Table 1

Summary of ESMS analysis of phosphopeptides from in vivo– and in vitro–phosphorylated Sic1p (17).

SitesHPLC fractionMolecular size measured/ calculatedPeptide*Number of PO3 groups (mol%)
In vitro
T519–201909.8/? (?) … efmaps T ppr(8)1
T22–S2516–172048.8/2048.3§ (14)ylaqpsgn TSSS almqgqk(32)1–2|
T33, 45, 4816–172163.2/2163.5 (33)T pqkpsqnlvpv T ps T tk(50)3#
S69, 76, 8024 3509.5/3510.1(54) napllappnsnmgmtspfngl tspqrspfpk(84)3–4
T17316–171349.6/1349.6(166)iikdvpg T psdk(177)1
S19112–131053.2/1053.4(186)nwnnn S pk (193)1
In vivo
T524, 251048.0/1047.6§ (2)aps T ppr (8)** 0 (ND)
1 (100%)
T33†† 53–554032.0/4031.1§ (14)ylaqpsgntsssalmqgqk T pqk 0 (58%)
kpsqnlvpvtpsttk(50)1 (28%)
2 (14%)
S76‡‡ 69–733348.0/3347.6§ (54)napllappnsnmgmtspfngl 0 (67.7%)
[TS]pqrspfpk(84)1 (23%)
2 (4.5%)
3 (4.8%)
  • * Numbers in parentheses refer to the first and last amino acid of each peptide. Amino acids indicated in large boldface type were deduced to be phosphorylated on the basis of ESMS-based sequencing.

  • Number of PO3 groups detected per peptide; mol% indicates the fraction of a given peptide that was detected in a particular phosphorylation state with positive-ion ESMS. N.D.: not detected.

  • Calculated mass is unknown because we do not know the exact amino acid sequence across the MBP-Sic1p junction. Nevertheless, phosphorylation of T5 was directly confirmed by sequencing residues from the COOH-terminus of the peptide.

  • § Masses given are those of the singly phosphorylated peptide.

  • | Two phosphate residues are distributed over T21-S25, with no single site phosphorylated at high stoichiometry.

  • Masses given are those of the triply phosphorylated peptide.

  • # T21-S25, T33, T45, and T48 were also recovered on peptide 14–50, which was detected in triply (10.5%), quadruply (26.3%), and quintuply (63.2%) phosphorylated states.

  • ** The initiator methionine was not present.

  • †† T33 was also recovered on peptides 11–50, 11–53, and 14–53. In each case, the peptides were recovered with 0, 1, and 2 mol of PO3 in ratios similar to those reported for the 14–50 peptide.

  • ‡‡ ESMS-based sequencing was unable to distinguish whether phosphate resided on T75 or S76.