No abstract

Trypsin autolysis fragments and matrix clusters are often observed as intense peaks in mass spectra of protein digests. It is demonstrated that these can be exploited to improve the mass calibration of a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometer. Interpretation of some of the autolysis masses is complicated by the existence of disulfide bonds. Surprisingly large matrix clusters are often visible for alpha-cyano-4-hydroxy-cinnamic acid. The fractional part of their masses differentiates them from protein digestion fragments.

A chemical derivatization method, amidination, that has recently been effectively employed in peptide mass spectrometry experiments is used to covalently modify lysines in several standard proteins. Protein and peptide mass spectra identify sites at which the reaction does or does not occur. This is therefore a rapid approach to elucidate solvent-accessible regions of folded proteins.

No abstract

Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4 nM), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles.