Proteins present PPI SCL Function

Mass Spectrometry

Western blotting

GFP tagging

Immuno- histochemistry

Enzyme assay

Arrays

Abundance

Quantitative Mass Spec

Western blotting

GFP tagging

Immuno- histochemistry

Enzyme assay

Arrays

Isoform status

Mass Spectrometry

Western blotting

Functional arrays

Y2H

Tagging + Mass Spectrometry

Western blotting

Structural studies

Protein Arrays

Biophysical assays (e.g.ITC,

AUC)

Mass Spectrometry

GFP tagging

Immuno- histochemistry

Enzyme assay

Functional arrays

Enzyme assay

Genetic approaches

Protein Isoform Analysis

Proteins may be:

– Covalently modified– Truncated– Dimerised

Isoform status

Mass Spectrometry

Western blotting

Functional arrays

Post Translational Modifications

100s of different PTMs

Most commonly characterised

– Phosphorylation xxx– Acetylation/Methylation x– Ubiquitination x– Sumoylation xx– Glycosylation– S-nitrosylation xxxx– …………………….

Isoform status

Mass Spectrometry

Western blotting

Functional arrays

Phosphorylation

Phosphorylation is a very important PTM

Signalling pathwaysProtein conformational changes

Serine, threonine and tyrosine are the most frequently phosphorylated residues

Phosphorylation

Most popular approaches

• 32P incorporation to track peptides and quantify recovery

• Isolate / enrich phosphopeptides by metal-chelation chromatography

• Use triple-quad and hybrid-Tof instruments to look for neutral mass loss

• Prediction algorithms

Problems with Phosphoproteomics

• Phospho groups are highly dynamic

• Phospho tyrosine is very rare

• Phosphopeptides ionise poorly, they tend to be very acidic

• The phosphate group tends to fall off pSer and pThr during MS/MS

Phospho-protein and -peptide enrichment

• Phospho-tyrosine

– good antibodies

• Phospho-serine and phospho-threonine

– Metal chelate chromatography– Ion exchange chromatography

AgaroseBead

Fe3+

NTA

OH

OPO

O

COOH

NH2

Immobilized metal affinity chromatography Titanium (IMAC) Dioxide

Enrichment methods for phosphopeptides

Ferric or Gallium columns most usually employed

Mass Spectrometry of Phosphopeptides

• Standard methods

• Neutral loss

• ETD

Precursor ion Loss of PO3- group Intense fragment ion peak at m/z = 747.94 (2+) m = 98 Da, z = 2+ m/z = 698.94 (747.94 – 49)

m/z = 49

RLSIELTNSLLRP P

RLSIELTNSLLR

Neutral Loss

Electron Transfer Dissociation (ETD).

Gentler fragmentation than CID Preserves post-translational modifications, such as

phosphorylation Produces c and z ions Better sequence coverage than CID

[M + 3H]2+•

[M + 3H]3+ + A- [M + 3H]2+• + A

Electron Transfer Dissociation (ETD)

[C+2H]1+ + [Z+H]1+•

C Z

R

R

>1 eV Electron “Thermal”

e-

-

+

FluorantheneRadical Anion (Good Electron Donor/ETD Reagent)

CID: Prominent loss of phosphate

Parent ion = 571.22

538.25 = loss of phosphoric acid

200 300 500 700 800 1100 1300 1500 1600 1800 1900 2000m/z

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100538.25

559.02

742.61

Cambridge_A1 T:

400 600 900 1000 1200 1400 1700

Cambridge_A1T:Cambridge_A1

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Cambridge_A1

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Rel

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bund

ance

(M+3H-H3PO4)3+

KKSSLSSNVGSTVKPPTKLSSNVGSTVKPPTK

ETD:ETD: KKSSLSSNVGSTVKPPTKLSSNVGSTVKPPTK

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

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Rel

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856.18

1711.71

571.03

791.141694.83

1581.70957.40600.31

754.38

1479.611112.54870.421157.58714.42554.32233.32 1058.49

653.44513.29 1399.721286.90

Methylation/Acetylation

Pang et al (2010) Identification of arginine- and lysine-methylation in the proteome of Saccharomyces cerevisiae and its functional implicationsBMC Genomics 2010, 11:92

Choudhary, et al. (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions, Science 325, 834-840.

More straightforward, but will be issues with digestion rates if trypsin is used

Can enrich, antibody affinity capture to acetyl-lysine

Ubiquitin

MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG

Kirkpatrick D.S., Denison C., and Gygi S.P., Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics. Nat Cell Biol, 2005. 7(8): p. 750-7

Ubiquitin is a small highly conserved eucaryote protein, it attaches to other protein via lysine residues by ubiqutin ligases, often marking proteins for degradation by the proteasome system

………….X-X-X-K-X-X……………….

..R-L-R-G-G-

UBIQUITIN

SUBSTRATE PROTEIN

Tryptic digestion

R G D E L Q K G A F LI

GG

SUMO

Small Ubiquitin-like Modifier

3 (4) versions

Many functions including stability, nuclear-cytosolic transport, and transcriptional regulation

Sadly there is no well placed tryptic site or site for any other common protease near the point at which it attaches to its modification target

MSMS spectra are thus a mess, as two sets of –b and –y ions will be produced per with SUMO modification

Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software. Pedrioli PG, et al. Nat Methods. 2006 3(7):533-9.

Galisson et al (2011) Mol. Cell Prot. A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells

Engineered SUMO is Hek293 cells to have strategically located tryptic site and (His)6 for purification

Bruderer et al EMBO Rep. 2011 Feb;12(2):142-8..Purification and identification of endogenous polySUMO conjugates.

E3 ligase inactive RNF4 fragment binds polySUMO

Glycosylation

Common, up to 50% of human proteins are glycosylated

Need to fond site of attached, using N-linked (Asn) or O-linked (Ser)

Also need to determine structure of glycosyl group

Very complex, highly combinatorial

The most challenging PTM for high throughput proteomics

Enrichment possible with lectin affinity chromatography

Typical schema used in large scale glycoproteome analysis

Sialylation

Dephosphorylation using a phosphatase

Palmisano et al (2010) Nat. Protocols 5

Sialyl groups are sometimes found as end caps of glycan chains.

They are extremely important in recognition between molecules. Sialyl lewis x, for instance, is important in ABO blood antigen determination and correct functioning of the immune response

Sialylation status has also been implicated in metastasis

Truncations

• Easiest way look at peptide coverage

• N-terminal peptide analysis– Edman degradation

COFRADICCombined fractional diagonal

chromatographyAcetylate total proteins with acetic anhydride. All amino

groups acetylatedDigest with trypsin

Carry out liquid chromatography (usually reverse phase) and collect peptides in fractions

Modify all new N-termini generated with trypsin with TNBS (this makes the peptide very hydrophobic).

Rerun all fractions using same LC conditions as before. Peptide which eluted in the same place are the original N-terminus, those that move are internal peptides.

AcAc

Ac

******Gevaert et al (2003) Nat. Biotech 21:566

This week

• Tuesday:– 1pm Seminar by Matthias Mann– 2.15pm Q and A session with Prof. Mann and

lecture for me

• Wednesday:– 9am – Practical Class– 4pm Lecture from me