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

Mass

Spectrometry

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

Advances for Proteomics

• Soft ionization

• High resolution/mass accuracy mass spectrometers

• Database and informatic tools

• Separation science

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

Electrospray Approaches

www.wikipedia.com

www.newobjective.com

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

High Flow ESI?

Agilent Jet Stream (AJS) Electrospray

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

Towards Label-Free Proteomics

Pergande, et al., Proteomics. 2019.

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

Pergande, et al., Proteomics. 2019.

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

AJS vs ChipCube NanoESI

Pergande, et al., Bioanalysis. 2019.

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

Research Program Question

What chemical signal(s) drive a neuron

(or other cell in the CNS) to survive or

die?

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

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

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

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).

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

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

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

NPC1 and cholesterol transport

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

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

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

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

+/+

-/-

X IX

Courtesy of Ian Williams, Ph.D., NIH

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

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

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

Differential Proteomics in the Symptomatic Mouse

1 – 4 weeks

(Pre-symptomatic)

5 – 6 weeks

(Tremors)

7 – 8 weeks

(Ataxia)

10 – 12 weeks

(Terminal)

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

Label-Free Quantitation in NPC1 Mouse

Cerebellum

Pergande, et al., Proteomics. 2019.

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

Pathway Analysis – Cerebellum

Pergande, et al., Proteomics. 2019.

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

Pergande, et al., Proteomics, 2019

Secondary Validation of Differential Proteins

Fatty Acid Transport Lysosomal Proteins

Purkinje Neuron

Proteins

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

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

What have we learned?

• Label-free differential proteomics with AJS

• New markers CNS pathophysiology?

• LIMP2 changes with progressive neurodegeneration

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

Altered Fatty Acid Metabolism in NPC

22

Untargeted Proteomics

Untargeted Lipidomics

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

Differential Proteome and Lipidome of the Liver

23

Phagosom

e

Lysoso

me

Peroxis

ome

Stero

id horm

one biosy

nthesis

Retinol m

etabolis

m

Prote

in pro

cess

ing in e

ndoplasmic

retic

ulum

Arach

idonic acid

meta

bolism

Carbon m

etabolis

m

Oxidativ

e phosp

horylatio

n

Chemica

l carci

nogenesis

Infla

mm

atory

mediato

r regulatio

n of T

RP channels

Seroto

nergic

synapse

Purine m

etabolis

m

Glycolys

is / G

luconeogenesis

PPAR signalin

g path

way

Amino su

gar and n

ucleotid

e sugar m

etabolis

m

Biosynth

esis o

f am

ino acid

s

Linoleic acid

meta

bolism

Valine

Bile se

cretio

n0

10

20

30

Diffe

rentia

l pro

tein

s

Pergande, et al., Bioanalysis. 2019

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

24

Fads2

Dietary PUFAs

Fads2

Fads1

Elov5

Elov2/Elov5

-3-6

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

Elov5

-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

Fads2

Eicosatrienoic acid (ERA)

C20:3 Δ11,14,17

Elov5

Docosapentaenoic acid (DPA)

C22:5 Δ7,10,13,16,19

Elov2

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

Fads2

Liver: Fabp5, Fabp7

Cerebella: Fabp3, Fabp5, Fads2

Cortex: Fabp3Cerebella: Fabp3, Fabp5, Fads2

Cortex: Fabp3

Fads2

Fads1

Elov5

Elov2/Elov5

Legend

Upregulated proteins

Downregulated proteins

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

Lipidomic Results

25

Pergande, et al., Proteomics. 2019

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

Free Fatty Acids in NPC

26

Pergande, et al., Proteomics. 2019

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

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

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

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/

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