Acid-labile isotope-coded extractants: a class of reagents for quantitative mass spectrometric analysis of complex protein mixtures.
Quantitative mass spectrometry using stable isotope-labeled tagging reagents such as isotope-coded affinity tags has emerged as a powerful tool for identification and relative quantitation of proteins in current proteomic studies. Here we describe an integrated approach using both automated two-dimensional liquid chromatography/ mass spectrometry (2D-LC/MS) and a novel class of chemically modified resins, termed acid-labile isotope-coded extractants (ALICE), for quantitative mass spectrometric analysis of protein mixtures. ALICE contains a thiol-reactive group that is used to capture all cysteine (Cys)-containing peptides from peptide mixtures, an acid-labile linker, and a nonbiological polymer. The acid-labile linker is synthesized in both heavy and light isotope-coded forms and therefore enables the direct relative quantitation of peptides/proteins through mass spectrometric analysis. To test the ALICE method for quantitative protein analysis, two model protein mixtures were fully reduced, alkylated, and digested in solution separately and then Cys-containing peptides covalently captured by either light or heavy ALICE. The reacted light and heavy ALICE were mixed and washed extensively under rigorous conditions and the Cys-containing peptides retrieved by mild acid-catalyzed elution. Finally, the eluted peptides were directly subjected to automated 2D-LC/MS for protein identification and LC/MS for accurate relative quantitation. Our initial study showed that quantitation of protein mixtures using ALICE was accurate. In addition, isolation of Cys-containing peptides by the ALICE method was robust and specific and thus yielded very low background in mass spectrometric studies. Overall, the use of ALICE provides improved dynamic range and sensitivity for quantitative mass spectrometric analysis of peptide or protein mixtures.
Qiu
Yongchang
Y
Proteomics/Protein Chemistry Department, Wyeth, Cambridge, Massachusetts 02140, USA. ycqiu@wyeth.com
Sousa
Eric A
EA
Hewick
Rodney M
RM
Wang
Jack H
JH
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D016208:Databases, Factual; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry*; D011868:Radioisotopes / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012997:Solvents
D007202:Indicators and Reagents; D011506:Proteins; D011868:Radioisotopes; D012997:Solvents; D012710:Serum Albumin, Bovine