Table I. Histone deacetylase 5 phosphorylation site mapping by complementary mass spectrometry analysis. The presence (+) or absence (−) of the phosphopeptide identification is indicated for each instrument configuration. n.a. indicates Lys-N-generated peptides that were analyzed only by the LTQ-Orbitrap Velos configuration.
| pSTa | Phosphopeptide sequenceb | pScc | Enzd | Xce | z | MALDI LTQ Orbi XLf NE IM | LC-ESI LTQ Orbi XL CID | LC-ESI LTQ Orbi Velos HCD ETD | ||
|---|---|---|---|---|---|---|---|---|---|---|
| S3 | (-)MNS3PNESDGMSGREPSLEILPR(T) | 103 | T | 4.66 | 3 | − | + | + | + | + |
| S3/S7 | (-)MNS3PNES7DGMSGREPSLEILPR(T) | 55 | T | 3.85 | 3 | − | − | − | − | + |
| S53 | (R)AMoxPSSMoxGGGGGGS53PSPVELR(G) | 61 | T | 3.41 | 2 | − | − | + | + | + |
| S55 | (R)AMoxPSSMoxGGGGGGSPS55PVELR(G) | 38 | T | 2.85 | 2 | − | − | + | + | + |
| S66 | (R)GALVGS66VDPTLR(E) | 49 | T | 3.63 | 2 | − | − | + | − | − |
| S206 | (K)SKEPTPGGLNHS206LPQHPK(C) | 32 | T | 2.64 | 3 | − | − | + | − | − |
| T234 | (P)KCamWGAHHASLDQSSPPQSGPPGT234PPSY(K) | 58 | L | 5.18 | 4 | − | n.a. | n.a. | + | + |
| S259 | (R)KTAS259EPNLK(V) | 90 | T | 2.44 | 2 | + | + | + | + | − |
| S259 | (R)KTAS259EPNL(K) | 53 | L | 2.27 | 2 | + | n.a. | n.a. | + | − |
| S278 | (Q)KVAERRS278SPLLRR(K) | 26 | L | 4.06 | 4 | + | n.a. | n.a. | − | + |
| S278/S279 | (Q)KVAERRS278S279PLLRR(K) | n.c. | L | 4.61 | 4 | − | n.a. | n.a. | − | + |
| S278/S279 | (K)VAERR S278S279PLLR(R) | n.c. | T | 3.94 | 3 | + | + | − | + | − |
| S368 | (R)ALPLDSSPNQFSLYTSPS368LPNISLGLQATVTVTNSHLTASPK(L) | 23 | T | 5.47 | 3 | − | − | + | − | − |
| S498 | (R)TQS498SPLPQSPQALQQLVMQQQHQQFLEK(Q) | 24 | T | 5.69 | 4 | + | + | + | + | + |
| S611 | (K)DEEGES611GAEEGPDLEEPGAGYK(K) | 154 | T | 7.40 | 3 | + | + | + | + | + |
| S611 | (V)KDEEGES611GAEEGPDLEEPGAGY(K) | 155 | L | 6.95 | 3 | + | n.a. | n.a. | + | + |
| S661 | (R)TQS661SPAAPGGMK(S) | 58 | T | 2.78 | 2 | − | + | + | + | − |
| S671 | (R)TQSSPAAPGGMKS671PPDQPVK(H) | 193 | T | 4.52 | 3 | − | + | + | + | + |
| S661/671 | (R)TQS661SPAAPGGMKS671PPDQPVK(H) | 28 | T | 3.89 | 3 | + | + | + | + | + |
| S755 | (R)KATLDEIQTVHSEYHTLLYGTS755PLNRQ(K) | 29 | L | 7.10 | 4 | − | n.a. | n.a. | + | + |
| S755 | (R)KATLDEIQTVHSEYHTLLYGTS755PLNR(Q) | 32 | T | 5.54 | 4 | + | + | + | − | + |
| S1108 | (M)ALLSVGAEQAQAAAAREHS1108PRPAEEPmEQEPAL(-) | 40 | L | 4.47 | 4 | − | n.a. | n.a. | − | + |
| Amb | (H)HASLDQSS225PPQSGPPGTPPSYK(L) | Amb | T | 4.39 | 3 | − | − | − | − | + |
| Amb | (P)KCamWGAHHASLDQSS225PPQSGPPGT234PPSY(K) | Amb | L | 4.64 | 3 | − | n.a. | n.a. | + | + |
a Phosphorylated residue in human HDAC5.
b Phosphopeptide sequences; methionine oxidation (Mox) and cysteine carbamidomethylation (Cam) are specified.
c Phosphosite confidence score were calculated by SLoMo (Bailey, JPR 2009). Scores ≥19 were considered confidently localized, corresponding to an estimated error rate of ≤ 1.3%, while phosphopeptide spectra below this threshold are indicated as “Amb”. n.c. indicates no site localization was required.
d Enzyme, trypsin (T) or Lys-N (L), which generated the phosphopeptide.
e SEQUEST cross-correlation score (Xc) and charge state (z).
f MALDI LTQ-Orbitrap XL analysis was conducted with (IM) or without (NE) IMAC.