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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
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.