Phosphopeptides fractionation

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. 

Figure 7. PAGE of reaction mixture after plasmin hydrolysis of B-casein for 0, 20, 30, 40, and 70 min (Slots 1-5) and Fractions 1 ana 2 from hydroxyapatite chromatography of hydrolysis products after plasmin treatment of fi-casein for 70 min (Slots 6 and 7). Fraction 2 contains a further phosphopeptide that is not visible in Slot 7 but appears on disc gels in the expected position for proteose peptone component 8F (cf. Ref. 32) (28).

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). 

Having isolated the phosphopeptide-containing fractions, it becomes possible to try to locate the site of phosphorylation by tandem mass spectrometry. 

In a next step, blends or mixtures of these gases were employed in the FAIMS method, and results were surprisingly different from the prediction of Blanc s law. In Blanc s law, the mobility of an ion in a binary mixture of the purified gases is a linear combination of fractional composition times the mobility coefficients in individual gases. This deviation was exploited with planar FAIMS separation of biological molecules. Helium-rich gas mixtures with up to 74% He in nitrogen permitted the separation of phosphopeptides with variant modification sites, and the results were superior in resolution possible with pure gases alone. 

Figure 4.4. Precursor-ion scan of miz 79 for selective detection of phosphopeptides. Two tyrosine phosphorylated peptides (1303.2 and 1686.7 Da) are detected in an LC fraction. (Reproduced from ref. 5 by permission of the American Chemical Society, Washington, DC, copyright 1993.)...

As mentioned above, main concerns in the mass spectrometry analysis of phosphopeptides in a complex protein digest are their low amounts and signal suppression in the presence of unphosphorylated peptides. To alleviate these concerns, the isolation and enrichment of phosphopeptides from the bulk of unphosphorylated peptides are highly desirable. A convenient means to fractionate phosphopeptides is RP-HPLC. Offline and online procedures with mass spectrometry have... 

Once the identity of phosphopeptides in a digestion mixture is revealed, it is imperative to recognize which amino acid residue (Ser, Thr, or Tyr) is phospho-rylated. This task is accomplished by determining the amino acid sequence of peptide fragments in the protein digest after their fractionation into individual components by RP-HPLC, either off- or online with tandem mass spectrometry [34], The tandem MS methods described below have proved to be practical for this purpose. 

Acidic peptides are released in the pH range 5.5-6.2 and phoshorylated peptides are eluted in the pH range 6.9-7.5. Elution of retained peptides can also be performed with sodium phosphate. IMAC has been successfully used for the characterization of casein phosphopeptides in cheese extracts. Phosphoproteins can be separated under very similar conditions as phosphopeptides. IMA sorbents were already used for fractionation of proteins... 

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