title: Quantitative analysis of deamidation and isomerization in β2-microglobulin by 18O labeling.
abstract: Deamidation of asparagine residues in proteins via the formation of a 5-membered succinimide ring intermediate is a nonenzymatic intramolecular reaction and, in general, occurs most rapidly at an Asn-Gly sequence. A protein containing this sequence would, therefore, be susceptible to modification, and the result would produce a structural alteration in the molecule. An Asn would be replaced with an Asp, resulting in an increase in the overall negative charge on the molecule but also an isomerization to isoAsp. Despite the fact that such a structural replacement could affect the functional properties of a protein, estimating the susceptibility of the Asn-Gly sequence to deamidation/isomerization remains a difficult task. This is especially true for proteins that are subjected to enzymatic digestion during their characterization, since the above transformation could occur spontaneously during this treatment. To address this issue, we applied a stable-isotope (18)O-labeling method combined with nano-LC-MS/MS to examine the susceptibility of two Asn-Gly sites in β2-microglobulin (β2m) to the reaction. The method permits the reaction occurring in a protein to be distinguished from that during enzymatic treatment. When β2m was incubated for 60 days at 37 °C, deamidation at Asn17-Gly and Asn42-Gly with half-lives of 33 and 347 days occurred, respectively. Moreover, a comparison of the deamidated products to synthetic peptides revealed that 44% of the Asp17 and 96% of the Asp42 had been converted into isoAsp forms. Interestingly, such structurally altered β2m showed a specific affinity for divalent Cu(2+) ions, which is thought to be a candidate for initiating fibril formation.
authors.affiliation: Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, Yamadaoka, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Fukuda
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Takao
mesh_terms: D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010103:Oxygen Isotopes / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis*; D013237:Stereoisomerism; D013388:Succinimides / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D001613:beta 2-Microglobulin / Q000032:analysis*
chemical_list: D000577:Amides; D010103:Oxygen Isotopes; D010446:Peptide Fragments; D013388:Succinimides; D001613:beta 2-Microglobulin; D001224:Aspartic Acid; D001216:Asparagine; D003300:Copper