Analysis of protein glycation products by MALDI-TOF/MS.
Matrix-assisted laser desorption ionization-mass spectrometry with time-of-flight detection (MALDI-TOF/MS) is a promising tool to analyze advanced glycation end product (AGE)-modified proteins. The combination of soft ionization (MALDI) with time-of-flight mass detection allows analysis of peptides and proteins of a molecular mass up to 300 kDa with minimal sample workup. Because the direct structural analysis of intact AGE proteins is not possible due to the formation of broad and poorly resolved peaks, peptide mapping was introduced into the analysis of AGE proteins by MALDI-TOF/MS, allowing site-specific analysis of defined AGEs. When methylglyoxal-modified lysozyme was subjected to MALDI-TOF/MS peptide mapping, methylimidazolone and argpyrimidine attached to the arginine residue and carboxyethyl (CEL) bound to the lysine were detected on peptide(aa1-7) (KVFGRCE). In contrast, only one methylimidazolone was found on peptide(aa8-35) (LAAAMKRHGLDNYRGYSLGNWVCAAKFE) and peptide(aa120-129) (VQAWIRGCRL), respectively. The analysis of AGE protein, which had been incubated with glucose, revealed the presence of an Amadori product and a carboxymethyl residue (CML) on peptide(aa1-7) and peptide(aa8-35), as well as an imidazolone A on peptide(aa120-129). Furthermore, the early Maillard reaction of lysozyme, which had been glycated by seven different sugars, was monitored by MALDI-TOF/MS peptide mapping. Finally, this approach was successfully applied for site- and product-specific relative quantification of AGEs. For example, kinetics of CML and Amadori product formation on peptide(aa1-7), as well as imidazolone A formation on peptide(aa120-129), were determined.
Kislinger
Thomas
T
Institute of Pharmacy and Food Chemistry, Schuhstr. 19, 91052 Erlangen, Germany.
Humeny
Andreas
A
Peich
Carlo C
CC
Becker
Cord-Michael
CM
Pischetsrieder
Monika
M
D000595:Amino Acid Sequence; D017127:Glycation End Products, Advanced / Q000032:analysis / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D017127:Glycation End Products, Advanced; D011506:Proteins