Enzymatic digestion and mass spectrometry in the study of advanced glycation end products/peptides.
An extensive study was carried out on HSA and non-enzymatically glycated HSA by enzymatic digestion with trypsin and endoproteinase Lys-C, with the aim of identifying specific glycated peptides deriving from enzymatic digestion of glycated HSA. They may be considered, in pectore, as advanced glycation end products/peptides. These compounds, important at a systemic level in diabetic and nephropathic subjects, are produced by enzymatic digestion of in vivo glycated proteins: They are related to the pathological state of patients and have been invoked as responsible for tissue modifications. The digested mixtures obtained by the two enzymes were analyzed by MALDI/MS and LC/ESI/MSn, and clear cut differences were found. First of all, the digestion products of glycated HSA are generally less abundant than those observed in the case of unglycated HSA, accounting for the lower proclivity of the former to enzymatic digestion. MS/MS experiments on doubly charged ions, comparisons with a protein database, and molecular modeling to identify the lysine NH2 groups most exposed to glycation, identified some glycated peptides in digestion mixtures obtained from both types of enzymatic digestion. Residues 233K, 276K, 378K, 545K, and 525K seem to be privileged glycation sites, in agreement with the fractional solvent accessible surface values calculated by molecular modeling.
Lapolla
Annunziata
A
Dipartimento di Scienze Mediche e Chirurgiche, Cattedra di Malattie del Metabolismo, Università degli Studi di Padova, Padova, Italy.
Fedele
Domenico
D
Reitano
Rachele
R
Aricò
Nadia Concetta
NC
Seraglia
Roberta
R
Traldi
Pietro
P
Marotta
Ester
E
Tonani
Roberto
R
D000595:Amino Acid Sequence; D017127:Glycation End Products, Advanced / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012709:Serum Albumin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*
D017127:Glycation End Products, Advanced; D010455:Peptides; D012709:Serum Albumin; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase