Quantitative proteomics is challenging and various stable isotope based approaches have been developed to meet the challenge. Hereby we describe a simple, efficient, reliable, and inexpensive method named reductive alkylation by acetone (RABA) to introduce stable isotopes to peptides for quantitative analysis. The RABA method leads to alkylation of N-terminal and lysine amino groups with isopropyl moiety. Using unlabeled (d0) and deuterium labeled (d6) acetone, a 6 Da mass split is introduced to each isopropyl modification between the light and heavy isotope labeled peptides, which is ideally suited for quantitative analysis. The reaction specificity, stoichiometry, labeling efficiency, and linear range of the RABA method have been thoroughly evaluated in this study using standard peptides, tryptic digest of proteins, as well as human cell lysate. Reliable quantitative results have been consistently obtained in all experiments. We also applied the RABA method to quantitative analysis of proteins in spinal cords of transgenic mouse models of amyotrophic lateral sclerosis. Highly homologous proteins (transgenic human SOD1 and endogenous mouse SOD1) were distinguished and quantified using the method developed in this study. In addition, the quantitative results using the RABA approach were independently validated by Western blot.
|Number of pages||12|
|Journal||Journal of the American Society for Mass Spectrometry|
|State||Published - Jul 2009|
Bibliographical noteFunding Information:
The authors thank Ping Shi and Rujuan Liu for technical assistance with cell culture and transgenic mice, Dr. Anna-Lena Ström for suggestions, and Dr. Jozsef Gal for critically reading the manuscript. This study was in part supported by NH grant R01NS49126 (to H.Z.). The authors acknowledge support from NIH/NCRR Center of Biomedical Research Excellence in the Molecular Basis of Human Disease (COBRE, P20RR020171) and NIH/NIEHS Superfund Basic Research Program (P42ES007380). The NIH Shared Instrumentation Grant S10RR023684 (to H.Z.) is acknowledged for purchasing the 4800 Plus MALDI-TOF-TOF mass spectrometer.
ASJC Scopus subject areas
- Structural Biology