Direct identification of tyrosine sulfation by using ultraviolet photodissociation mass spectrometry

Abstract

Sulfation is a common post-translational modification of tyrosine residues in eukaryotes; however, detection using traditional liquid chromatography-mass spectrometry (LC-MS) methods is challenging based on poor ionization efficiency in the positive ion mode and facile neutral loss upon collisional activation. In the present study, 193 nm ultraviolet photodissociation (UVPD) is applied to sulfopeptide anions to generate diagnostic sequence ions, which do not undergo appreciable neutral loss of sulfate even using higher energy photoirradiation parameters. At the same time, neutral loss of SO₃ is observed from the precursor and charge-reduced precursor ions, a spectral feature that is useful for differentiating tyrosine sulfation from the nominally isobaric tyrosine phosphorylation. LC-MS detection limits for UVPD analysis in the negative mode were determined to be around 100 fmol for three sulfated peptides, caerulein, cionin, and leu-enkephalin. The LC-UVPD-MS method was applied for analysis of bovine fibrinogen, and its key sulfated peptide was confidently identified.

Figure 1

MS/MS spectra for 2- charged cholecystokinin (RDsYTGWNleDF-NH₂), m/z 624.24, using (A) CID at normalized collision energy (NCE) 35% and (B) UVPD using 3 pulses at 2 mJ. “-W” denotes sidechain loss from tryptophan (C₉H₇N, 129 Da). Products ion that have lost SO₃ are annotated in red font. UVPD data for the other model sulfopeptides is summarized in part (C) with sequence coverage listed for the most abundant charge state of each peptide. Product ions from which SO₃ loss is observed are also listed.

Figure 2

UVPD mass spectra of singly deprotonated peptides: (A) sulfated leu-enkephalin and (B) phosphorylated P60c-src substrate II Ac-IpYGEF-NH₂ using 3 laser pulses at 2 mJ. (C) Average percentage of the neutral loss product observed from the selected precursor and charge reduced precursor ions. Cionin is excluded from the sulfated peptide averages.

Figure 3

Impact of laser parameters: Optimization of abundances of a/x sequence ions and minimization of loss of sulfate. For each peptide, the abundances of three singly charged Y-containing a/x product ions were monitored in both their sulfated and non-sulfated forms. Specifically these were a4, a7, a8 from caerulein; a3, a6, a7 from cholecystokinin; x8, x9, a10 from hirudin; and a2, a3, a4 from leu-enkephalin. A) The summed abundances of sulfated product ions (normalized to 100%) is charted as a function of both laser energy (1–5 mJ) and number of pulses (1–6). B) Percent sulfate retention is determined for each set of laser conditions based on the total abundance of the sulfated products ions divided by the total abundance of both sulfated and non-sulfated (neutral loss) product ions. Heat maps for 1- and 2- precursor ions (top and middle, respectively) contain data from all four peptides, whereas heat maps for 3- precursor ions (bottom) contain data only from hirudin and caerulein. The deepest red shade indicates the most optimum outcome.

Figure 4

MS² LOD determined for leu-enkephalin (1-), caerulein (2-), and cionin (2-) using three 2 mJ pulses for photodissociation. A) Extracted ion chromatograms (XIC) representing the summed abundance of three product ions from each peptide were generated for each dilution and overlaid. B) The area under the curve was plotted as a function of femtomoles of peptide injected to generate a calibration curve for each peptide. C) Precursor ion peak area (MS1) was divided by product ion abundance (MS2) and plotted against the number of femtomoles injected. Above the MS2 LOD, the MS1:MS2 ratio should remain consistent as represented by the flat region of the graph.

Figure 5

A) Base peak chromatogram of tryptic bovine fibrinogen (red) with extracted ion chromatogram for sulfopeptide GlpFPTDsYDEGQDDRPK (2-) from the fibrinogen beta chain (green) overlaid. B) Negative UVPD mass spectrum (three pulses at 2 mJ) of GlpFPTDsYDEGQDDRPK (2-) from the average of 18 MS/MS scans acquired over 12 seconds. SO₃ neutral loss products are annotated in red.