Identifying Sites of Phosphorylation in Proteins
Overview
Two techniques can be used to map the precise sites of phosphorylation on a protein.
The first approach, Method A, requires 10-50 pmol of total protein which following enzymatic digestion, is analyzed by LC-MS/MS. In general this is the preferred method as it requires less sample, avoids the handling of radioactive material and yields data more rapidly.
The alternative technique, Method B, requires 20-50 pmol of protein labeled with [32P]-ATP, chromatographic separation of the digested protein, followed by Edman sequencing to identify the phosphorylated residues.
Method A - Mass Spectrometry (10-50 pmol)
This method uses non-radiolabeled protein that is digested with trypsin or another proteolytic enzyme and then analyzed in precursor ion scan mode (loss of 79 amu) on the QStar or QTrap mass spectrometers (Fig.1).
Following this initial analysis the crude protein digest is then analyzed by MS/MS to obtain sequence information to identify the residue with the phosphate group (Fig. 2). An explanantion of the fragmentation patterns generated in the MS can be found here.
To improve our ability to identify phosphorylation sites, especially low abundance sites, an LC/MS/MS experiment can be performed (Fig. 3). The best data will be obtained when stoichiometry for the phosphorylation event approaches 1.
An estimation of the degree of phosphorylation should be determined before submitting samples. Assuming the stoichiometry is high, the phosphorylated peptide may be identified by MALDI-MS by comparing the MS profile of the phosphorylated sample (+80 amu) with that obtained after treatment with alkaline phosphatase.
Method B - Edman Sequencing of [32P]-labeled protein (20-50 pmol)
Depending on the amount of protein available and the stoichiometry of phosphorylation some advantages are offered by using [32P]-labeled ATP, as the radioisotope provides a useful way to identify and track the modified peptide(s).
As with Method A, this approach begins with an in-gel digest of the Coomassie Blue stained protein. It is recommended that 20-50 pmol protein, containing >100,000 cpm/expected site be submitted for analysis. An estimation of stoichiometry of phosphorylation should also be determined before sample submission.
Standard capillary HPLC/peptide fractionation with [32P]-labeled protein
The sample is digested with trypsin, the tryptic peptides extracted from the gel, then separated by reversed-phase HPLC and collected on a 96-well ELISA plate. Fractions containing radioactivity are identified on a scintillation counter and prepared for additional analysis.
Mass spectrometric identification of [32P]-labeled phosphopeptides
A small aliquot of each [32P]-containing fraction is analyzed by MALDI-MS. The masses obtained will correspond to singly-charged (M+H) ions and since the presence of phosphate adds 80 amu to the mass of the expected tryptic peptide, its identity can be tentatively assigned.
Edman sequencing of [32P]-labeled phosphopeptides
Since the phosphorylated PTH derivatives are too hydrophilic to be extracted from the sequencing support, it is necessary to perform a partial modification of the phosphorylated residue with aminoethanethiol, which is stable to Edman chemistry.
