TABLE 3.
Top five identifications of a protein biomarker of C. lari strain RM2100 at m/z 11253.3 based on all in silico fragment ions and on D-, E-, and P-specific in silico fragment ionsa
| Fragment ions | In silico identification | Identifier | Sample name | Protein (MW) | USDA score | P value |
|---|---|---|---|---|---|---|
| All in silico fragment ions | 9623 | >Q4HJN3 Q4HJN3_CAMLA | Campylobacter lari RM2100 | Thioredoxin PTM N-Met (11,246.88) | 53.62 | 6.4E−9 |
| 9224 | >A7H3X2 A7H3X2_CAMJD | Campylobacter jejuni subsp. doylei strain ATCC BAA-1458 (=RM4099 = 269.97) | Putative uncharacterized protein PTM N-Met (11,252.08) | 44.93 | 3.0E−6 | |
| 8933 | >Q8XUD3 Q8XUD3_RALSO | Ralstonia solanacearum | Putative uncharacterized protein (11,251.85) | 39.13 | ||
| 9223 | >A7H3K3 A7H3K3_CAMJD | Campylobacter jejuni subsp. doylei strain ATCC BAA-1458 (=RM4099 = 269.97) | Transcriptional regulator, Cro/CI family (11,249.82) | 2.1E−4 | ||
| 9223 | >A7H3K3 A7H3K3_CAMJD | Campylobacter jejuni subsp. doylei strain ATCC BAA-1458 (=RM4099 = 269.97) | Transcriptional regulator, Cro/CI family (11,249.82) | 39.13 | ||
| 9233 | >A7JU22 A7JU22_PASHA | Mannheimia hemolytica PHL213 | Putative uncharacterized protein PTM N-Met (11,251.98) | 4.3E−4 | ||
| 9233 | >A7JU22 A7JU22_PASHA | Mannheimia hemolytica PHL213 | Putative uncharacterized protein PTM N-Met (11,251.98) | 39.13 | ||
| 8933 | >Q8XUD3 Q8XUD3_RALSO | Ralstonia solanacearum | Putative uncharacterized protein (11,251.85) | 7.4E−4 | ||
| D-, E-, and P-specific in silico fragment ions | 9623 | >Q4HJN3 Q4HJN3_CAMLA | Campylobacter lari RM2100 | Thioredoxin PTM N-Met (11,246.88) | 37.68 | 2.2E−11 |
| 9224 | >A7H3X2 A7H3X2_CAMJD | Campylobacter jejuni subsp. doylei strain ATCC BAA-1458 (=RM4099 = 269.97) | Putative uncharacterized protein PTM N-Met (11,252.08) | 23.19 | 6.6E−5 | |
| 8398 | >A6E0L1 A6E0L1_9RHOB | Roseovarius sp. strain TM1035 | Putative uncharacterized protein (11,253.5) | 21.74 | ||
| 9294 | >A9HBE8 A9HBE8_N-METNO | Methylobacterium nodulans ORS 2060 | Thioredoxin PTM N-Met (11,253.06) | 1.2E−4 | ||
| 9294 | >A9HBE8 A9HBE8_N-METNO | Methylobacterium nodulans ORS 2060 | Thioredoxin PTM N-Met (11,253.06) | 21.74 | ||
| 9223 | >A7H3K3 A7H3K3_CAMJD | Campylobacter jejuni subsp. doylei strain ATCC BAA-1458 (=RM4099 = 269.97) | Transcription regulator, Cro/CI family, PTM N-Met (11,249.82) | 1.7E−4 | ||
| 9540 | >Q2CJW4 Q2CJW4_9RHO | Oceanicola granulosus HTCC2516 | Thioredoxin PTM N-Met (11,252.78) | 21.74 | ||
| 8819 | >Q4ZTY4 Q4ZTY4_PSEU2 | Pseudomonas syringae pv. syringae strain B728a | Putative uncharacterized protein (11,245.79) | 2.9E−4 |
A protein marker at m/z 11253.3 was analyzed by MS-MS using MALDI-TOF-TOF MS and compared to all in silico fragment ions and D-, E-, and P-specific in silico fragment ions of bacterial protein sequences having the same molecular mass as the biomarker (within 5 Da). This corresponded to 1,548 in silico bacterial protein sequences. The protein biomarker had been identified previously by bottom-up proteomics techniques as thioredoxin (12). The top identification of both algorithms was the correct identification of the protein and its source microorganism. In addition, the top identification scores of the two algorithms are enhanced compared with the “runner-up” identification scores. The parameters for comparison of MS-MS data and in silico data were as follows: intensity threshold, 4%; number of MS-MS peaks with an intensity of ≥4%, 69; m/z range for comparison, 0 to 10,000 Th; fragment ion tolerance, 2.5 Th; and protein MW, 11,252 ± 10. “PTM N-Met” indicates that the in silico protein sequence was modified to remove the N-terminal methionine. The algorithm computation times for all in silico fragment ions and for the D-, E-, and P-specific in silico fragment ions were as follows: USDA peak-matching algorithm, 44.7 and 22.0 s, respectively; and P value algorithm, 73.6 and 75.3 s, respectively.