Detection of tyrosine phosphorylated peptides by precursor ion scanning quadrupole TOF mass spectrometry in positive ion mode

Phosphorylation is a common form of protein modification. To understand its biological role, the site of phosphorylation has to be determined. General ly, only limited amounts of phosphorylated proteins are present in a cell, thus demanding highly sensitive procedures for phosphorylation site determi nation. Here, a novel method is introduced which enables the localization o f tyrosine phosphorylation in gel-separated proteins in the femtomol range, The method utilizes the immonium ion of phosphotyrosine at m/z 216.043 for positive ion mode precursor ion scanning combined with the recently introd uced Q2-pulsing function on quadrupole TOF mass spectrometers. The high res olving power of the quadrupole TOF instrument enables the selective detecti on of phosphotyrosine immonium ions without interference from other peptide fragments of the same nominal mass. Performing precursor ion scans in the positive ion mode facilitates sequencing, because there is a no need for po larity switching or changing pH of the spraying solvent. Similar limits of detection were obtained in this approach when compared to triple-quadrupole mass spectrometers but with significantly better selectivity, owing to the high accuracy of the fragment ion selection. Synthetic phosphopeptides cou ld be detected at 1 fmol/muL, and 100 fmol of a tyrosine phosphorylated pro tein in gel was sufficient for the detection of the phosphorylated peptide in the unseparated digestion mixture and for unambiguous phosphorylation si te determination. The new method can be applied to unknown protein samples, because the identification and localization of the modification is perform ed on the same sample.