TABLE 1.
Top five identifications of a protein biomarker of C. upsaliensis strain RM3195 at m/z 11138.9 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 | 1262 | >Q4HNM5 Q4HNM5_CAMUP | Campylobacter upsaliensis RM3195 | Thioredoxin PTM N-Met (11,136.76) | 82.69 | 7.7E−19 |
| 323 | >B1WRD3 B1WRD3_CYAA5 | Cyanothece sp. strain ATCC 51142 | Carbon dioxide-concentrating mechanism protein (11,133.85) | 53.85 | 3.6E−6 | |
| 71 | >A4T659 A4T659_MYCGI | Mycobacterium gilvum strain PYR-GCK (Mycobacterium flavescens strain ATCC 700033 [=PYR-GCK]) | Putative uncharacterized protein (11,142.24) | 46.15 | 3.1E−4 | |
| 697 | >Q1LMQ8 Q1LMQ8_RALME | Ralstonia metallidurans strain CH34 (=ATCC 43123 = DSM 2839) | Putative uncharacterized protein PTM 23SigPep (11,128.81) | 44.23 | 1.1E−3 | |
| 1154 | >Q18XI2 Q18XI2_DESHD | Desulfitobacterium hafniense strain DCB-2 | Small multidrug resistance protein PTM N-Met (11,138.67) | 44.23 | 1.6E−3 | |
| D-, E-, and P-specific in silico fragment ions | 1262 | >Q4HNM5 Q4HNM5_CAMUP | Campylobacter upsaliensis strain RM3195 | Thioredoxin PTM-N-Met (11,136.76) | 61.54 | 3.0E−21 |
| 143 | >A7A905 A7A905_BIFAD | Bifidobacterium adolescentis L2-32 | Putative uncharacterized protein (11,134.94) | 25.00 | ||
| 942 | >A8LX75 A8LX75_SALAI | Salinira arenicola strain CNS-205 | Putative uncharacterized protein PTM N-Met (11,140.56) | 9.0E−5 | ||
| 136 | >A6QD71 A6QD71_STAAE | Staphylococcus aureus strain Newman | Putative uncharacterized protein (11,138.98) | 23.08 | ||
| 143 | >A7A905 A7A905_BIFAD | Bifidobacterium adolescentis L2-32 | Putative uncharacterized protein (11,134.94) | 9.4E−5 | ||
| 145 | A7BQP4 A7BQP4_9GAMM | Beggiatoa sp. strain PS | Putative uncharacterized protein (11,132.06) | 23.08 | ||
| 136 | >A6QD71 A6QD71_STAAE | Staphylococcus aureus strain Newman | Putative uncharacterized protein (11,138.98) | 1.5E−4 | ||
| 271 | >B1BI09 B1BI09_CLOPE | Clostridium perfringens C strain JGS1495 | Putative uncharacterized protein (11,140.05) | 23.08 | ||
| 449 | >Q2G1R1 Q2G1R1_STAA8 | Staphylococcus aureus strain NCTC 8325 | Putative uncharacterized protein (11,138.98) | 1.5E−4 |
The protein marker at m/z 11138.9 (Fig. 2) was analyzed by MS-MS using MALDI-TOF-TOF MS (Fig. 3; see Fig. S1 in the supplemental material) 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 1,409 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 relatively enhanced compared to the “runner-up” identification scores. The parameters for comparison of MS-MS data and in silico data were as follows: intensity threshold, 2%; number of MS-MS peaks with an intensity of ≥2%, 52; m/z range for comparison, 0 to 14,000 Th; fragment ion tolerance, 2.5 Th; and protein MW, 11,138 ± 10. “PTM N-Met” indicates that the in silico protein sequence was modified to remove the N-terminal methionine.“PTM 23SigPep” indicates that the in silico protein sequence was modified to remove a signal peptide. 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, 35.7 and 18.4 s, respectively; and P value calculation, 48.8 and 39.7 s, respectively.