Discovery of Undefined Protein Cross-Linking Chemistry: A Comprehensive Methodology Utilizing ¹⁸O-Labeling and Mass Spectrometry

Abstract

Characterization of protein crosslinking, particularly without prior knowledge of the chemical nature and site of crosslinking, poses a significant challenge due to their intrinsic structural complexity and the lack of a comprehensive analytical approach. Towards this end, we have developed a generally applicable workflow—XChem-Finder that involves four stages. (1) Detection of crosslinked peptides via 18O-labeling at C-termini. (2) Determination of the putative partial sequences of each crosslinked peptide pair using a fragment ion mass database search against known protein sequences coupled with a de novo sequence tag search. (3) Extension to full sequences based on protease specificity, the unique combination of mass, and other constraints. (4) Deduction of crosslinking chemistry and site. The mass difference between the sum of two putative full-length peptides and the crosslinked peptide provides the formulas (elemental composition analysis) for the functional groups involved in each cross- linking. Combined with sequence restraint from MS/MS data, plausible crosslinking chemistry and site were inferred, and ultimately, confirmed by matching with all data. Applying our approach to a stressed IgG2 antibody, ten cross-linked peptides were discovered and found to be connected via thioether originating from disulfides at locations that had not been previously recognized. Furthermore, once the crosslink chemistry was revealed, a targeted crosslink search yielded four additional crosslinked peptides that all contain the C-terminus of the light chain.

Site‐specific Bioconjugation and Convergent Click Chemistry Enhances Antibody–Chromophore Conjugate Binding Efficiency

“…It is worth noting that the intrinsic charge heterogeneity of the unmodified antibody contributed to the multiple bands observed for both enzymatic and chemical conjugates. As is well documented in the literature, these antibody charge variants are due to various post‐translational modifications, such as deamidation, oxidation, glycosylation, or other reactive metabolites (51‐59).…”

Detection and Quantitation of Succinimide in Intact Protein via Hydrazine Trapping and Chemical Derivatization

“…Moreover, once a protein is introduced to a human subject, that is, to the blood at pH 7.4, succinimide will rapidly hydrolyze into Asp and isoAsp . Succinimide formation, together with Asn deamidation and Asp isomerization, introduces heterogeneity into proteins and may lead to a number of issues including alterations in structure, intermolecular cross‐linking, aggregation, loss of activity, and even immunogenicity, thereby affecting both efficacy and toxicity …”

“…[49][50][51][52] Moreover, once a protein is introduced to a human subject, that is, to the blood at pH 7.4, succinimide will rapidly hydrolyze into Asp and isoAsp. 40,53 Succinimide formation, together with Asn deamidation and Asp isomerization, introduces heterogeneity into proteins and may lead to a number of issues including alterations in structure, 9,54 intermolecular cross-linking, [55][56][57][58][59] aggregation, 60 loss of activity, 48,61,62 and even immunogenicity, 63,64 thereby affecting both efficacy and toxicity. 36,42,65 Detection methods for succinimide are limited both in number and practicality, mainly because of its intrinsic instability, that is, it is rapidly hydrolyzed during tryptic digestion typically carried out at or above neutral pH.…”

Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5

“…Therefore, no appreciable isomerization was expected at pH 4.5 in ammonium acetate, but was investigated in calmodulin nonetheless. Once more, 18 O-labeling is the best approach to monitor isomerization during sample preparation (Liu et al 2013b; Yao et al 2003). Among 17 aspartic acid residues, 12 were detected by mass spectrometry, including five Asp-Gly hotspots.…”