TABLE 1.
List of peptides found in the combined TAILS and HYTANE data sets and subsequently validated as ASP5 substratesa
| Gene name | Gene ID (TGME49_) |
Sequence preceding (and location) |
Peptide found | Up in WT or Δasp5 |
Expt found |
Ratio of WT/Δasp5 or if HYTANE, no. of samples detected in |
|---|---|---|---|---|---|---|
| Genes found following RRL (validated) |
||||||
| LCAT | 272420 | RRL476-478 | (fi)DAVLTDEVGGPESGAR | WT | SILAC (TAILS) | 5.61/0.14 |
| GRA46 | 208370 | RRL894-896 | (fi)LSSSAILTGQQIGTYR | WT | SILAC (TAILS) | 6.65/0.21 |
| GRA46 | 208370 | RRL894-896 | (n)LSSSAILTGQQIGTYR | WT | LFQ (HYTANE) | 8/9 WT, 0/9 Δasp5 |
| GRA44 (IMC2A) | 228170 | RRL83-85 | (Ac)SGIIKTLVLWDPVQR | WT | LFQ (HYTANE) | 4/9 WT, 0/9 Δasp5 |
| WNG2 (ROP34) | 240090 | RRL109-111 | (n)DSLIPGFLKR | WT | LFQ (HYTANE) | 6/9 WT, 0/9 Δasp5 |
| IST | 240060 | RRL137-139 | _(ac)AAEGGSESEDEQGVAR | No ratiob | Dimethyl (TAILS) |
Unable to determine* |
| Genes found following RRL (not validated) |
||||||
| Hypothetical | 233695 | RRL115-117 | QAGVYFSEEDR | WT | LFQ (HYTANE) | 2/9 WT, 0/9 Δasp5 |
| Hypothetical | 297890 | RRL183-185 | (n)TTLASTLSLSR | WT | LFQ (HYTANE) | 2/9 WT, 0/9 Δasp5 |
| Hypothetical (zinc finger-containing) |
248450 | RRL345-347 | (Ac)YAPGASVVESPVFGTPPSR | WT | LFQ (HYTANE) | 1/9 WT, 0/9 Δasp5 |
| Hypothetical | 286530 | RRL27-29
SP-cleavedc |
(n)MFAAAPLQSFSVTNKQFHPE- GLEAQAPRPHQGLDMR |
Neither | LFQ (HYTANE) | 4/9 WT, 2/9 Δasp5 |
| Genes found following predicted signal peptide cleavage site |
||||||
| WNG2 (ROP34) | 240090 | AVA62-64
(SP cleaved) |
(n)AHAEHPEDSATNFLFSFAENS- LANREPPEDSAARPSSR |
Δasp5 | LFQ (HYTANE) | 0/9 WT, 7/9 Δasp5 |
| WNG2 (ROP34) | 240090 | AVA61-64
(SP cleaved) |
(fi)AHAEHPEDSATNFLFSFAE- NSLANR |
Neither | SILAC (TAILS) |
WTH/KOL 1.49/0.67 (not significant) |
| WNG1 (ROP35) | 304740 | AGA68-70 (SP cleaved?)d |
(Ac)TVAAPQVETGPLLSVR | WT | Dimethyl (TAILS) |
WT light dimethyl 5.31/0.18 |
| WT heavy dimethyl 4.17/0.81 |
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Locations of the peptide and three preceding amino acids were obtained from Toxodb.org v34. (fi), fixed dimethylation from SILAC experiments; (n), fixed dimethylation in HYTANE experiment; (Ac), acetylation occurring within parasites and/or host cell. The HYTANE experiment did not use differential labeling, so we could not directly compare ratios between WT versus Δasp5; therefore, results are displayed as number of samples the peptide was detected in per condition (n = 9) (3 independent biological samples, performed in triplicate, with the HYTANE procedure performed once to reduce variation).
We were unable to determine which samples (WT or Δasp5) the RRL-cleaved peptide [(ac)AAEGGSESEDEQGVAR] originated from, as this was found only in the dimethyl experiment and contains no differential heavy/light dimethylation, as the N terminus is blocked and there are no lysine residues.
Peptide for TGME49_286530 found in both WT and Δasp5 parasites and maps within predicted SP, suggesting that this processing is mediated by signal peptidase.
We have annotated the peptide mapping to WNG1 [(Ac)TVAAPQVETGPLLSVR] as potentially SP cleaved; however, SignalP 4.1 (58) does not recognize a signal peptide within this protein. This annotation is based on the peptide being N acetylated, a modification observed predominantly at the initiator methionine, SP cleavage site, and the ASP5 cleavage site. Spectra for HYTANE peptides can be found in Fig. S5 in the supplemental material.