Comprehensive Analysis of Tryptic Peptides Arising from Disulfide Linkages in NISTmAb and Their Use for Developing a Mass Spectral Library

• DOI: 10.1021/acs.jproteome.0c00823
• Publication Date: 2021-02-08T18:44:58Z
• Authors: Qian Dong; Xinjian Yan; Yuxue Liang; Sanford P. Markey; Sergey L. Sheetlin; Concepcion A. Rem...; William E. Wallace; Stephen E. Stein
• Keywords: Spectral Analysis

This work presents methods for identifying and then creating a mass spectral library disulfide-linked peptides originating from the NISTmAb, reference material of humanized IgG1k monoclonal antibody (RM 8671). Analyses involved both partially reduced non-reduced samples under neutral weakly basic conditions followed by nanoflow liquid chromatography tandem spectrometry (LC-MS/MS). Spectra containing disulfide bonds are identified MS1 ion MS2 fragment data in order to completely map all linkages NISTmAb. led detection 383 distinct peptide ions, arising fully tryptic cleavage, missed irregular complex Met/Trp oxidation mixtures, metal adducts. Fragmentation features low-energy collision dissociation were examined. These include (1) bond cleavage leaving intact; (2) often leading extensive fragmentation; (3) double products resulting breakages two or bonds. Automated annotation various MS/MS fragments enabled identification with high confidence. Peptides each nine native along 86 additional shuffling. The presence shuffled disulfides was nearly abrogated refining digest conditions. A curated 702 spectra created this analysis is publicly available free download. Since IgG1 antibodies have same constant regions, can be used as tool facile "hard-to-find" disulfide-bonded peptides. Moreover, we show that one may identify such proteins human serum, thereby serving means monitoring completeness protein reduction proteomics studies. Data via ProteomeXchange identifier PXD023358.