Program

Fundamental Sessions

Day 1, June 10（Wed.）　14:08-14:26　Room D (5F 511+512)

1D-O1-1408（1P-03）

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Development of a cysteine modification workflow using 2-ME/DMSO and its application to proteomics

(¹Kitasato Univ., ²Kitasato Univ., ³Kitasato Univ., ⁴Kitasato Univ.)
^oArisa Suto¹, Yoshihiro Ishikawa¹, Toshihide Matsumoto², Makoto Itakura^3,4, Yoshio Kodera^1,4, Takashi Matsui^1,4

Cysteine is a highly nucleophilic residue that forms disulfide bonds and undergoes oxidative modifications both in vivo and in vitro. Therefore, reduction of disulfide bonds and alkylation of reactive thiol groups are commonly performed prior to mass spectrometry–based proteomic analysis. Iodoacetamide (IAA), a widely used alkylating reagent, has been reported to cause nonspecific modifications that may negatively affect identification reliability and quantitative reproducibility. To overcome this problem, we developed a cysteine-selective modification method using 2-mercaptoethanol (2-ME) coupled with dimethyl sulfoxide (DMSO). We optimized the reaction conditions using β-galactosidase and found that 20% DMSO provided the most efficient 2-ME modification of cysteine residues. We then applied this method to protein extracts from mouse liver and compared our 2-ME/DMSO workflow with conventional IAA modification. Both modification methods showed no substantial difference in the number of protein identifications. In contrast, peptide identifications and sequence coverage were significantly increased with the 2-ME/DMSO workflow, along with improved signal intensity and reproducibility of cysteine-modified peptides. Here, we present the optimized 2-ME/DMSO workflow and its application to proteomic profiling of cisplatin-treated ovarian clear cell carcinoma.

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