Determination of primary structure and microheterogeneity of a β-amyloid plaque-specific antibody using high-performance LC–tandem mass spectrometry

Abstract

Using the "bottom-up" approach and liquid chromatography in combination with mass spectrometry, the primary structure and sequence microheterogeneity of a plaque-specific anti-β-amyloid (1-17) monoclonal antibody (clone 6E10) was characterized. This study describes the extent of structural information directly attainable by a high performance LC-tandem mass spectrometric method in combination with both protein database searching and de novo sequence determination. Using trypsin and chymotrypsin for enzymatic digestion, 95% sequence coverage of the light chain and 82% sequence coverage of the heavy chain of the 6E10-antibody were obtained. The primary structure determination of a large number of peptides from the antibody variable regions was obtained through de novo interpretation of the data. In addition, N-terminal truncations of the heavy chain were identified as well as low levels of pyro-glutamic acid formation. Surprisingly, pronounced sequence microheterogeneities were determined for the CDR 2 region of the light chain, indicating that changes at the protein level derived from somatic hypermutation of the Ig V L genes in mature B-cells might contribute to unexpected structural diversity. Furthermore, the major glycoforms at the conserved heavy chain N-glycosylation site, Asn-292, were determined to be core fucosylated, biantennary, complex type structures containing zero to two galactose residues.

Mass spectrometric identification of oxidative modifications of tryptophan residues in proteins: Chemical artifact or post-translational modification?

“…Following in-solution digestion with trypsin, LC-MS/MS and NCBInr database search (19), these residues were identified as unmodified, suggesting that this antibody is not primarily oxidized during storage, as previously reported for Trp residues in an MAb (18). Upon SDS-PAGE separation, pronounced molecular heterogeneity due to various oxidative modifications of the majority of Trp residues was observed.…”

“…The sequences determined from the MS/MS data were validated manually. The peptides were fit against antibody sequences from the NCBInr protein database (19). …”

IgG glycosylation analysis

“…For IgG glycosylation analysis, FA is therefore applied more often as acidic mobile phase additive. In addition, to ensure method stability in terms of separation, ionization, and detection, most studies have applied micro-LC [108,141,144] or capillary-LC [52,53,105,107] coupled to ESI-MS, while nano-LC is less frequently used [9,33,42,123,139,145].…”

Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry

“…For the peptides and proteins theoretical titration curves and diffusion coefficients were calculated based on amino acid composition and molecular mass, respectively, as described earlier . The amino acid sequence of the anti-β-amyloid 1-16 antibody (6E10) was obtained from the literature …”