Open Search of Peptide Glycation Products from Tandem Mass Spectra.

Identification of chemically modified peptides in mass spectrometry (MS)-based glycation studies is a crucial yet challenging task. There is a need to establish a mode for matching tandem mass spectrometry (MS/MS) data, allowing for both known and unknown peptide glycation modifications. We present an open search approach that uses classic and modified peptide fragment ions. The latter are shifted by the mass delta of the modification. Both provide key structural information that can be used to assess the peptide core structure of the glycation product. We also leverage redundant neutral losses from the modification side chain, introducing a third ion class for matching referred to as characteristic fragment ions. We demonstrate that peptide glycation product MS/MS spectra contain multidimensional information and that most often, more than half of the spectral information is ignored if no attempt is made to use a multi-step matching algorithm. Compared to regular and/or modified peptide ion matching, our triple-ion strategy significantly increased the median interpretable fraction of the glycation product MS/MS spectra. For reference, we apply our approach for Amadori product characterization and identify all established diagnostic ions automatically. We further show how this method effectively applies the open search concept and allows for optimized elucidation of unknown structures by presenting two hitherto undescribed peptide glycation modifications with a delta mass of 102.0311 and 268.1768 Da. We characterize their fragmentation signature by integration with isotopically labeled glycation products, which provides high validity for non-targeted structure identification.

Molecular characterization of sequence-driven peptide glycation.

Peptide glycation is an important, yet poorly understood reaction not only found in food but also in biological systems. The enormous heterogeneity of peptides and the complexity of glycation reactions impeded large-scale analysis of peptide derived glycation products and to understand both the contributing factors and how this affects the biological activity of peptides. Analyzing time-resolved Amadori product formation, we here explored site-specific glycation for 264 peptides. Intensity profiling together with in-depth computational sequence deconvolution resolved differences in peptide glycation based on microheterogeneity and revealed particularly reactive peptide collectives. These peptides feature potentially important sequence patterns that appear in several established bio- and sensory-active peptides from independent sources, which suggests that our approach serves system-wide applicability. We generated a pattern peptide map and propose that in peptide glycation the herein identified molecular checkpoints can be used as indication of sequence reactivity.

Insights into the Chemistry of Non-Enzymatic Browning Reactions in Different Ribose-Amino Acid Model Systems.

Reactions between sugars and amino acids in the Maillard reaction produce a multitude of compounds through interconnected chemical pathways. The course of the pathways changes depending on the nature of the amino acids and sugars as well as the processing conditions (e.g. temperature, water activity). Some partial pathways have been elucidated using labelled precursors but the process is very time intensive. Here, we use rapid, non-targeted analysis with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to deliver the molecular formulae and ion intensities of the compounds generated from reaction of four amino acids with ribose (10 h at 100 °C) to study the effect of amino acid side chains on the reaction pathways. Using van Krevelen diagrams, known chemical changes during the reaction (e.g. dehydration or decarboxylation) can be studied. Comparison of the data from the four amino acids studied, showed a common pathway, which involved 73 Maillard reaction products (MRPs) where the differences were due only to the nature of the amino acid side chain. From the more than 1400 different molecular formulae found, pathways unique to the amino acids were also identified and the order of reactivity was lysine >cysteine >isoleucine ≈ glycine. While unequivocal identification of the compounds cannot be achieved with FT-ICR-MS, applying known chemical transformations found in the Maillard reaction, not only identifies new and known pathways, but also integrates the MRPs into a general Maillard reaction scheme that better represents the totality of the Maillard reaction.