Extending the Coverage of Lys-C/Trypsin-Based Bottom-up Proteomics by Cysteine S-Aminoethylation
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Extending the Coverage of Lys-C/Trypsin-Based Bottom-up Proteomics by Cysteine S-Aminoethylation. / Tomioka, Ryota; Tomioka, Ayana; Ogata, Kosuke; Chan, Hsin Ju; Chen, Li Yu; Guzman, Ulises H.; Xuan, Yue; Olsen, Jesper V.; Chen, Yu Ju; Ishihama, Yasushi.
In: Journal of the American Society for Mass Spectrometry, Vol. 35, No. 2, 2024, p. 386-396.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Extending the Coverage of Lys-C/Trypsin-Based Bottom-up Proteomics by Cysteine S-Aminoethylation
AU - Tomioka, Ryota
AU - Tomioka, Ayana
AU - Ogata, Kosuke
AU - Chan, Hsin Ju
AU - Chen, Li Yu
AU - Guzman, Ulises H.
AU - Xuan, Yue
AU - Olsen, Jesper V.
AU - Chen, Yu Ju
AU - Ishihama, Yasushi
N1 - Publisher Copyright: © 2024 American Society for Mass Spectrometry. Published by American Chemical Society. All rights reserved.
PY - 2024
Y1 - 2024
N2 - To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.
AB - To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.
KW - bottom-up proteomics
KW - cysteine S-aminoethylation
KW - membrane proteomics
KW - T790 M EGFR
U2 - 10.1021/jasms.3c00448
DO - 10.1021/jasms.3c00448
M3 - Journal article
C2 - 38287222
AN - SCOPUS:85184516841
VL - 35
SP - 386
EP - 396
JO - Journal of The American Society for Mass Spectrometry
JF - Journal of The American Society for Mass Spectrometry
SN - 1044-0305
IS - 2
ER -
ID: 383398544