Mj. Huddleston et al., SELECTIVE DETECTION OF PHOSPHOPEPTIDES IN COMPLEX-MIXTURES BY ELECTROSPRAY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY, Journal of the American Society for Mass Spectrometry, 4(9), 1993, pp. 710-717
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.