Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of phosphopeptides by precursor ion scanning.
An API 3000 triple-quadrupole instrument and a QSTAR Pulsar quadrupole time-of-flight (TOF) mass spectrometer were compared for the determination of phosphopeptides by precursor ion scanning in both the positive and negative nanoelectrospray ionization modes. The limits of detection for synthetic phosphopeptides were similar (500 amol microl(-1)) for both types of instruments when monitoring precursors of -79 Da (PO(3)(-)). However, the quadrupole TOF system was approximately fivefold more sensitive (1 fmol microl(-1)) than the triple-quadrupole instrument (5 fmol microl(-1)) when monitoring precursors of 216 Da (immonium ion of phosphotyrosine). The recently introduced Q(2)-pulsing function, which enhances the transmission of fragment ions of a selected m/z window from the collision cell into the TOF part, improved the sensitivity of precursor ion scans on a quadrupole TOF instrument. The selectivity of precursor ion scans is much better on quadrupole TOF systems than on triple quadrupoles because the high resolving power of the reflectron-TOF mass analyzer permits high-accuracy fragment ion selection at no expense of sensitivity. This minimizes interferences from other peptide fragment ions (a-, b-, and y- type) of the same nominal mass but with sufficient differences in their exact masses. As a result, the characteristic immonium ion of phosphotyrosine at m/z 216.043 can be utilized for the selective detection of tyrosine phosphorylated peptides. Our data suggest that, in addition to their superior performance for peptide sequencing, quadrupole TOF instruments also offer a very viable alternative to triple quadrupoles for precursor ion scanning, thus combining high sensitivity and selectivity for both MS and MS/MS experiments in one instrument.
Steen
H
H
Protein Interaction Laboratory at the Center for Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark/Odense University, Campusvej 55, DK-5230 Odense M, Denmark.
Küster
B
B
Mann
M
M
D000595:Amino Acid Sequence; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods*; D010047:Ovalbumin / Q000032:analysis / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis*; D019000:Phosphotyrosine / Q000032:analysis / Q000737:chemistry; D011786:Quality Control; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry
D007477:Ions; D010748:Phosphopeptides; D019000:Phosphotyrosine; D012710:Serum Albumin, Bovine; D010047:Ovalbumin