RABA (reductive alkylation by acetone): a novel stable isotope labeling approach for quantitative proteomics.
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 (d(0)) and deuterium labeled (d(6)) 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.
Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, Kentucky 40536, USA.
Jianjun
J
Zhai
Xiaoyan
X
Liu
Zhenyu
Z
Huang
Haining
H
Zhu
D000096:Acetone / Q000737:chemistry*; D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D002457:Cell Extracts / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D016014:Linear Models; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000032:analysis / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013482:Superoxide Dismutase / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D000072105:Superoxide Dismutase-1; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
D002457:Cell Extracts; D009211:Myoglobin; D010446:Peptide Fragments; D011506:Proteins; C000606290:SOD1 protein, human; D000096:Acetone; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1