Synthetic peptide arrays for pathway-level protein monitoring by liquid chromatography-tandem mass spectrometry.
Effective methods to detect and quantify functionally linked regulatory proteins in complex biological samples are essential for investigating mammalian signaling pathways. Traditional immunoassays depend on proprietary reagents that are difficult to generate and multiplex, whereas global proteomic profiling can be tedious and can miss low abundance proteins. Here, we report a target-driven liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy for selectively examining the levels of multiple low abundance components of signaling pathways which are refractory to standard shotgun screening procedures and hence appear limited in current MS/MS repositories. Our stepwise approach consists of: (i) synthesizing microscale peptide arrays, including heavy isotope-labeled internal standards, for use as high quality references to (ii) build empirically validated high density LC-MS/MS detection assays with a retention time scheduling system that can be used to (iii) identify and quantify endogenous low abundance protein targets in complex biological mixtures with high accuracy by correlation to a spectral database using new software tools. The method offers a flexible, rapid, and cost-effective means for routine proteomic exploration of biological systems including "label-free" quantification, while minimizing spurious interferences. As proof-of-concept, we have examined the abundance of transcription factors and protein kinases mediating pluripotency and self-renewal in embryonic stem cell populations.
Hewel
Johannes A
JA
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.
Liu
Jian
J
Onishi
Kento
K
Fong
Vincent
V
Chandran
Shamanta
S
Olsen
Jonathan B
JB
Pogoutse
Oxana
O
Schutkowski
Mike
M
Wenschuh
Holger
H
Winkler
Dirk F H
DF
Eckler
Larry
L
Zandstra
Peter W
PW
Emili
Andrew
A
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D051379:Mice; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010455:Peptides; D011506:Proteins
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