Abstract
Non-enzymatic glycosylation or glycation involves covalent attachment of reducing sugar residues to proteins without enzyme participation. Glycation of glucose to human serum albumin in vivo is related to diabetes and many other diseases. We present an approach using liquid chromatography coupled to an electrospray ionization source of a hybrid ion trap-time of flight (IT-TOF-MS/MS) tandem mass spectrometer to identify the glycation sites on serum albumin from both a healthy person and a diabetic patient. The MetID software, which is commonly used for screening metabolites, is adapted for peptide fingerprinting based on both m/z values and isotopic distribution profiles. A total of 21 glycation sites from the healthy person and 16 glycation sites from the diabetic patient were identified successfully. We also demonstrate the use of matrix assisted laser desorption ionization-time of flight mass spectrometry to estimate the incorporation ratio of glucose to albumin during glycation. Results from this study show that the glycation in healthy person is more complicated than previously thought. Further analysis of incorporation ratio distribution may be necessary to accurately reflect the change of serum albumin glycation in diabetic patients.
Highlights
It is well known in the literature that enzymes are involved in the glycosylation process, as exemplified in the N-glycosylation at asparagine residues, the O-glycosylation at threonine or serine residues and the glycosylphosphatidylinositol-anchoring at the C-terminus of some proteins
We demonstrated the use of Ion trap-time of flight (IT-TOF) for study of glycation sites in glycated HSA (GA) from both diabetic patient and healthy person
Total human serum albumin (HSA) was firstly extracted from serum by polyethylene glycol (PEG) precipitation
Summary
It is well known in the literature that enzymes are involved in the glycosylation process, as exemplified in the N-glycosylation at asparagine residues, the O-glycosylation at threonine or serine residues and the glycosylphosphatidylinositol-anchoring at the C-terminus of some proteins. The glycation site-containing peptides were labeled by reduction with NaB3H3, purified by affinity HPLC and reversed phase HPLC with radioactivity monitor, and identified by amino acid composition analysis [16,17]. Quadrupoletime of flight (Q-TOF), one the most popular types of MS analyzer in peptide mapping fields, was reported to be applied in glycation sites study by different research groups [20,21]. The ratio of glucose incorporated to HSA, a potentially important character of the GA glycoconjugate which seemed overlooked by previous researchers, was successfully elucidated using MALDI-TOF-MS
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