Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS.
Isotopic labeling of cysteine residues with acrylamide was previously utilized for relative quantitation of proteins by MALDI-TOF. Here, we explored and compared the application of deuterated and (13)C isotopes of acrylamide for quantitative proteomic analysis using LC-MS/MS and high-resolution FTICR mass spectrometry. The method was applied to human serum samples that were immunodepleted of abundant proteins. Our results show reliable quantitation of proteins across an abundance range that spans 5 orders of magnitude based on ion intensities and known protein concentration in plasma. The use of (13)C isotope of acrylamide had a slightly greater advantage relative to deuterated acrylamide, because of shifts in elution of deuterated acrylamide relative to its corresponding nondeuterated compound by reversed-phase chromatography. Overall, the use of acrylamide for differentially labeling intact proteins in complex mixtures, in combination with LC-MS/MS provides a robust method for quantitative analysis of complex proteomes.
Faca
Vitor
V
Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109, USA. vfaca@fhcrc.org
Coram
Marc
M
Phanstiel
Doug
D
Glukhova
Veronika
V
Zhang
Qing
Q
Fitzgibbon
Matthew
M
McIntosh
Martin
M
Hanash
Samir
S
D000178:Acrylamides / Q000737:chemistry*; D000465:Algorithms; D000478:Alkylation; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis* / Q000235:genetics; D002250:Carbon Radioisotopes / Q000737:chemistry; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data
D000178:Acrylamides; D001798:Blood Proteins; D002250:Carbon Radioisotopes; D003903:Deuterium; D003545:Cysteine