Proteomics analysis peptides

The enormous dynamic range of proteins in the sample represents an additional difficulty in proteome analysis. The best example is semm with a protein abundance ranging over eleven orders of magnitude (Anderson and Anderson, 2002). To detect the low abundant species, one has to load a sufficient amount of digest on a column to meet the limit of detection (LOD) of the MS instrument. Some reports published used up to 2.5 L of plasma with an extensive fractionation of intact proteins prior to LC-MS analysis on the peptide level (Rose et al., 2004). 

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. 

Lee, D.-H., Kim, S.-G., Park, Y.-C. et al. (2007) Proteome analysis of recombinant Escherichia coli producing human glucagon-like peptide-1. Analytical Tools for Proteomics, 849, 323-330. 

Peptidic photoprobes can be based on the photoreactive amino acid p-benzoyl-L-phenylalanine inserted into a peptide in place of a natural aromatic residue by peptide synthesis [65] or by manipulation of the genetic code [66]. The use of p-benzoyl-L-phenylalanine for this purpose is not new, but the nature of peptide probes naturally offers opportunities for the location of linkage sites by proteomic analysis [67]. 

We conducted proteomic analysis of the KO mouse brain to identify proteins or peptides whose expression levels may change due to a lack of SCRAPPER. Imaging MS allowed us to statistically analyze location and expression intensities of many biomolecules and to extract molecules that exhibited region-specific expression. Groups of molecules whose expression patterns differed between WT mice and KO mice particularly attracted our attention. 

Y. An, J. W. Cooper, B. M. Balgley, and C. S. Lee. Selective Enrichment and Ultrasensitive Identification of Trace Peptides in Proteome Analysis Using Transient Capillary Isotachophoresis/Zone Electrophoresis Coupled with Nano-ESI-MS. Electrophoresis, 27(2006) 3599-3608. 

Proteome analysis in general involves two stages protein separation and subsequent identification and analysis. Multidimensional separations are required in order to result in an adequate resolution of complex protein or peptide... 

In general, these instruments are capable of measuring peptide masses. However, this kind of information has limited use in proteome analysis. 

Jensen, O. N. 1999. Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels. In 2-D Proteome Analysis Protocols, Link, A. I, ed., Humana Press (Totowa, New Jersey), 513-530. 

---