title: Synthetic peptide arrays for pathway-level protein monitoring by liquid chromatography-tandem mass spectrometry.
abstract: 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.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Hewel
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Kento
authors.initials: K
authors.lastname: Onishi
authors.forename: Vincent
authors.initials: V
authors.lastname: Fong
authors.forename: Shamanta
authors.initials: S
authors.lastname: Chandran
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Olsen
authors.forename: Oxana
authors.initials: O
authors.lastname: Pogoutse
authors.forename: Mike
authors.initials: M
authors.lastname: Schutkowski
authors.forename: Holger
authors.initials: H
authors.lastname: Wenschuh
authors.forename: Dirk F H
authors.initials: DF
authors.lastname: Winkler
authors.forename: Larry
authors.initials: L
authors.lastname: Eckler
authors.forename: Peter W
authors.initials: PW
authors.lastname: Zandstra
authors.forename: Andrew
authors.initials: A
authors.lastname: Emili
mesh_terms: 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*
chemical_list: D010455:Peptides; D011506:Proteins
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