Fractionation of Peptide Fragments in the Digest

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 

The nonspecific binding of acidic amino acid residues with the IMAC packing is a major concern of the IMAC approach. In addition, IMAC exhibits a preferential binding to multiply phosphorylated peptides and recovery of phos-phopeptide is variable. To prevent nonspecific binding of the COOH group. 

The second chemical-tagging approach, which in addition is also applicable to phosphotyrosine-containing peptides, uses a more complex chemistry to tag phosphopeptides [33], It involves the attachment of l-amine-2-thioethane to the phosphate group via a carbodiimide condensation reaction. The modified phosphopeptides are captured by an iodoacetyl resin and subsequently released with an acidic solution. 

Use of Antibodies Antibodies are useful tools to isolate relevant proteins from a complex mixture by immunoprecipitation, provided that specific antibodies are available for each protein of interest. Although antibodies have been raised against all three 0-phosphoryIated residues, successful use of this technique has been demonstrated primarily for phosphotyrosine-containing peptides and proteins [35-37]. 

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