Download - Top Down Proteomics: A Datasource for PRO
Top Down Proteomics:
A Datasource for PROPaul Thomas, Northwestern University
Ryan Fellers, Cecilia Arighi (UDel/PIR/UniProt)The Consortium for Top Down Proteomics
16 May 2013
Protein Variability in Higher Proteomes:The Age of Protein Isoforms
Insertion or Alternative Sequence
Sources of variability• Alternative
Splices• Endogenous
Cleavages• Alternative
Promoter Usage
Isoform: Large changes to amino acid sequence
Base Isoform
Protein Variability in Higher Proteomes:The Age of Protein Isoforms and Proteoforms
Proteoform: The complete set of modifications on an amino acid sequence
http://dx.doi.org/10.1038/nmeth.2369
What’s in a name?
You can’t please everyone!
Technical HypothesisMeasurement of intact proteins (isoforms and
proteoforms) will provide strong correlations between mass spectrometric data and complex phenotypes.
Differences: The measurement
challengeN CN C
@!$D%@Q%@L%@T%@E!@E!@Q!@I%@A%@E!@F!@K#@E#@A%@F!@S!@L!@F!@D!@K!@D%@G#@D!@G#@T#@I!@T#@T!@K!$E%@L!@G#@T!@V!@M!@R%@S!@L%@G#@Q;$N%@P!@T%@E%@A%E%@L%@Q#$D%@M!@I!@N#$E%V!D!A!D!G#@N!G!T!I!D!F!P!@E!@F!@L!@T!$M!@M!@A!@R#$K!$M!@K#$D!T#$D!$S!$E!$E#@E!I%@R#$E!@A!@F!$R!@V!@F#$D#:K#$D!@G#$N#$G!@Y!$I#$S!$A!$A!$E!@L#@R!@H!$V!@M!@T#$N!@L!@G!@E!@!@L!@T!@D!@E!@E!@V!@D!@E!@M!@I!@R!@E!@A!@D!@I!@D!G!@D!G!@Q!V!@N!@Y!E!E!:F!@V!:Q!M!M!T!@A#@K!
Top Down vs. Bottom Up Approaches to DNA-Predicted Protein Sequence Analysis
DEVELOPMENT OF A GENERAL MEASUREMENT PLATFORM FOR INTACT,
MODIFIED PROTEINS
2. Automated Instrumentation
1. “Front End” Separations
3. “Back end” data processing and informatics
Top Down Proteomics Work Flow
Sample cleanup(SDS removal)
GELFREE – MW based separation
Online nano-LC-MS/MS: 12 T/Orbitrap LTQ-FTMS
–
+
200 kDa
10 kDa
Cell lysateor nuclei
(Optional) isoelectricfocusing
+ _
Top Down Proteomics and Plants
A. thaliana Thylakoid Lumen PO:0020039
Similar Proteins
www.topdownproteomics.org
Proteoform Repository
PFR2217
Senescence
Chemotherapy-Induced Accelerated Senescence
Roberson, R. S et al. Cancer research 2005, 65, 2795-803.Wang, Q. Wu, P. C. et al. International journal of cancer. 2011, 128, 1546-58.
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 9 Day 11 Day 14 Day 170.00E+00
1.00E+07
2.00E+07
3.00E+07
4.00E+07
5.00E+07
6.00E+07
7.00E+07
8.00E+07
9.00E+07
1.00E+08
25 nM camptothecin DMSO
Cell
Coun
t (c
ells
/mL) Treatment: 25nM
camptothecin for 24 hours
Control
Senescent Early Escape?
CHEBI:27656
2Pi
+Me+Me
2Pi 3Pi
+Me
+2Me
4Pi
HMGA1a HMGA1b3Pi
2Pi
726 730 734 738 742 746m/z
3Pi
1PiN-Ac
4Pi
3Pi
2Pi
4Pi
705 710 715 720 725 730 735 740m/z
1PiN-Ac
726 730 734 738 742 746m/z 705 710 715 720 725 730 735
HMGA1 Isoforms
P17096-2 HMGA1b:
P17096-1 HMGA1a:
Control
Senescent
Escape
AcetylationPhosphorylationMethylation
PRIDE, Modified HMGA1
Glyco-Proteoforms
ApoC-IIIApoC-III
OH
Unmodified
ApoC-III
Linear Branched
MOD:?????MOD:?????
Proteomics Center of Excellence Northwestern University
http://pce.northwestern.edu