Posttranslational modifications protein identification

Figure 4.13. Summary of advantages and limitations of representative proteomic platforms for protein expression analysis. The capabilities and advantages (solid line) are compared to the drawbacks (dotted lines) for each proteomic platform. Explanation of advantages and limitations are briefly highlighted here and more thoroughly discussed in the text. Abbreviations for each platform are provided in the Figure 4.1 legend and in the text. Other abbreviations used are PTMs, posttranslational modifications ID, identification.

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. 

The combination of this top-down proteomics approach, which generates information on the structure of the intact protein, with a bottom-up approach for protein identification (using MS/MS data of tryptic peptides from the collected fractions) has been particularly useful for identifying posttranslational modifications, cotransla-tional processing, and proteolytic modifications in a number of proteins. Examples from our work will be shown to illustrate this hybrid methodology for proteomics analysis. 

ExPASy Proteomics tools (http //expasy.org/tools/), tools and online programs for protein identification and characterization, similarity searches, pattern and profile searches, posttranslational modification prediction, topology prediction, primary structure analysis, or secondary and tertiary structure prediction. 

Size-based analysis of SDS-protein complexes in polyacrylamide gels (SDS-PAGE) is the most common type of slab gel electrophoresis for the characterization of polypeptides, and SDS-PAGE is one of the most commonly used methods for the determination of protein molecular masses.117 The uses for size-based techniques include purity determination, molecular size estimation, and identification of posttranslational modifications.118119 Some native protein studies also benefit from size-based separation, e.g., detection of physically interacting oligomers. 

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). 

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).

Peptide sequence analysis (PSA) of the peptides that contains a PTM. If the amino-acid sequence of the protein is not known or the position is not unambiguously determined in PMF, sequence analysis of the modified peptides is necessary. Strategies for shotgun identification (Ch. 18.3.2 ) of posttranslation modifications are reviewed by Cantin and Yates [4]. 

The protein molecular mass is insufficient information for identification, but it is adequate to confirm identity therefore, MS is one of the preferred techniques for characterization and quality control of recombinant proteins and other biomolecules. In the same way, it has been used to study posttranslational modifications (like glycosylation and disulfide bonding pattern), and other processes that can modify protein mass.11... 

Sequence information can also be obtained using Edman degradation to remove amino-terminal residues from a peptide, to produce a collection of peptides. MALDI-TOF can then be used to obtain the peptides masses, and the sequence determined by mass difference between consecutive peptides. This methodology is called protein ladder sequencing, and allows information to be obtained for up to 30 residues. This method is useful for the identification of posttranslational modifications, such as phosphorylated amino acid residues.24... 

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