Posttranslational modifications mapping

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. 

DOR-1 encodes both delta and delta2 receptor subtypes. Therefore, the different properties of the delta and delta2 receptor subtypes could result from differential posttranslational modification of the DOR-1-encoded receptor protein, alterations in the molecular environment of the receptor protein. Alternatively, the understanding of the existed splice variants for DOR-1 must be important to accept the concept of the deltai and delta2 receptor subtypes, as suggested by DOR-1 antisense mapping studies [60]. 

Figure 4.3. Fields of proteomic research. Proteomic research can be classified into six general research fields. Proteomic mapping and proteomic profiling constitute the first tier of proteomic analysis based upon identification and quantitation of proteins within a defined space of interest that can range from the entire organism to the protein level. The second tier of proteomic analyses is shown below involving global characterization of structure, function, posttranslational modifications, and association with other proteins (or other biochemical components).

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." " ... 

Traditional methods to map posttranslational modification sites, like those of phosphorylation, have been anchored by protein digest and mass spectroscopic (MS) approaches (for a review on the classic evaluation and for MS analyses of O-glycans, see Reference (56)). Unfortunately, like many posttranslational modifications, O-GlcNAcylation occurs routinely on a protein population with substoichiometric frequency, which results in a very small detectable population of a O-GlcNAc-modified product. Also, much like O-phosphate additions, the protein-O-GlcNAc bond is labile and is detached by collision-induced dissociation (CID) during MS analysis. Often, the bond is lost before it can be detected on the peptides analyzed (57, 58). Phosphate modifications, however, can overcome this limitation by emiching the peptide mixtures... 

The interplay between posttranslational modifications and the ligand potentially leads to a myriad of functional outcomes for the nuclear receptors. We are only begiiming to map out the relationships between individual posttranslational events and to understand the specific effects of their combinations on receptor activity. Numerous studies highlight the importance of the histone code or how posttranslational modifications of histone proteins affect transcriptional state of the chromatin and dictate transcriptional competency of genes. The abundance of posttranslational modifications on nuclear receptors suggests a similar idea of regulation. 

The introduction of soft ionization techniques such as ESI and M ALDI brought tremendous progress in on- and offline characterization of electrophoretically separated peptides and proteins by MS [50]. Combination of CE with MS techniques allows not only high-accuracy molecular mass determination of peptides and proteins separated by CE, but also it provides important structural data on amino acid sequence, the sites of posttranslational modifications, peptide mapping, and the noncovalent interactions of peptides and proteins. 

Peptide Mapping. Peptide mapping is an important tool for protein identif-ication, primary structure determination, the detection of posttranslational modifications, the identification of genetic variants, and the determination of glycosylation and/or disulfide sites. For these reasons, peptide mapping is widely used for quality control and for the characterization of recombinant DNA-derived products. Moreover, the high resolution of CE makes it a powerful peptide mapping technique. 

Amino acid analysis and peptide mapping are standard methods in the course of protein identification processes [5, 6]. Molecular mass determination of whole molecules as well as peptide fragments with accuracies of about 0.01% by either matrix-assisted laser desorp-tion/ionization (MALDI)-MS for surface immobilized samples, or electrospray ionization (ESI)-MS for liquid samples, is another highly efficient protein identification method. These methods additionally support the identification of posttranslational modifications such as... 

Posttranslational modification profiling Specific biochemical methods are used to identify the abundance of targets with specific posttranslational modifications (such as phosphorylated proteins) (Zheng and Chan, 2002). Monitoring protein phosphorylations is particularly important in mapping the intracellular signaling network... 

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