Jet Stream Thermal Focusing Electrospray in Quantitative … · 2019. 10. 4. · Jet Stream Thermal...

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Jet Stream Thermal Focusing Electrospray in Quantitative

Proteomics

Stephanie M. Cologna, Ph.D.

Department of Chemistry, Laboratory for Integrative Neuroscience

For Research Use Only. Not for use in diagnostic procedures.

Molecular Processes of Neurodegeneration: Proteins and Lipids

Biochemistry/

Molecular Biology

Translational/Clinical Need

Analytical Chemistry

Spectrometry

Advances for Proteomics

• Soft ionization

• High resolution/mass accuracy mass spectrometers

• Database and informatic tools

• Separation science

Electrospray Approaches

www.wikipedia.com

www.newobjective.com

High Flow ESI?

Agilent Jet Stream (AJS) Electrospray

Towards Label-Free Proteomics

Pergande, et al., Proteomics. 2019.

AJS vs ChipCube NanoESI

Pergande, et al., Bioanalysis. 2019.

Research Program Question

What chemical signal(s) drive a neuron

(or other cell in the CNS) to survive or

Neurodegenerative Disorders• Class of diseases with progressive neuron loss

• Degeneration of the central nervous system

• Neurodegenerative disorders include:

• Alzheimer

• Parkinson

• Huntington

• Lysosomal storage disorders

• Oftentimes, mis-folded proteins, fibrilation or

deposits are observed

Cholesterol• In mammals, synthesized enzymatically from acetyl-CoA and

obtained from the diet peripherally

• Brain cholesterol is synthesized endogenously and tightly regulated

• Required for cell membrane structure and function

• Cholesterol has been implicated in neurodegenerative disorders

J.E. Vance, Disease Models and Mechanisms, 2013, Vol 5 (6).

Niemann Pick Disease, Type C (NPC)

• A fatal, autosomal recessive neurodegenerative disease

• Accumulation of unesterified cholesterol and

glycosphingolipids in the endosomal/lysosomal system

• Due to mutations in the NPC1 or NPC2 gene

Mutations in the NPC1 gene account for approximately

95% of diagnoses

www.mayoclinic.org

NPC1 and cholesterol transport

Kwon,et al., Cell 137, (2009)1213–1224

Cologna, S.M., Rosenhouse-Dantkster, in revision.

NPC Neuropathology

• Neuronal apoptosis in the cerebral cortex and

cerebellum

• Progressive neuronal loss, especially of cerebellar

Purkinje neurons

• Neuron degeneration mechanism is unclear

• Some pathological overlap with Alzheimer

Disease, such neurofibrillary tangles and

tau hyperphosphorylation

Courtesy of Ian Williams, Ph.D., NIH

Clinical Presentations of NPC

• Large variation in age of onset, and severity

• NPC has a wide spectrum of clinical presentations including

neurological symptoms such as:

• Ataxia

• Progressive dementia

• Seizures

• Vertical gaze palsy

• Currently no FDA-approved therapy for NPC

• Off-label miglustat, Gaucher Disease

• Clinical Trial: 2-hydroxypropyl-ß-cyclodextrin

Differential Proteomics in the Symptomatic Mouse

1 – 4 weeks

(Pre-symptomatic)

5 – 6 weeks

(Tremors)

7 – 8 weeks

(Ataxia)

10 – 12 weeks

(Terminal)

Label-Free Quantitation in NPC1 Mouse

Cerebellum

Pathway Analysis – Cerebellum

Pergande, et al., Proteomics, 2019

Secondary Validation of Differential Proteins

Fatty Acid Transport Lysosomal Proteins

Purkinje Neuron

Proteins

What have we learned?

• Label-free differential proteomics with AJS

• New markers CNS pathophysiology?

• LIMP2 changes with progressive neurodegeneration

Altered Fatty Acid Metabolism in NPC

Untargeted Proteomics

Untargeted Lipidomics

Differential Proteome and Lipidome of the Liver

Phagosom

Lysoso

Peroxis

id horm

one biosy

nthesis

Retinol m

etabolis

in pro

ing in e

ndoplasmic

idonic acid

bolism

Carbon m

etabolis

Oxidativ

e phosp

horylatio

Chemica

l carci

nogenesis

mediato

r regulatio

n of T

RP channels

Seroto

nergic

synapse

Purine m

etabolis

Glycolys

is / G

luconeogenesis

PPAR signalin

g path

Amino su

gar and n

ucleotid

e sugar m

etabolis

Biosynth

esis o

ino acid

Linoleic acid

bolism

Valine

Bile se

cretio

rentia

Pergande, et al., Bioanalysis. 2019

Dietary PUFAs

Elov2/Elov5

Linoleic acid (LA)

C18:2 Δ9,12

γ-Linoleic acid (GLA)

C18:3 Δ6,9,12

Dihomo-γ-Linoleic acid (DGLA)

C20:3 Δ8,11,14

Arachidonic acid (AA)

C20:4 Δ5,8,11,14

Docosatetraenoic acid (DTA)

C22:4 Δ7,10,13,16

Eicosadienoic acid (EDA)

C20:2 Δ11,14

-Linoleic acid (ALA)

C18:3 Δ9,12,15

Stearidonic acid (SDA)

C18:4 Δ6,9,12,15

Eicosatetraenoic acid (ETA)

C20:4 Δ8,11,14,17

Eicosapentaenoic acid (EPA)

C20:5 Δ5,8,11,14,16

Tetracosapentaenoic acid (TPA)

C24:5 Δ9,12,15,18,21

Eicosatrienoic acid (ERA)

C20:3 Δ11,14,17

Docosapentaenoic acid (DPA)

C22:5 Δ7,10,13,16,19

Fads2Tetracosahexaenoic acid (THA)

C24:6 Δ6,9,12,15,18,21

Docosahexaenoic acid (DHA)

C22:6 Δ4,7,10,13,16,19

β-oxidation enzymes

Transport across

BBB by FABPsTransport across

BBB by FABPs

Docosapentaenoic acid (DPA)

C22:5 Δ7,10,13,16,19

Liver: Fabp5, Fabp7

Cerebella: Fabp3, Fabp5, Fads2

Cortex: Fabp3Cerebella: Fabp3, Fabp5, Fads2

Cortex: Fabp3

Elov2/Elov5

Legend

Upregulated proteins

Downregulated proteins

Lipidomic Results

Pergande, et al., Proteomics. 2019

Free Fatty Acids in NPC

Pergande, et al., Proteomics. 2019

Summary and Outlook

• NPC1 is a fatal, lysosomal storage disease without an approved

therapy

• Jet Stream (AJS) has been used for differential proteomic and

lipidomics studies in NPC1

• Proteomic studies reveal new NPC1 biomarkers in the cerebellum

and liver

• Integrated studies reveal altered -3 and -6 fatty acid metabolism

in the liver

Acknowledgements

Cologna Laboratory – UICChathurika RathnayakeMelissa PergandeThu (Mi) NguyenChandimal PathmasiriWill LaFonEstefania Zarate Fidel Serna-Perez

NIHForbes (Denny) PorterCristin Davidson

Carol Haney-BallVadi BhatBen Owen

Christine MillerSandy YatesChris KleinBob WalkerMark Hoppe

Sheher Mosin

Graduate Studies: http://chem.uic.edu/graduate-

studies/

Jet Stream Thermal Focusing Electrospray in Quantitative … · 2019. 10. 4. · Jet Stream Thermal...

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