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![Page 1: Jet Stream Thermal Focusing Electrospray in Quantitative … · 2019. 10. 4. · Jet Stream Thermal Focusing Electrospray in Quantitative Proteomics Stephanie M. Cologna, Ph.D. Department](https://reader031.vdocuments.mx/reader031/viewer/2022011916/5fde752426c83d65247f70fb/html5/thumbnails/1.jpg)
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.
![Page 2: Jet Stream Thermal Focusing Electrospray in Quantitative … · 2019. 10. 4. · Jet Stream Thermal Focusing Electrospray in Quantitative Proteomics Stephanie M. Cologna, Ph.D. Department](https://reader031.vdocuments.mx/reader031/viewer/2022011916/5fde752426c83d65247f70fb/html5/thumbnails/2.jpg)
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.
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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.
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Electrospray Approaches
www.wikipedia.com
www.newobjective.com
For Research Use Only. Not for use in diagnostic procedures.
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High Flow ESI?
Agilent Jet Stream (AJS) Electrospray
For Research Use Only. Not for use in diagnostic procedures.
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Towards Label-Free Proteomics
Pergande, et al., Proteomics. 2019.
For Research Use Only. Not for use in diagnostic procedures.
![Page 7: Jet Stream Thermal Focusing Electrospray in Quantitative … · 2019. 10. 4. · Jet Stream Thermal Focusing Electrospray in Quantitative Proteomics Stephanie M. Cologna, Ph.D. Department](https://reader031.vdocuments.mx/reader031/viewer/2022011916/5fde752426c83d65247f70fb/html5/thumbnails/7.jpg)
Pergande, et al., Proteomics. 2019.
For Research Use Only. Not for use in diagnostic procedures.
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AJS vs ChipCube NanoESI
Pergande, et al., Bioanalysis. 2019.
For Research Use Only. Not for use in diagnostic procedures.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Label-Free Quantitation in NPC1 Mouse
Cerebellum
Pergande, et al., Proteomics. 2019.
For Research Use Only. Not for use in diagnostic procedures.
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Pathway Analysis – Cerebellum
Pergande, et al., Proteomics. 2019.
For Research Use Only. Not for use in diagnostic procedures.
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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.
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For Research Use Only. Not for use in diagnostic procedures.
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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.
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Altered Fatty Acid Metabolism in NPC
22
Untargeted Proteomics
Untargeted Lipidomics
For Research Use Only. Not for use in diagnostic procedures.
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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.
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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.
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Lipidomic Results
25
Pergande, et al., Proteomics. 2019
For Research Use Only. Not for use in diagnostic procedures.
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Free Fatty Acids in NPC
26
Pergande, et al., Proteomics. 2019
For Research Use Only. Not for use in diagnostic procedures.
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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.
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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.