Comparison of modification sites in glycated crystallin in vitro and in vivo.

Glycation of α-crystallin is responsible for age- and diabetic-related cataracts, which are the main cause of blindness worldwide. We optimized the method of identification of lysine residues prone to glycation using the combination of LC-MS, isotopic labeling, and modified synthetic peptide standards with the glycated lysine derivative (Fmoc-Lys(i,i-Fru,Boc)-OH). The in vitro glycation of bovine lens α-crystallin was conducted by optimized method with the equimolar mixture of [(12)C6]- and [(13)C6]D-glucose.

Nonenzymatic modification of Ubiquitin under high-pressure and -temperature treatment: mass spectrometric studies.

The effect of high-pressure and/or high-temperature on the glycation of a model protein (ubiquitin) was investigated by mass spectrometry. This paper reports the impact of high pressure (up to 1200 MPa) on the modification of a ubiquitin using ESI-MS measurements. The application of glucose labeled with stable isotope allows a quantitative assessment of modification under the conditions of high-pressure (HPG) and high-temperature (HTG) glycation.

A synthesis of new, bi-labeled peptides for quantitative proteomics.

Unlabelled: Isotopically labeled peptides are often used in proteomics as internal reference allowing quantification of peptides by isotopic dilution method. Although the synthesis of peptides labeled with stable isotopes is relatively simple, there are several factors limiting application of these standards in proteomic research: cost of labeled derivatives of amino acids, time needed to obtain labeled peptide and problems with quantification of the standard. To solve these problems we developed a method of synthesis of peptides labeled with heavy oxygen and with a dabsyl moiety.

Hydrogen-deuterium exchange in imidazole as a tool for studying histidine phosphorylation.

Isotope exchange at the histidine C2 atom of imidazole in D2O solution is well known to occur at a significantly slower rate than the exchange of amide protons. Analysis of the kinetics of this isotope-exchange reaction is proposed herein as a method of detecting histidine phosphorylation. This modification of His-containing peptides is challenging to pinpoint because of its instability under acidic conditions as well as during CID-MS analysis.

A study on human serum albumin influence on glycation of fibrinogen.

Although in vivo glycation proceeds in complex mixture of proteins, previous studies did not take in consideration the influence of protein-protein interaction on Maillard reaction. The aim of our study was to test the influence of human serum albumin (HSA) on glycation of fibrinogen. The isotopic labeling using [(13)C6] glucose combined with LC-MS were applied as tool for identification possible glycation sites in fibrinogen and for evaluation the effect of HSA on the glycation level of selected amino acids in fibrinogen.

Testing isotopic labeling with [¹³C₆]glucose as a method of advanced glycation sites identification.

The Maillard reaction occurring between reducing sugars and reactive amino groups of biomolecules leads to the formation of a heterogeneous mixture of compounds: early, intermediate, and advanced glycation end products (AGEs). These compounds could be markers of certain diseases and of the premature aging process. Detection of Amadori products can be performed by various methods, including MS/MS techniques and affinity chromatography on immobilized boronic acid.