SELECTIVE DETECTION OF PHOSPHOPEPTIDES IN COMPLEX-MIXTURES BY ELECTROSPRAY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY

A mass spectrometry-based method that does not involve the use of radi olabeling was developed for selective detection of phosphopeptides in complex mixtures. Mixtures of phosphorylated and nonphosphorylated pep tides at the low picomole level are analyzed by negative ion electrosp ray liquid chromatography/mass spectrometry using C-18 packed fused-si lica columns (less-than-or-equal-to 320-mum i.d.). Peptides and phosph opeptides in the chromatographic eluant undergo collision-induced diss ociation in the free-jet expansion region prior to the mass analyzing quadrupole. Using relatively high collisional excitation potentials, p hosphopeptides containing phosphoserine, phosphothreonine, and phospho tyrosine fragment to yield diagnostic ions at m/z 63 and 79 correspond ing to PO2- and PO3-, respectively. Chromatographic peaks containing p hosphopeptides are indicated where these diagnostic ions maximize. The highest sensitivity for phosphopeptide detection is obtained using se lected-ion monitoring for m/z 63 and 79. Full-scan mass spectra that e xhibit the diagnostic phosphopeptide fragment ions, together with pseu domolecular ions, may be obtained by stepping the collisional excitati on potential from a high value during the portion of each scan in whic h the low-mass-to-charge ratio diagnostic marker ions are being detect ed to a lower value while the upper mass-to-charge ratio range is bein g scanned. Good sensitivity for phosphopeptide detection was achieved using standard trifluoroacetic acid containing mobile phases for rever sed-phase high-performance liquid chromatography. Data illustrating th e selectivity and sensitivity of the approach are presented for mixtur es of peptides and phosphopeptides containing the three commonly phosp horylated amino acids.