Table 2. LC-MS2 Identification of 2-DE-Separated F. hepatica CL Zymogen Sub-Proteomesa.
| MS/MS
ion search |
highest-scoring GenBank hit |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Sequence coverage | ||||||||||
| ID | GenBank hit | MASCOT score (Av) | peptides matched (non-duplicate) | average percentage coverage (%) | collective residue coverage (aa) | exponentially modified protein abundance index (emPAI) | protein (length, aa) | organism | accession | E-value |
| live | gi|116488416a | 116.5 ± 58.5 | 4.0 ± 0.0 | 19.5 ± 1.5 | 57–81, 91–106, 116–124, 186–205, 231–253, 289–298, 308–324 | 0.365 ± 0.155 | secreted cathepsin L1 (1–326) | F. hepatica | AAB41670.2 | 0.0 |
| gi|157862759b | 101.0 ± 54.0 | 2.0 ± 0.0 | 11.5 ± 0.5 | 12–35, 70–78, 140–152, 185–207 | 0.280 ± 0.070 | cathepsin L, partial (1–280) | F. gigantica | ABV90502.1 | 0.0 | |
| gi|211909240b | 67.5 ± 20.5 | 3.0 ± 0.0 | 14.5 ± 1.5 | 58–81, 116–124, 186–198, 231–253, 289–298, 308–324 | 0.200 ± 0.010 | cathepsin L1D (1–326) | F. hepatica | ACJ12893.1 | 0.0 | |
| dead | gi|31558997 | 458.0 ± 260.0 | 10.5 ± 0.5 | 52.5 ± 6.5 | 42–81, 84–147, 186–205, 215–298 | 2.360 ± 1.830 | cathepsin L (1–326) | F. hepatica | AAP49831.1 | 0.0 9E–180 |
| gi|41152540 | 384.5 ± 286.5 | 8.0 ± 1.0 | 52.5 ± 6.5 | 4–60, 99–118, 128–166, 202–211, 221–237 | 3.930 ± 3.510 | cathepsin L protein (1–239) | F. hepatica | AAR99519.1 | 0.0 | |
| gi|148575301 | 237.0 ± 152.0 | 10.5 ± 0.5 | 51.0 ± 2.0 | 50–81, 84–97, 106–115, 151–209, 215–302, 308–324 | 1.020 ± 0.840 | secreted cathepsin L2 (1–326) | F. hepatica | ABQ95351.1 | 0.0 | |
| gi|190350155 | 153.5 ± 62.5 | 10.5 ± 0.5 | 33.0 ± 7.0 | 0.335 ± 0.025 | enolase | F. hepatica | CAK47550.1 | 0.0 | ||
| gi|684403575 | 135.5 ± 37.5 | 14.0 ± 1.0 | 51.5 ± 7.5 | 0.440 ± 0.020 | hypothetical protein T265_09499 | Opisthorchis viverrini | XP 009173845.1 | |||
| gi|684403578 | 135.5 ± 37.5 | 14.0 ± 1.0 | 46.0 ± 7.0 | 0.440 ± 0.020 | hypothetical protein T265_09500 | O. viverrini | XP 009173846.1 | 0.0 | ||
| gi|684415044 | 135.5 ± 37.5 | 7.5 ± 0.5 | 54.0 ± 11.0 | 0.440 ± 0.020 | hypothetical protein T265_09500 | O. viverrini | XP 009178086.1 | 3E-128 | ||
| gi|8547325 | 126.0 ± 70 | 10.5 ± 0.5 | 33.5 ± 0.5 | 42–81,84–102, 116–124, 151–165, 186–198, 206–214, 254–266, 289–302 | 0.475 ± 0.295 | Cathepsin L (1–326) | F. hepatica | AAF76330.1 | 0.0 | |
CL zymogens in 2-DE-separated whole ES from untreated live and ethyl 4-aminobenzoate-terminated dead adult flukes (Figure 4A,Bi) were investigated by LC-MS. Protein hits are shown following identification against the GenBank database (v204) using an in-house Ma(Matrix Science) server, with consistent hits reported with average scores between duplicate sample submissions by two LC-MS2 methods (Agilent 6550 iFunnel Q-TOF (a) and Orbitrap Fusion Tribrid mass spectrometer (b)). Significant hits identified with an average score of 67 or greater (P < 0.05) are shown, including reliable error tolerance, reporting significant hits consistent between duplicate sample submissions, average abundance indices per hit (exponentially modified protein abundance index, emPAI), and showing protein family groupings in bold. Superscripts refer to consistent proteins identified as top hits from analyses by each LC-MS2 method.