Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line liquid chromatography followed by MALDI analysis on a time-of-flight/curved field reflectron tandem mass spectrometer.
Glycated peptides arising from in vivo digestion of glycated proteins, usually called advanced glycation end (AGE) product peptides, are biologically relevant compounds due to their reactivity towards circulating and tissue proteins. To investigate their structures, in vitro glycation of human serum albumin (HSA) has been performed and followed by enzymatic digestion. Using different MALDI based approaches the digestion products obtained have been compared with those arising from enzymatic digestion of the protein. Results obtained using 2,5-dihydroxybenzoic acid (DHB) indicate this as the most effective matrix, leading to an increase in the coverage of the glycated protein. Off-line microbore liquid chromatography prior to MALDI analysis reveals that 63% of the free amino groups amenable to glycation are modified. In addition, the same approach shows the co-presence of underivatised peptides. This indicates that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Tandem mass spectrometric data suggest that the collision induced dissociation of singly charged glycated peptides leads to specific fragmentation pathways related to the condensed glucose molecule. The specific neutral losses derived from the activated glycated peptides can be used as signature for establishing the occurrence of glycation processes.
Brancia
Francesco L
FL
Shimadzu Research Laboratory (Europe), Manchester, UK. Francesco.Brancia@srlab.co.uk
Bereszczak
Jessica Z
JZ
Lapolla
Annunziata
A
Fedele
Domenico
D
Baccarin
Lorenzo
L
Seraglia
Roberta
R
Traldi
Pietro
P
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D017127:Glycation End Products, Advanced / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*; D012709:Serum Albumin / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D017127:Glycation End Products, Advanced; D010455:Peptides; D012709:Serum Albumin; D014357:Trypsin