Mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry

Guedes, Sofia; Vitorino, Rui; Domingues, M. Rosário M.; Amado, Francisco; Domingues, Pedro

doi:10.1016/j.jasms.2009.03.004

Cited by 26 publications

(26 citation statements)

References 24 publications

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“…No modification on the lysine residues and N-terminal were detected by our experimental approach. Insulin glycation by D -glucose also led to the coexistence of protein molecules glycated at different residues [34]. In opposition to our results, the N-terminus of both chains and the lysine residue 29 were modified upon glucose glycation.…”

Section: Discussioncontrasting

confidence: 99%

“…In contrast, no evidences of glycation in the N-terminal and the lysine residue were observed by our mass spectrometry analysis. Although the N-terminal of insulin was found to be the major glycation target when using glucose as glycation agent [33,34], it is well known that methylglyoxal preferentially reacts and modify arginine residues [35]. …”

Section: Resultsmentioning

confidence: 99%

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Insulin glycation by methylglyoxal results in native-like aggregation and inhibition of fibril formation

et al. 2011

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BackgroundInsulin is a hormone that regulates blood glucose homeostasis and is a central protein in a medical condition termed insulin injection amyloidosis. It is intimately associated with glycaemia and is vulnerable to glycation by glucose and other highly reactive carbonyls like methylglyoxal, especially in diabetic conditions. Protein glycation is involved in structure and stability changes that impair protein functionality, and is associated with several human diseases, such as diabetes and neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Familiar Amyloidotic Polyneuropathy. In the present work, methylglyoxal was investigated for their effects on the structure, stability and fibril formation of insulin.ResultsMethylglyoxal was found to induce the formation of insulin native-like aggregates and reduce protein fibrillation by blocking the formation of the seeding nuclei. Equilibrium-unfolding experiments using chaotropic agents showed that glycated insulin has a small conformational stability and a weaker dependence on denaturant concentration (smaller m-value). Our observations suggest that methylglyoxal modification of insulin leads to a less compact and less stable structure that may be associated to an increased protein dynamics.ConclusionsWe propose that higher dynamics in glycated insulin could prevent the formation of the rigid cross-β core structure found in amyloid fibrils, thereby contributing to the reduction in the ability to form fibrils and to the population of different aggregation pathways like the formation of native-like aggregates.

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Section: Discussioncontrasting

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Insulin glycation by methylglyoxal results in native-like aggregation and inhibition of fibril formation

et al. 2011

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“…[26 -28] Recently, the characterization of the glycation sites of bovine insulin was determined by MALDI tandem mass spectrometry. [29] However, reports on detailed structural elucidation of the synthesized neoglycoconjugate vaccines by mass spectrometry have been quite meager. Little work has been done with regard to localizing the lysine residues where the carbohydrate haptens are attached in protein carriers.…”

Section: Introductionmentioning

confidence: 99%

Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O‐PS of Vibrio cholerae O1, serotype Ogawa, and BSA revealed by matrix‐assisted laser desorption–ionization tandem mass spectrometry

et al. 2010

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We present the MALDI-TOF/TOF-MS analyses of various hapten-bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer-equipped, terminal monosaccharide of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten-BSA neoglycoconjugates with different hapten-BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI-TOF/TOF-MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI-TOF/TOF-MS/MS of the glycated peptides. The product-ion scans of the protonated molecules were carried out with a MALDI-TOF/TOF-MS/MS tandem mass spectrometer equipped with a high-collision energy cell. The high-energy collision-induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y-series product ions was very useful for the sequencing of various peptides. The series of a- and b-product ions confirmed the sequence of the conjugated peptides.

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“…The detailed characterization as well as the localization of such modifications in a protein sequence has been a key point in many reports 6–9. The characterization of the so‐called advanced glycation end products (AGE) has been extensively studied,10, 11 and some pioneering mass spectrometric work related to the glycation of plasma proteins has been reported by Lapolla et al 12, 13.…”

mentioning

confidence: 99%

Glycation pattern of peptides condensed with maltose, lactose and glucose determined by ultraviolet matrix-assisted laser desorption/ionization tandem mass spectrometry

Montgomery

Tanaka

Belgacem

2010

Rapid Commun. Mass Spectrom.

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Protein glycation is the non-enzymatic condensation of sugars with proteins. Although commonly occurring in both the therapeutic and food/beverage industries, protein glycation has not been the focus of many proteomic investigations. This study aims to establish a reliable mass spectrometric method for screening large tandem mass spectrometric (MSMS) datasets for protein glycation with glucose, lactose and maltose. Control experiments using a standard peptide containing a single glycation site led to the discovery of characteristic neutral loss fragmentation patterns in MSMS analysis for glucose, lactose and maltose condensed with peptides. Valid in both tandem time-of-flight (TOFTOF) and quadrupole ion trap time-of-flight matrix-assisted laser desorption/ionization (QIT TOF MALDI) mass spectrometers, these neutral loss signatures were then applied to elucidation of modified peptides from a complex human serum albumin (HSA) digest glycated with each of the proposed sugars. Screening of these large datasets was made possible by specifically designed software solutions that enable the input of detailed user-defined post-translational modifications that are not included in the universally available databases such as Unimod.

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Mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry

Cited by 26 publications

References 24 publications

Insulin glycation by methylglyoxal results in native-like aggregation and inhibition of fibril formation

Insulin glycation by methylglyoxal results in native-like aggregation and inhibition of fibril formation

Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O‐PS of Vibrio cholerae O1, serotype Ogawa, and BSA revealed by matrix‐assisted laser desorption–ionization tandem mass spectrometry

Glycation pattern of peptides condensed with maltose, lactose and glucose determined by ultraviolet matrix-assisted laser desorption/ionization tandem mass spectrometry

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