Investigation of products arising from enzymatic digestion of advanced glycated albumin by high‐performance liquid chromatography/mass spectrometry

Abstract

The structural investigation of the products arising from 28 days incubation of albumin with high glucose concentration and further enzymatic hydrolysis has been carried out by means of high-performance liquid chromatography/mass spectrometry (HPLC/MS) under plasmaspray conditions. By this approach many different compounds have been detected, and for most of them, possible structures have been proposed on the basis of literature data and molecular weight assignments.

Accurate mass measurements by Fourier transform mass spectrometry in the study of advanced glycation end products/peptides

“…In vitro glycation of pure HSA was carried out with glucose under pseudo‐physiological conditions, according to a published procedure 24. Briefly, HSA (Sigma, St. Louis, MO, USA) (100 mg ml −1 in 0.2 M phosphate buffer, pH 7.4, containing 5 m M sodium azide as a bacteriostatic) was incubated with 0.5 M D ‐glucose (Sigma) at 37° C for 28 days.…”

“…For new information on AGE peptides, we undertook an investigation based on liquid chromatography (LC), LC/electrospray ionization mass spectrometry (ESI‐MS) and matrix‐assisted laser desorption/ionization (MALDI) of in vitro glycated HSA, followed by digestion with proteinase K 17. We chose these instrumental approaches in view of the interesting results they had given, in previous studies, on the glycation levels of proteins glycated both in vitro and in vivo 18–24. Furthermore, digestion with proteinase K was chosen considering the results of Gugliucci and Bendayan6 on in vitro glycated BSA.…”

The role of mass spectrometry in the study of non-enzymatic protein glycation in diabetes

“…Because the data previously obtained on products that arise by acid hydrolysis of glycated proteins indicated the possibility of artifact production, it was thought of interest to undertake a similar study based on enzymatic digestion of glycated albumin (Lapolla et al, 1991b). HPLC/MS was believed to be the mass spectrometric approach most suitable for this aim, and some products that originate from further dehydration of 3-and 4-deoxyosone (methylfurfurale and hydroxyacetyl furan) were identi®ed.…”

Advanced glycation end products: a highly complex set of biologically relevant compounds detected by mass spectrometry

“…In view of the valid results obtained by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) and high‐performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI‐MS) in the study of the glycation levels of both in vitro and in vivo glycated proteins,23–29 and considering that HPLC/ESI‐MS was useful in the identification of AGE compounds of very low molecular mass under both in vitro and in vivo conditions,30, 31 the present mass spectrometric investigation was based on both MALDI‐MS and HPLC/ESI‐MS. The data used to evaluate in vitro LMM‐AGE‐peptides were also compared with those derived from the commonly employed UV/visible and fluorimetric detectors.…”