Post-Translational Modifications

Mass spectrometry has a particular strength in its capability for characterizing covalent modifications of proteins, either natural posttranslational modifications or chemical modifications introduced by the investigator to probe the structure and function of the protein. Among the more common posttranslational modifications are disulfide bonds. To identify the locations of disulfide bonds, cleavage of the protein is carried out without prior reduction. Molecular weights of the fragments with disulfide bonds intact are determined. The disulfide bonds are then cleaved with observance of the masses of the peaks which disappear and the masses of the cleavage products which appear. If the amino acid sequence of the protein is already known, the locations of the disulfide bonds can usually be deduced simply from molecular weight measurements. If necessary, sequencing of cleavage products can be done by tandem mass spectrometry. It may also be necessary to carry out multiple cleavages if an initial cleavage yields fragments with more than one disulfide bond.

Post-translational modifications (e.g. phosphorylation) or chemical modifications (e.g. photoaffinity labeling) which result in covalent modifications of amino acid residues can also be characterized by mass spectrometry. Cleavage fragments containing such modifications can be identified by characteristic mass shifts. The location of the modification within the peptide cleavage fragment can be determined from mass shifts in the tandem mass spectrum. For example, phosphorylation of a serine or threonine residue yields a characteristic mass increment of 80 units in the fragment molecular weight as well as 98 mass unit losses (loss of phosphoric acid) in the tandem mass spectrum. Glycosylation of proteins can also be characterized by mass spectrometry. Molecular weight changes for intact proteins after chemical or enzymatic deglycosylation can be used to determine the presence and extent of such modifications. Location of glycosylated sites within proteins can be determined by cleaving the protein and observing shifts in cleavage fragment masses from the masses predicted for the unmodified peptide fragment. Often, glycosylation of a peptide cleavage fragment will shift its molecular weight beyond the range normally observed. Appearance of a new peak after deglycosylation is indicative that the peptide fragment was initially glycosylated. Carbohydrate moieties can also be characterized by mass spectrometry after cleavage from the peptide or protein. Molecular weight measurements give information on the possible composition as well as the heterogeneity of the carbohydrate. Since carbohydrates tend to fragment at glycosidic bonds in the mass spectrometer, structural information can also be observed.

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Biomolecular Mass Spectrometry Facility Department of Pharmacology Medical University of South Carolina 173 Ashley Avenue, CRI 305 Charleston, SC 29425	Telephone Numbers: 843-792-5849 (CRI 305) 843-792-2471 (department office) FAX: 843-792-2475