Posttranslational modifications spectrometry

Arnold, R. J. Reilly, J. P. Observation of Escherichia coli ribosomal proteins and their posttranslational modifications by mass spectrometry. Anal. Biochem. 1999, 269,105-112. 

The results for bacterial whole-cell analysis described here establish the utility of MALDI-FTMS for mass spectral analysis of whole-cell bacteria and (potentially) more complex single-celled organisms. The use of MALDI-measured accurate mass values combined with mass defect plots is rapid, accurate, and simpler in sample preparation then conventional liquid chromatographic methods for bacterial lipid analysis. Intact cell MALDI-FTMS bacterial lipid characterization complements the use of proteomics profiling by mass spectrometry because it relies on accurate mass measurements of chemical species that are not subject to posttranslational modification or proteolytic degradation. 

Larsen, M.R., Trelle, M.B., Thingholm, T.E., and lensen, O.N. 2006. Analysis of posttranslational modifications of proteins by tandem mass spectrometry. BioTechniques 40, 790-798. 

J. J. Lennon and K. A. Walsh. Locating and Identifying Posttranslational Modifications by In-Source Decay During MALDI-TOF Mass Spectrometry. Protein Sci., 8(1999) 2487-2493. 

IV. Characterization of Posttranslational Modifications with Mass Spectrometry... 

Annan R.S. and Carr S.A. (1997), The essential role of mass spectrometry in characterizing protein structure mapping posttranslational modifications, J. Protein Chem. 16(5), 391-402. 

Carr SA, Annan RS, Huddleston MJ. Mapping posttranslational modifications of proteins by MS-based selective detection Application to phosphoproteomics. In Burlingame AL, ed.. Mass Spectrometry Modified Proteins and Glycoconjugates, Vol. 405, New York Academic Press, 2005, 82-115. 

HSP27 and Tau protein, respectively. 2D immunoblotting combined with mass spectrometry-based identification methods has been widely applied to the characterization of 2D electrophoretic cross-reactive isoforms of the same protein, e.g., resulting from alternative splicing, co- and/or posttranslational modifications and proteolytic cleavages (15-19). 

S. Udiavar, A. Apffel, J. Chakel, S. Swedberg, W. S. Hancock, and E. Pungor, The use of multidimensional liquid-phase separations and mass spectrometry for the detailed characterisation of posttranslational modifications in glycoproteins, Anal. Chem., 70 (1998) 3572-3578. 

Mass spectrometry is more than 100 times more accurate than gel electrophoresis in molecular mass determination, and obtaining the data for sequence analysis requires only a fraction of the time needed for Edman degradation. Moreover, mass spectrometry is well suited for analysis of posttranslational modifications, which cannot be determined by Edman degradation. 

F. Kjeldsen, K. F. Haselmann, B. A. Budnik, E. S. Sorensen, and R. A. Zubarev, Complete characterization of posttranslational modification sites in the bovine milk protein PP3 by tandem mass spectrometry with electron capture dissociation as the last stage, Anal. Chem., 75 (2003) 2355-2361. 

The dioxygen reduction site of the key respiratory enzyme, cytochrome c oxidase [E.C. 1.9.3.1], is a bimetallic catalytic center comprised of a heme iron adjacent to a Type 2 mononuclear copper center (see Cytochrome Oxidase). The recent solution of the X-ray crystal structure of this enzyme revealed an entirely unanticipated covalent modification of the protein structure, a cross-link between a histidine and tyrosine side chain (23) within the active site (Figure 2)." This extraordinary posttranslational modification has been confirmed by peptide mapping and mass spectrometry, and has been detected as a conserved element in cytochrome c oxidases isolated from organisms ranging from bacteria to cows. The role of the cross-linked structure in the function of cytochrome c oxidase is still controversial." " ... 

Understanding the action mechanism of flavoenzymes heavily relies on the combination of different chemical tools and techniques. First, it is of utmost importance to have a pure and stable (recombinant) protein. Size exclusion chromatography will provide information about the enzyme quaternary structure, and mass spectrometry can establish posttranslational modifications. For a detailed insight into the protein structure, well-diffracting crystals are needed to determine the X-ray structure. 

Fatty acylation of proteins is a common posttranslational modification that blocks sequencing of the N-terminal amino acids by Edman techniques. Mass spectrometry can play an essential role in such sequencing, but it is not always trivial to locate the blocked N-terminal peptide. Besides acetylation, acylation of amino acids with longer-chain fatty acids (e.g., myristic acid) has been reported. 

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