Proteogenomic analysis of granulocyte macrophage colony- stimulating factor autoantibodies in the blood of a patient with autoimmune pulmonary alveolar proteinosis.

Recently, attempts to reveal the structures of autoantibodies comprehensively using improved proteogenomics technology, have become popular. This technology identifies peptides in highly purified antibodies by using an Orbitrap device to compare spectra from liquid chromatography-tandem mass spectrometry against a cDNA database obtained through next-generation sequencing. In this study, we first analyzed granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies in a patient with autoimmune pulmonary alveolar proteinosis, using the trapped ion mobility spectrometry coupled with quadrupole time-of-flight (TIMS-TOF) instrument. The TIMS-TOF instrument identified peptides that partially matched sequences in up to 156 out of 162 cDNA clones. Complementarity-determining region 3 (CDR3) was fully and partially detected in nine and 132 clones, respectively. Moreover, we confirmed one unique framework region 4 (FR4) and at least three unique across CDR3 to FR4 peptides via de novo peptide sequencing. This new technology may thus permit the comprehensive identification of autoantibody structure.

Aldehyde stress-mediated novel modification of proteins: epimerization of the N-terminal amino acid.

Various kinds of aldehyde-mediated chemical modifications of proteins have been identified as being exclusively covalent. We report a unique noncovalent modification: the aldehyde-mediated epimerization of the N-terminal amino acid. Epimerization of amino acids is thought to cause conformational changes that alter their biological activity. However, few mechanistic studies have been performed, because epimerization of an amino acid is a miniscule change in a whole protein. Furthermore, it does not produce a mass shift, making mass spectrometric analysis difficult. Here, we have demonstrated epimerization mediated by endogenous aldehydes. A model peptide, with an N-terminal l- or d-FMRFamide, was incubated with an endogenous or synthetic aldehyde [acetaldehyde, methylglyoxal, pyridoxal 5'-phosphate (PLP), 4-oxo-2(E)-nonenal, 4-hydroxy-2(E)-nonenal, d-glucose (Glc), 4- or 2-pyridinecarboxaldehyde] under physiological conditions. Each reaction mixture was analyzed by liquid chromatography with ultraviolet detection and/or electrospray ionization mass spectrometry. Considerable epimerization occurred after incubation with some endogenous aldehydes (PLP, 40.6% after 1 day; Glc with copper ions, 6.5% after 7 days). Moreover, the epimerization also occurred in whole proteins (human serum albumin and PLP, 26.3% after 1 day). Tandem mass spectrometric studies, including deuterium labeling and sodium borohydride reduction, suggested that the epimerization results from initial Schiff base formation followed by tautomerization to ketimine that causes the chirality to be lost. This suggests that the epimerization of the N-terminal amino acid can also occur in vivo as a post-translational modification under a high level of aldehyde stress.