Tandem mass spectrometry of non-enzymatically glycated peptides and proteins

Doctoral thesis English OPEN
Lopez-Clavijo, Andrea F.
  • Subject: QD | QP

The thesis presents the study of the reaction of glyoxal (ethanedial) with polypeptides. This\ud reaction is important in the food industry as well as during ageing and diabetes mellitus. To\ud study this reaction a Fourier transform ion cyclotron resonance mass spectrometer coupled with\ud electron capture dissociation and collisionally activated dissociation was used. Initially this\ud reaction was carried out in the neuropeptide Substance P to set up the reaction conditions,\ud sample preparation, as well as the instrumental parameters in the mass spectrometer.\ud The results in Substance P revealed two compounds, with mass additions assigned as C2O and\ud C2H2O2 from glyoxal, were formed. MS/MS results showed that the modification site for both\ud species could be located at either the arginine residue or at the N-terminus. Thus, in order to\ud distinguish N-terminus from arginine modification the position of the arginine was varied in\ud four model peptides. The results indicated that both mass additions C2O, C2H2O2 were located\ud at the arginine residue. Interestingly, two of those model peptides showed an unusual mass\ud addition of 21.9843 Da, which was assigned as a new type of glyoxal modification at the arginine\ud residue showing the addition of two carbon atoms from glyoxal and the loss of two hydrogen\ud atoms from the peptide (C2-H2), herein referred to as 2-imino-imidazole.\ud In order to assess the involvement of other residues in the reaction with glyoxal a new set of\ud experiments in acetylated and non-acetylated undecapeptides were carried out. Unexpectedly,\ud these experiments revealed that two species with the same mass (16.01092 Da) were being\ud formed in the non-acetylated peptide. One of the species corresponded to diglycation, where the\ud results suggest that the glyoxal binding at the lysine residue is crosslinked with the N-terminus.\ud The second species showing the addition of 116.01092 Da was formed at the arginine residue\ud forming a species, here called a glyoxal dimer, at the arginine residue. The formation of the\ud glyoxal dimer species was also observed in the acetylated peptide. Although is clear that\ud crosslinking between the lysine residue and the N-terminus is not possible in the acetylated\ud peptide, the results seem to indicate that crosslinking between the amino group of the lysine\ud and the amide group of glutamine could occur. However, a systematic study varying the position\ud of the lysine relative to the glutamine residue and also relative to the N-terminus needs to be\ud addressed in the future in order to determine the extent of the involvement of the N-terminus\ud and amide group in the glyoxal glycation reaction.
  • References (114)
    114 references, page 1 of 12

    [111] Cotham, W. E. and Metz, T. O. and Ferguson, P. L. and Brock, J. W. C. and Hinton, D. J. S. and Thorpe, S. R. and Baynes, J. W. and Ames, J. M. Proteomic analysis of arginine adducts on glyoxal-modified ribonuclease. Mol.Cell. Proteomics, 3 (12):1145-53, 2004.

    [112] Ames, J. M. Mass spectrometry to detect the site specificity of advanced glycation/lipoxidation end-product formation on protein: some challenges and solutions. Biochem. Soc. Trans., 36(Pt 5):1051-4, 2008.

    [113] Thornalley, P. J. and Battah, N. and Ahmed, N. and Karachalias, S. and Agalou, R. Babaei-Jadidi, A. and Dawnay, A. Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry. Biochem. J., 375:581-592, 2003.

    [114] Glomb, M. A. and Lang, G. Isolation and characterization of glyoxal-arginine modifications. J. Agr. Food Chem., 49(3):1493-501, 2001.

    [115] Brock, J. W. C. and Hinton, D. J. S. and Cotham, W. E. and Metz, T. O. and Thorpe, S. R. and Baynes, J. W. and Ames, J. M. Proteomic Analysis of the Site Specificity of Glycation and Carboxymethylation of Ribonuclease. J. Proteome Res., 2:506-513, 2003.

    [116] Ahmed, M. U. and Thorpe, S. R. and Baynes, J. W. Identification of N-epsiloncarboxymethyllysine as a degradation product of fructoselysine in glycated protein. J. Biol. Chem., 261(11):4889-4894, 1986.

    [117] Al-Abed, Y. M. and Bucala, R. N -carboxymethyllysine formation by direct addition

    [118] Hayashi, C. M. and Nagai, R. and Miyazaki, K. and Hayase, F. and Tomohiro, A. and Tomomichi, O. and Horiuchi, S. Conversion of Amadori Products of the Maillard Reaction to N - (carboxymethyl)lysine by Short-Term Heating : Possible Detection of Artifacts by Immunohistochemistry. Lab. Invest., 82(6):795-807, 2002.

    [119] Shangari, N. and O'Brien, P. J. The cytotoxic mechanism of glyoxal involves oxidative stress. Biochem. Pharmacol., 68(7):1433-42, 2004.

    [120] Mera, K. and Takeo, K. and Izumi, M. and Maruyama, T. and Nagai, R. and Otagiri, M. and Masaki, O. Effect of reactive-aldehydes on the modification and dysfunction of human serum albumin. J. Pharm. Sci., 99(3):1614-1625, 2010.

  • Metrics
    0
    views in OpenAIRE
    0
    views in local repository
    12
    downloads in local repository

    The information is available from the following content providers:

    From Number Of Views Number Of Downloads
    Warwick Research Archives Portal Repository - IRUS-UK 0 12
Share - Bookmark