Analysis of protein phosphorylation by capillary liquid chromatography coupled to element mass spectrometry with 31P detection and to electrospray mass spectrometry

A method for phosphopeptide identification by capillary liquid chromatography (mu LC) interfaced alternatively to element mass spectrometry (inductively coupled plasma mass spectrometry, ICPMS) and to electrospray ionization mass spectrometry (ESI-MS) is described, ICPMS is used for P-31 detection and ESI-MS provides the corresponding molecular weight-information. Alignment of the two separate mu LC runs is performed using the baseline distortion at the elution front, which shows up in both mu LC-ICPMS and mu LC-ESI-MS, Both a quadrupole and a magnetic sector field mass analyzer were used in combination with ICP, The detection limit achieved for the mu LC-ICP-HRMS runs is similar to0.1 pmol of phosphopeptide injected. Without any further precautions, contamination by phosphate-containing compounds at this level was found to be uncritical. The method is demonstrated for the analysis of a complex mixture of synthetic phosphopeptides and a set of tryptic digests of three phosphoproteins, These include beta -casein, activated human MAP kinase ERK1, and protein kinase A catalytic subunit, The tryptic:phosphopeptides of these proteins could all be detected and identified by our new strategy. Analysis of three fractions of protein kinase A catalytic subunit with different phosphorylation status gives direct access to the order in which the phosphorylation of the four phosphorylation sites occurs, The two most important aspects of using mu LC-ICPMS with P-31 detection for phosphopeptide identification are (i) that a high selectivity is achieved and (ii) that the signal intensity is independent of the chemical form of phosphorus and directly proportional to the molar amount of P-31 in the mu LC eluate. Thus, mu LC-ICPMS with P-31 detection is introduced as a new, robust, and specific method in phosphoproteomics.