Peptides fraction enrichment

Generically, sample preparation in bottom-up proteomics involves the solubilization of proteins from the biological source, protein fractionation, subsequent enzymatic digestion of the proteins, and separation of the resulting peptides. Unique enrichment techniques can be applied in many of these preparatory steps (Figure 5). 

On the basis of the large number of nonhomologous bands in both cases, we concluded that a great number of peptide bonds was cleaved and formed due to considerable transpeptidation. It is interesting to note that, in this respect, the samples both with and without methionine addition behaved in an analogous way. The methionine content of the EPM product with Met enrichment was higher by 2.6% only than that of the product without amino acid enrichment. Nevertheless, the peptide fractions of the EPM product without amino acid incorporation and the Met-enriched product differed from each other remarkably. This is why we suppose that the presence of methionine in the reaction mixture and its incorporation give rise to a new type of transpeptidation, namely, they modify the sequential position of the cleaved and formed peptide bonds. 

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

A similar investigation was carried out by Kfirkela and Kulonen (1959) who partially hydrolyzed elastin by acids, alkalies, and enzymes under varying conditions and fractionated the products by adsorption on alumina and elution with ammonia. They obtained fractions in which the yellow color was enriched and determined the infrared and ultraviolet absorption spectra. They concluded that the yellow pigment was tightly bound to the peptide chain and that it did not appear to be a bile pigment. 

Attempts to develop the sieving process as a tool for the isolation of a pure peptide containing the chromophore were not successful because it was found that the resins did not act as pure ionic sieves (Partridge, 1952) the colored peptides displayed strong adsorptive effects which appeared to be nonionic in character. However it was found that the strong adsorptive affinity of the colored peptides could itself be utilized for further enrichment of the fractions. 

Fractionation of proteins by strong cation exchange (SCX) chromatography, followed by IMAC enrichment of phosphopeptides from SCX fractions, led to a comprehensive identification of phosphoproteins of PSD isolated from mouse brain using LC-MS/MS (Trinidad et al. 2006). In this study, phosphorylation site(s) were mapped to 287 proteins from a total of 1,264 unique proteins identified. This translates into a 23% phosphorylation rate, comparable to an expected 33% rate in the general proteome (Johnson et al. 2005). The 287 phosphoproteins were derived from a total of 998 unique phosphorylated peptides, and the phosphorylations were mapped to 723 unique sites. Most of these occurred on serines, to a lesser extent on threonines, and only minimally on tyrosines (Figure 5A). 

Figure 6. MALDI mass spectrum of fraction 13 from RP-HPLC. The H,K-ATPase-enriched vesicles were trypsinized and centrifuged to separate supernatant from pellet. The supernatant was subjected to RP/HPLC and individual fractions collected and subjected to MALDI/MS. The MALDI mass spectrum (reflectron-ion mode) was obtained using a-cyano-4-hydroxy cinnamic acid as a matrix (Panel A). The signals were assigned to a-subunit peptides (Table 2). The signal at m/z 1798, indicated by an arrow was next subjected to PSD-analysis. The PSD-spectrum of MH+ 1798.4 is shown in Panel B. Only the peaks for the b and y fragment ions are labeled. The deduced amino acid sequence is shown at the top of the panel.

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