RT Journal Article
SR Electronic
T1 Synthetic Peptide Arrays for Pathway-Level Protein Monitoring by Liquid Chromatography-Tandem Mass Spectrometry
JF Molecular & Cellular Proteomics
JO Mol Cell Proteomics
FD American Society for Biochemistry and Molecular Biology
SP 2460
OP 2473
DO 10.1074/mcp.M900456-MCP200
VO 9
IS 11
A1 Hewel, Johannes A.
A1 Liu, Jian
A1 Onishi, Kento
A1 Fong, Vincent
A1 Chandran, Shamanta
A1 Olsen, Jonathan B.
A1 Pogoutse, Oxana
A1 Schutkowski, Mike
A1 Wenschuh, Holger
A1 Winkler, Dirk F. H.
A1 Eckler, Larry
A1 Zandstra, Peter W.
A1 Emili, Andrew
YR 2010
UL http://www.mcponline.org/content/9/11/2460.abstract
AB 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.