Analyses of the in vitro non-enzymatic glycation of peptides/proteins by matrix-assisted laser desorption/ionization mass spectrometry

Abstract Non-enzymatic glycation of proteins with the reducing agent glucose is implicated to be responsible for diabetes-derived complications, food browning, and aging. However, the non-enzymatic glycation process of peptides/proteins is not well understood and further research is needed to gain an understanding of the underlying principles involved in diabetes-related complications. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is used to analyze the in vitro glycation of peptides/proteins. In addition to the physiological conditions, harsh conditions (higher concentration of glucose, higher or lower pH, and higher temperature) are also used in this study. Peptides/proteins are reacted with glucose for up to 120 h at 4 °C, 37 °C, or 65 °C. Single and/or multiple glycations are observed using broad pH conditions (from 10% TFA with p K a of 0.5 to pH 10) at various glucose concentrations (from 0.01 M to 1 M). Data suggest that glucose reacts readily with both peptides and proteins, and the efficiency of the glycation increases with higher temperature, higher pH, higher glucose concentration, or longer incubation time. However, influence of the buffer pH on the efficiency of the glycation of peptides is less pronounced compared to that of proteins. This effect could result from denaturation of proteins at higher pH and the resultant exposure of potential glycation sites. This data could lead to the inference that the glycation process of peptides/proteins would occur but proceed very slowly under the diabetes conditions in vivo (37 °C, ∼neutral pH, ∼0.007 M glucose). Postsource decay and MS/MS results of singly glycated angiotensin I, P 14 R (PPPPPPPPPPPPPPR), and human adrenocorticotropic hormone (ATCH) fragments 1–13 indicate that glucose reacts with the amino group of the N-terminal of ATCH 1–13 and the guanidino group of the arginine residue of both angiotensin I and P 14 R.