Communicated by Paul Talalay, Johns Hopkins University School of Medicine, Baltimore, MD, December 25, 2006 (received for review November 1, 2006)
A schematic depicting the strategy for analysis of phosphopeptides carried out in this study. All fractions were analyzed by LC-MS/MS. A total of 30 Lys-C digested fractions were analyzed by LC-MS/MS (ETD); 12 of the selected Lys-C fractions (1/3 of each sample was used) were subjected to a replicate analysis, with one part of a sample analyzed by ETD and the other by CID; the remaining 2/3 of the 12 protein fractions were digested by using trypsin or Glu-C, respectively, and analyzed in replicate by LC-MS/MS (CID) and LC-MS/MS (ETD). A fraction (7%) of the digested samples before enrichment of phosphopeptides was also analyzed by CID.
MS/MS spectra of four phosphorylated Lys-C peptides identified by ETD. Phosphopeptides with charge states of + 5 (A), +4 (B), +3 (C) and +2 (D) are shown. The four peptides originate from splicing factor, arginine/serine-rich 2 interacting protein, splicing coactivator subunit SRm300, Bcl2-associated transcription factor 1 and DnaJ homolog, subfamily C, member 9, respectively. The peptide sequence with the fragmentation pattern is shown in each panel. The signs: “/”, “ /”, and “|” designate that the C-terminal type fragments, N-terminal fragments, or both types of fragments, respectively, were identified. For all spectra, except A, the intensity axis has been enlarged by a factor of ≈5. Fragment ions resulting from charge stripping of the precursor ion are assigned with charge states (in bold). Small letters indicate phosphorylated residues.
Phosphopeptide identifications from ETD and CID experiments. (A) The overlap among phosphopeptides identified from Lys-C digested samples. (B) The corresponding data for nonphosphorylated peptides from the same samples.
Direct comparison of phosphopeptides subjected to alternating CID and ETD experiments. The CID and ETD experiments identified the exact same sites for the two phosphopeptides show in A and B (from tumor protein D52-like 2 isoform E and tripartite motif-containing 28 protein, respectively) whereas C and D show phosphopeptides with identical amino acid sequence but different assignments of the phosphorylated residues (PDZ and LIM domain 5 isoform a and tumor protein D52 isoform 1, respectively). The peptide sequence with the fragmentation pattern is shown in each panel. The signs: “/”, “ /”, and “|” designate that the C-terminal type fragments, N-terminal fragments, or both type of fragments, respectively, were identified. All intensity axes have been enlarged ≈5 times.
A summary of phosphopeptide analysis by CID and ETD. Identical samples digested with three proteases were analyzed by CID and ETD. The number of phosphopeptide ions identified in each of the sample sets digested with trypsin, Glu-C, and Lys-C is shown.
| Novel motif | Occurrence in phosphopeptides | |
|---|---|---|
| 1 | pS[E/D]X[E/D][E/D] | 55 |
| 2 | pSPXXXP | 31 |
| 3 | pSPXXXT | 27 |
| 4 | DXXXp[S/T]P | 14 |
| 5 | GGpS | 13 |
| 6 | p[S/T]PPP | 12 |
| 7 | QXp[S/T]P | 12 |
| 8 | PSp[S/T]P | 11 |
| 9 | PPXp[S/T]P | 9 |
| 10 | PPp[S/T]P | 9 |
| 11 | EXSXp[S/T]P | 9 |
| 12 | PXpSPX[R/K] | 8 |
| 13 | PpSXL | 7 |
| 14 | PLp[S/T]P | 6 |
| 15 | TpTP | 5 |
| Category | No. of examples |
|---|---|
| No. of unique phosphorylation sites | 1,435 |
| Novel phosphorylation sites identified | 1,141 |
| No. of unique phosphorylated proteins | 500 |
| Phosphopeptides with 1, 2, 3, or 4 phosphorylation sites | 868; 374; 93; 24 |
| Phosphorylated (serine:threonine:tyrosine) residues | 1,096:266:73 |
| Phosphopeptides identified by both CID and ETD | 129 |
| Protein with the most phosphorylation sites identified: SRm300 (serine/arginine repetitive matrix 2) | 119 total (71 novel, 48 known) |
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Molina et al. 10.1073/pnas.0611217104. |
Fig. 6. Reproducibility of LC-MS/MS runs of Lys-C (A-D), Glu-C (E), and trypsin (F) digested samples subjected to CID (red) and ETD (blue) analysis. Representative base peak chromatograms are depicted to show the reproducibility of the replicate analyses. The base peak chromatograms obtained are from four different samples (2, 5, 9, and 11).
