Posttranslational modifications analysis

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. 

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. 

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. 

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. 

Krishna R.G. and Wold F. (1998), Posttranslational modifications, in Proteins — Analysis and Design, Angeletti R.H. (Ed.), 121-126, Academic Press, San Diego. 

How do we then envision the protein microarray as a proteomics tool We now estimate the human genome to comprise around 30,000 genes. For gene expression analysis using DNA microarrays, 1000 to 10,000 gene elements are often used. Since proteins undergo posttranslational modification (>200 different types see McDonald and Yates, 2000, Reference 40) and can occur as isoforms and multiprotein complexes, the number of protein expression elements needs to be much larger. 

Despite the fact that only 20 amino acids (plus selenocys-teine and formylmethionine in prokaryotic systems) are known to be directly specified by the genetic code, chemical analysis of mature proteins has revealed hundreds of different amino acids, all of them structural variants on the original 20. This structural diversity, which greatly expands the chemical lexicon of proteins, results from posttranslational modification of the primary products of translation. Our knowledge of the nature and significance of enzymatic reactions that bring about these important alterations is still very incomplete. 

The protein posttranslational modifications (PTMs) play a crucial role in modifying the end product of expression and contribute towards biological processes and diseased conditions. Important posttranslational modifications include phosphorylation, acetylation, glycosylation, ubiquitination, and nitration [Mann and Jensen, 2003], The analysis of posttranslational modifications on a proteome scale is still considered an analytical challenge [Zhou et al., 2001] because of the extremely low abundance of modified proteins among very complex proteome samples. 

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