Protein tyrosine-O-sulfation analysis by exhaustive product ion scanning with minimum collision offset in a NanoESI Q-TOF tandem mass spectrometer.
Tyrosine-O-sulfated peptides were studied by nanoESI Q-TOF mass spectrometry and were found to exhibit an abundant loss of SO3 in positive ion mode under the usually nonfragmenting conditions of survey spectrum acquisition. A new strategy for the detection of tyrosine-O-sulfated peptides in total protein digests was designed based on exhaustive product ion scanning at the collision offset conditions typical for the recording of survey spectra (minimum collision offset). From these data, Q-TOF neutral loss scans for loss of 80/z and Q-TOF precursor ions scans were extracted. The specificity of this approach for analysis of tyrosine-O-sulfation was tested using a tryptic digest of bovine serum albumin spiked with sulfated hirudin (1:1 and 1000:1 molar ratio of BSA to sulfated hirudin, respectively) and using an in-solution digest of the recombinant extracellular domain of thyroid stimulating hormone receptor (ECD-TSHr). For both examples, the combination of in silico neutral loss scans for 80/z and subsequent in silico precursor ion scans resulted in a specific identification of sulfated peptides. In the analysis of recombinant ECD-TSHr, a doubly sulfated peptide could be identified in this way. Surprisingly, approximately 1/4 of the product ion spectra acquired from the tryptic digest of ECD-TSHr at minimum collision offset exhibited sequence-specific ions suitable for peptide identification. Complementary ion pairs were frequently observed, which either were b2/y(max-2) pairs or were induced by cleavage N-terminal to proline. MS/MS analysis at minimum collision offset followed by extraction of neutral loss and precursor ion scans is ideally suited for highly sensitive detection of analyte ions which exhibit facile gas-phase decomposition reactions.
Salek
Mogjiborahman
M
Central Spectroscopy Unit, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Costagliola
Sabine
S
Lehmann
Wolf D
WD
D000818:Animals; D002417:Cattle; D036103:Nanotechnology / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011989:Receptors, Thyrotropin / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D010446:Peptide Fragments; D010455:Peptides; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; C005662:tyrosine O-sulfate; D014443:Tyrosine; D014357:Trypsin