posted on 2021-02-08, 19:06authored byQian Dong, Xinjian Yan, Yuxue Liang, Sanford P. Markey, Sergey L. Sheetlin, Concepcion A. Remoroza, William E. Wallace, Stephen E. Stein
This
work presents methods for identifying and then creating a
mass spectral library for disulfide-linked peptides originating from
the NISTmAb, a reference material of the humanized IgG1k monoclonal
antibody (RM 8671). Analyses involved both partially reduced and non-reduced
samples under neutral and weakly basic conditions followed by nanoflow
liquid chromatography tandem mass spectrometry (LC–MS/MS).
Spectra of peptides containing disulfide bonds are identified by both
MS1 ion and MS2 fragment ion data in order to completely map all the
disulfide linkages in the NISTmAb. This led to the detection of 383
distinct disulfide-linked peptide ions, arising from fully tryptic
cleavage, missed cleavage, irregular cleavage, complex Met/Trp oxidation
mixtures, and metal adducts. Fragmentation features of disulfide bonds
under low-energy collision dissociation were examined. These include
(1) peptide bond cleavage leaving disulfide bonds intact; (2) disulfide
bond cleavage, often leading to extensive fragmentation; and (3) double
cleavage products resulting from breakages of two peptide bonds or
both peptide and disulfide bonds. Automated annotation of various
complex MS/MS fragments enabled the identification of disulfide-linked
peptides with high confidence. Peptides containing each of the nine
native disulfide bonds were identified along with 86 additional disulfide
linkages arising from disulfide bond shuffling. The presence of shuffled
disulfides was nearly completely abrogated by refining digest conditions.
A curated spectral library of 702 disulfide-linked peptide spectra
was created from this analysis and is publicly available for free
download. Since all IgG1 antibodies have the same constant regions,
the resulting library can be used as a tool for facile identification
of “hard-to-find” disulfide-bonded peptides. Moreover,
we show that one may identify such peptides originating from IgG1
proteins in human serum, thereby serving as a means of monitoring
the completeness of protein reduction in proteomics studies. Data
are available via ProteomeXchange with identifier PXD023358.