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