RABA (Reductive Alkylation By Acetone): A novel stable isotope labeling approach for quantitative proteomics

Abstract

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. (J Am Soc Mass

Quantitative Phosphoproteomics Using Acetone-Based Peptide Labeling: Method Evaluation and Application to a Cardiac Ischemia/Reperfusion Model

“…Similarly, two additional RABA-labeling reactions were performed to ensure near complete labeling of peptides. The reactions were quenched by adding glycine (1 M, 150 μL) as previously described [13]. The samples were then dried using a vacuum centrifuge system.…”

A Novel Method for Relative Quantitation ofN-Glycans via Acetone Stable Isotopic Labeling and ESI-MS Analysis

“…Pronase E 酶解糖蛋白会使糖链 带上一个氨基活性基团, 获得的糖氨酸只带有一个氨基 基团, 相对于蛋白质定量中遇到的多氨基情况, 定量结 果更易分析. 丙酮作为广泛使用的烷化试剂已被成功应用于蛋 白质的定量研究中 [17] . [18] .…”

“…按文献 [17]方法, 将 4.2 节中糖链溶于 50 μL 90%乙 腈溶液(含 0.1%甲酸), 与新鲜配制的 100 µL 0.1 mol/L NaCNBH 3 的丙酮溶液(100 μg NaCNBH 3 溶于 100 μL d 0 /d 6 丙酮)混合于 2 mL 离心管中, 室温震荡反应 1 h. 然 后将反应产物用氮气吹干, 溶于 1 mL 去离子水中, 然 后加载到石墨碳柱中, 用 2 mL 去离子水除盐, 被丙酮 标记上的 Glycan-Asn 用 2 mL 的 25%的乙腈洗脱, 洗脱 速度为 1.5 mL/min, 将洗脱物用氮气吹干后溶解于 200 μL 去离子水中进行 ESI-MS 分析.…”

Relative Quantification of Biomarkers Using Mixed-Isotope Labeling Coupled With Ms

“…A similar procedure, reductive alkylation, has been used to isolate highly homologous proteins such as isoforms within samples [44,56]. Reductive methylation consists of the addition of alkyl groups to the amino residue of the proteins from an alkyl-donating compound.…”