Y l U i it S h l f M di iYale University School of MedicineW.M. Keck Foundation
Biotechnology Research Laboratory
Christopher Colangelo, Ph.D.
Quantitative Protein Profiling &Quantitative Protein Profiling &Quantitative Protein Profiling & Quantitative Protein Profiling & Targeted Protein Expression Targeted Protein Expression
NIDA NIDA External Advisory Board (EAB) MeetingExternal Advisory Board (EAB) Meeting
December 3, 2008December 3, 2008
Goals of Protein Profiling Core
• Improve and develop new protein profiling technologies to p p p p g gidentify proteins that play key roles and/or are biomarkers in response to substance abuse
• 1 Use existing DIGE MudPIT iTRAQ and SILAC pipelines1. Use existing DIGE, MudPIT, iTRAQ, and SILAC pipelines on larger number of samples.
• 2. Better Protein Identification• 3. More Accurate Protein Quantitation (replicates)• 4. Faster Sample Turnaround• 5 Verification of potential markers (Targeted Proteomics)5. Verification of potential markers (Targeted Proteomics)
MudPITProtein ID& PTM
Phosphoprotein Profiling
Discovery
Protein ID
TiO2
PP
2D-ProteinSeparation DIGEiTRAQ
ICATSILAC
Quantitative Proteomics
MRM T t d P t iTargeted Proteomics Verification/
Validation
Validation
20
BioanalyticalQuantitation(small molecule)
Bioanalytical
Small Molecule & Med. Chem. MS
0C
onc
Bioanalytical
Growth of NIDA iTRAQ Protein Profiling Service35
64%
30
35
i ( l )68%
20
25
ples
iTRAQ (4‐plex)iTRAQ (8‐plex)
61%
15
20
er of S
amp
50%
57%
61%
5
10
Num
b
100%
0
5
2005 (April ‐ Dec) 2006 2007 20082005 (April Dec) 2006 2007 2008Year
Red Values = Percentage (%) of NIDA samples/Total samples run during calendar year
iTRAQ® Reagents - 8plex
Isobaric Tag: Total mass = 305
Reporter Group
N
N
O
ON
O
Balance GroupPeptide Reactive
N N N N113 114 115 116
OPeptide Reactive
Group (PRG)
N N N13
2CHN
15
+
N N+ + +
N
117 118 119 121N
133CH
15
+++ +
N15
iTRAQ® Reagents Workflow
8 samplesidentical m/z
Dig
est
Mix MS113
114
192
191
PRG
PRG
+
+
+
+S1
S2S3
S4
S5 S7 Den
atur
e &
D 114
115
116
117
118
191
190
189
188
187
PRG
PRG
PRG
PRG
PRG
+
+
+
+
+
+
+
+
+
+
1197.61
S5S6
S7S8
Para
llel
D 118
119
121
187
186
184
PRG
PRG
PRG
+
+
+
+
+
+MS/MS
iTRAQ® Reagents
[Figure used by permission from ABI]
Workflow for iTRAQ Protein Profiling Analysis
Control Treated
100 ug of control/treated samples are reduced, alkylated, and digested with trypsin
iTRAQ labeling
Control Treated
114 115 116 117
g
SCX
2D chromatographicti f tid
Strong cation exchange
RP
separation of peptidesReverse-phase
QSTAR Elite Mass Spectrometer
Yale Protein Expression Database (YPED)
Database Searching and Quantitation
Yale Protein Expression Database (YPED): Key FeaturesKey Features
Web-based Client Built on Open-Source PlatformWeb based Client Built on Open Source Platform Web-based viewing of results
Data and Technology IntegrationData Upload from multiple technologies integration of theData Upload from multiple technologies, integration of the results, enables data review
Data Archive and AnalysisIntuitive Web interface for users to query analyze and visualizeIntuitive Web interface for users to query, analyze and visualize dataAllows comparison of results across samples
Public Repository and Data Preservation (Sept 2008)Public Repository and Data Preservation (Sept 2008)Stores and Exports data in industry standard format (MIAPE)Chosen by Yale Digital Landscape as the best Yale scientific database for development and beta testing
“YPED: a web-accessible database system for protein expression analysis.”Shifman, M.A., Li, Y., Colangelo, C.M., Stone, K.L., Wu, T.L., Cheung, K.H., Miller, P.L., Williams, K.R. J Proteome Research 2007, 6(10), 4019-4024.
Postsynaptic Density (PSD)
• Multi-protein complex organized into functional assemblies
• Site of synaptic plasticity• Receptor complexes localized by scaffold proteins, which
links to signaling moleculeslinks to signaling molecules• Proteomic studies of the rodent PSD reveals diverse
classes of proteins (Li et al. 2004, Jordan et al. 2004, Yoshimura et al. 2002, Collins et al. 2005), , )
• Large-scale changes to the PSD after drug exposure is poorly studied
iTRAQ Workflow for Rat Brain PSD samples
Saline treated Exposed: 30 mg/kg cocaineSaline-treated Exposed: 30 mg/kg cocaine
Cortex Striatum Hippocampus Cortex Striatum Hippocampus
iTRAQ Analysis
iTraq Summary of PSD analysis
• Cortical PSD:Cortical PSD:– 323 proteins identified– 29 proteins , 63 proteins
Cortical Hippocampal2760 127
• Striatal PSD:– 560 proteins identified– 57 proteins , 67 proteins
175
25160
57 proteins , 67 proteins
• Hippocampal PSD:– 580 proteins identified Striatal
112
– 33 proteins , 45 proteins
YPED Data Results 8-plex iTRAQ on Cortical, Striatal, and Hippocampal PSD from naïve rats
Principal Component Analysis on 8‐plex iTRAQ dataset from Mouse Brain PSD from various brain regions.
StriatumStriatum
i
Cortex
Hippocampus
iTRAQ ProteinPilot False Discovery Rate
8-plex iTRAQ on Cortical, Striatal, and Hippocampal PSD from naïve ratsfrom naïve rats
Scatter plot for the cortical PSD
11611
118
121119Calmodulin peptide
Hippocampal PSD
33.5
114 113 ti
Cortical PSD Striatal PSD
Scatter plot for the cortical PSD replicates
113
114
116 115 117
11.5
22.5
114:113 ratio
115:113 ratio
Additional calmodulin peptides were reviewed to substantiate this expression change.
00.5
1
0 100 200 300p g
Ratios shows that the expected deviation is ±20%
Upgraded Protein Identification and Quantitation Software
• Upgraded to MASCOT Cluster version 2.2 • Offers improved Phosphopeptide Identification• Migrated MASCOT to an duo quadcore LINUX cluster• Migrated MASCOT to an duo quadcore LINUX cluster• This allows processing on 8 nodes, which was needed given the
additional data generated from the QSTAR Elite, LTQ-Orbitrap, and 4000 QTRAP mass spectrometersOrbitrap, and 4000 QTRAP mass spectrometers
• ProteinPilot 2.0.1• Protein Identification and Quantitation software• Enables simultaneous identification and quantitation for
ICAT™, iTRAQ™, and SILAC™ reagents• Performs Protein Grouping and Calculates False Discovery Rates
• Mascot Distiller – Quantitation Toolbox (SILAC)• We were a beta test site for the MASCOT Distiller Quantitation
T lb d h l d h d l t f SILAC b dToolbox and helped push development for SILAC based quantitation
Targeted Proteomics Platform
• Utilizes Multiple Reaction Monitoring (MRM)
• Uses– Utilizes information from MS based discovery– Rapid develop of an assay to monitor proteinRapid develop of an assay to monitor protein
dynamics (eg. Expression changes or PTMs in experimental systems)
• AdvantagesAdvantages– High dynamic ranges (105)– Hundreds of samples– Quantitation – Relative or Absolute (heavy
idi t i tid )ABI 4000 QTRAP and idiotypic peptides)ABI 4000 QTRAP and Waters NanoAcquity UPLC
Yale Center for Clinical Investigation
Schematic of the ESI Triple Quadrupole MS System in MRM Operation Mode
Fragment Filter
MS Orifice y12
1243.8+ &1236 8+Filter
p
All Ionse-
gpeptides
840.9++ & 837.5++
fragment
Electrospray Ion Detector
1236.8+peptides
Ion Guide
CollisionCell
Q1st
AnalyticalQuadrupole
AmplifiedSignal
(current)
3rd
AnalyticalQuadrupole
Fragment peptideSelect Peptide Select Fragment Detect Fragment
– Highest specificity and sensitivity for detecting components in a complex mixture– Largest linear dynamic range for quantitation– Requires triple quadrupole MS capability– Well accepted as the MS technique for quantitation (Pharmaceutical Industry)
Targeted Proteomics Workflow
Peptide synthesisHPLC purification
Proteinmixture
Enzymatic Digest
Heavy idiotypic peptide
pAmino acid analysis
LC-MRMChromatogramMix
Heavy Internal MRM
Analysis
Heavy Internal Standard
Analyte
Schematic of Multiple Reaction Monitoring Scan (MRM)
YPED Synthetic Peptide Database
MRM targeted Proteomics of Rho GEF LFC Ser646 (WIL11613 )
Heavy internal standard peptide calibration curve for the 534.3/849.4 and 534.3/720.4 MRM transitions of ser 646 peptide L*ESpFESLR (L* = Heavy Leucine Amino Acid (13C6, 15N),
Sp = phosphoserine)
ser 646L*ESpFESLR
534 3 / 849 4 (y7 H3PO4 )
534.3 / 720.4 (y6‐H3PO4)
Sp = phosphoserine)534.3 / 849.4 (y7‐H3PO4 )
5342+ 849+
(Internal Std) 5342+ 720+
(Internal Std)Heavy Internal Standard
L*ESpFESLRp
5302+ 849+
(LFC ctrl)5302+ 720+
(LFC ctrl)LFC control mouse brain sample
ser 646LESpFESLR
Integration of MultiQuant Results in YPED
Small Molecule Bioanalytical Chemistry
Plasma levels of Lipitor in Mouse Plasma18
12
14
16
g/m
l)8
10
cent
ratio
n (n
gPE Series 275 HRes LC System
2
4
6
Con
c
0
1 hr, 10 mg/kg
1 hr, 30 mg/kg
1hr, 100 mg/kg
24 hr, 10 mg/kg
24 hr, 30 mg/kg
24 hr, 100 mg/kg
API 5000Yi T, Rao DA, Tang PC, Wang Y, Cuchara LA, Bothwell AL, Colangelo
CM, Tellides G, Pober JS, Lorber MI. Transplantation. 2008 Sep 15;86(5):719-27.
Acknowledgements
Ken Williams (Director)Kathy Stone Erol Gulcicek T Kiet LamTuKiet Lam Terence Wu
Mary Lopresti Tom AbbottJean KanyoJean Kanyo
Kathrin Wilczak-Havill Edward Voss
Matt Berberich Mark ShifmanMark Shifman Ji Young LeeHongyu Zhao Erika Andrade
Alex StipanovichAlex Stipanovich
Yale Center for Clinical Investigation
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