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Protein glycation in diabetes as determined by mass spectrometry.

ABSTRACT: Diabetes is a common endocrine disorder characterized by hyperglycemia leading to nonenzymatic glycation of proteins, responsible for chronic complications. The development of mass spectrometric techniques able to give highly specific and reliable results in proteome field is of wide interest for physicians, giving them new tools to monitor the disease progression and the possible complications related to diabetes, as well as the effectiveness of therapeutic treatments. This paper reports and discusses some of the data pertaining protein glycation in diabetic subjects obtained by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). The preliminary studies carried out by in vitro protein glycation experiments show clear differences in molecular weight of glycated and unglycated proteins. Then, the attention was focused on plasma proteins human serum albumin (HSA) and immunoglobulin G (IgG). Enzymatic degradation products of in vitro glycated HSA were studied in order to simulate the in vivo enzymatic digestion of glycated species by the immunological system leading to the highly reactive advanced glycation end-products (AGEs) peptides. Further studies led to the evaluation of glycated Apo A-I and glycated haemoglobin levels. A different MALDI approach was employed for the identification of markers of disease in urine samples of healthy, diabetic, nephropathic, and diabetic-nephropathic subjects.

SUBMITTER: Lapolla A

PROVIDER: S-EPMC3610347 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Protein glycation in diabetes as determined by mass spectrometry.

Lapolla Annunziata A Molin Laura L Traldi Pietro P

International journal of endocrinology 20130313

Diabetes is a common endocrine disorder characterized by hyperglycemia leading to nonenzymatic glycation of proteins, responsible for chronic complications. The development of mass spectrometric techniques able to give highly specific and reliable results in proteome field is of wide interest for physicians, giving them new tools to monitor the disease progression and the possible complications related to diabetes, as well as the effectiveness of therapeutic treatments. This paper reports and di ...[more]

PMID: 23573087

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Project description:Reovirus is an enteric virus comprising eight structural proteins that form a double-layered capsid. During reovirus entry into cells, the outermost capsid layer (composed of proteins sigma3 and mu1C) is proteolytically processed to generate an infectious subviral particle (ISVP) that is subsequently uncoated to produce the transcriptionally active core particle. Kinetic studies suggest that protein sigma3 is rapidly removed from virus particles and then protein mu1C is cleaved. Initial cleavage of mu1C has been well described and generates an amino (N)-terminal delta peptide and a carboxyl (C)-terminal phi peptide. However, cleavage and removal of sigma3 is an extremely rapid event that has not been well defined. We have treated purified reovirus serotype 1 Lang virions with a variety of endoproteases. Time-course digestions with chymotrypsin, Glu-C, pepsin, and trypsin resulted in the initial generation of two peptides that were resolved in SDS-PAGE and analyzed by in-gel tryptic digestion and MALDI-Qq-TOFMS. Most tested proteases cut sigma3 within a "hypersensitive" region between amino acids 217 and 238. In addition, to gain a better understanding of the sequence of subsequent proteolytic events that result in generation of reovirus subviral particles, time-course digestions of purified particles were performed under physiologic salt conditions and released peptide fragments ranging from 500 to 5000 Da were directly analyzed by MALDI-Qq-TOFMS. Trypsin digestion initially released a peptide that corresponded to the C-terminus of mu1C, followed by a peptide that corresponded to amino acids 214-236 of the sigma3 protein. Other regions of mu1C were not observed until protein sigma3 was completely digested. Similar experiments with Glu-C indicated the hypersensitive region of sigma3 was cut first when virions were treated at pH values of 4.5 or 7.4, but treatment of virions with pepsin at pH 3.0 released different sigma3 peptides, suggesting acid-induced conformational changes in this outer capsid protein. These studies also revealed that the N-terminus of sigma3 is acetylated.

Project description:BackgroundCertain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated.ResultsFlour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction.ConclusionsThis is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the analysis revealed additional layers of complexity to the wheat flour proteome that should be considered when evaluating quantitative 2-DE data.

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Protein glycation in diabetes as determined by mass spectrometry.

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Protein glycation in diabetes as determined by mass spectrometry.

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