Table 1.
Identities for selected polypeptide spots from 2-D gels for M. jannaschii cell extracts
| Spot number* (mass, kDa)* (digesting enzyme) | Polypeptide identified (Calculated mass, Da/pI), Accession number | MALDI mass† (MH+, average), Da | The peptide sequence consistent with observed mass‡ (start-end position in the parent polypeptide) {calculated average size, Da} | Δ, Da (observed mass −calculated mass) | Coverage§ [pro-peptide region excluded from calculations]¶ |
|---|---|---|---|---|---|
| LH178 (30 kDa) (Trypsin) | MJ0892/FlaB2 (21,155/4.73) Q58302 | 3681.31 2887.26 2327.35 | TTVTGSVIPEFGAPAVIEFTTPAAYLSTQEVIQLQ (183–217) {3681.174} AMATGKESTEQVASGLSTLQVIGIHDNK (47–74) {2887.234} ESTEQVASGLSTLQVIGIHD NK (53–74) {2327.553} | 0.136 0.026 −0.203 | 29% [pro-peptide, residues 1–12] |
| LH179 (29 kDa) (Trypsin) | MJ0893/FlaB3 (21,720/4.86) Q58303 | 2935.11 2283.28 1998.02 1743.88 1416.52 | LAILVSPNVGDEIDLSSTIVTISNGDYK (79–106) {2935.298} ERIYGEVIPEFGASGIIEFR (184–203) {2283.588} IYGEVIPEFGASGIIEFR (183–203) {1998.285} AIIAINVGDVFGGIMPR (167–183) {1744.105} APSTFSEHVVTLQ (204–216) {1416.573} | −0.188 −0.308 −0.568 −0.225 −0.053 | 37% [pro-peptide, residues 1–11] |
| LH174 (16 kDa) (Trypsin) | MJ0895/FlaD (39,950/5.91) Q58305 | 2198.28 1958.09 1714.84 1405.55 1152.35 | LAGRPIDSEILEMLEIEIR (312–330) {2198.582} DKLSPSDHIVSLLYIEK (295–311) {1958.262} LSPSDHIVSLLYIEK (297–311) {1714.999} ITFDEEELRPR (284–294) {1405.550} ITFDEEELR (284–292) {1152.246} | −0.302 −0.172 −0.159 0.0 0.104 | 14% (or 27% for a C-terminal ∼16 kDa fragment; 207–342 stretch; pI, 5.14) |
| LH175 | MJ0896/FlaE | 1797.94 | LNITDHIISLLFIER (95–109) {1798.135} | −0.195 | 27% |
| (15 kDa) | (15,892/5.02) | 1715.84 | ISAELLDKIEWELR (115–128) {1715.987} | −0.147 | |
| (Trypsin) | Q58306 | 1013.06 | KGAEQFYGI (132–140) {1013.138} | −0.078 | |
| LH176 | MJ1035/MTD | 2483.90 | MVEEVKPDFIIYIGPNPAAPGPK (55–77) {2483.928} | −0.028 | 35% |
| (30 kDa) | (30,306/5.86) | 2234.64 | AREILSQSGIPAVIIGDAPGLR (79–100) {2234.603} | 0.037 | |
| (Trypsin) | Q58441 | 2136.37 | REFLDPVEMALFNADVIR (125–142) {2136.473} | −0.103 | |
| 2007.29 | EILSQSGIPAVIIGDAPGLR (81–100) {2007.337} | −0.047 | |||
| 1980.08 | EFLDPVEMALFNADVIR (126–142) {1980.286} | −0.206 | |||
| 1958.09 | EAEKYIPIVASAHEMIR (220–236) {1958.283} | −0.193 | |||
| 1500.73 | YIPIVASAHEMIR (1500.799) {224–236} | −0.069 | |||
| 1471.65 | AVEAMEFTNPYAK (182–194) {1471.668} | −0.018 | |||
| 1053.19 | AMAAFTIAEK (197–206) {1053.261} | −0.071 | |||
| HH023 | MJ0715/HMDX | 2477.83 | QILISLQTIASIIETSGMEGLMK (236–258) {2477.977} | −0.147 | 42% |
| (37 kDa) | (36,854/5.69) | 2384.55 | AGHDVVLSEPNRDVMSDDLWK (42–62) {2384.625} | −0.075 | |
| (Lys-C) | Q58125 | 2208.3 | RRDVGISSMHPTGVPGTPSQK (135–155) {2208.500} | −0.2 | |
| 1967.14 | ISIYGAGNQRLYLEQLK (3–19) {1967.275} | −0.135 | |||
| 1916.08 | NMLIDERQEDLNLALK (271–286) {1916.199} | −0.119 | |||
| 1800.03 | FGGEPPYGGAGMAIEFAK (24–41) {1800.038} | −0.008 | |||
| 1660.86 | DISQTYLVAPQALIK (300–314) {1660.951} | −0.091 | |||
| 1435.59 | SAVEGMIRRSSNK (323–335) {1435.641} | −0.051 |
See Fig. 1 C–J for the location of a spot in the gels; the apparent molecular mass was derived from the migration distances in these gels.
Masses not matched: LH178 (4153.87, 4111.14, 3855.81, 3719.01, 3270.14, 3313.42, 3270.14, 2954.86, 2674.74, 2082.78); LH179 (4095.09, 3680.52, 3556.75, 3395.36, 1759.52, 1416.52); LH174 (2214.39, 1754.47, 1737.81, 1441.46, 1425.48, 1225.76); LH175 (2551.73, 2198.11, 1844.01, 1405.52); LH176 (1861.52, 1516.34, 1483.84, 1068.88); HH023 (2400.67, 2322.55, 2288.5, 2225.72, 1989.44, 1932.11, 1821.8, 1682.87, 1319.47).
Matched by use of the ProFound and the MS-Fit programs. The general search parameters were: MW of the intact polypeptide, a range based on the second dimension electrophoresis data (or a higher value, if the first search did not yield a good match; this could happen due to cleavage or processing of the full-length polypeptide or due to an error in assigning an ORF length or a start site in the databases); digest chemistry or type, trypsine or Lys-C (as applicable); cysteine modification, by acrylamide; maximum number of missed cleavages, 1 for trypsin and 0 for Lys-C; peptide mass type, average. The program-specific parameters were as follows. For ProFound: taxonomic category, archaebacteria; search for, single protein only; number of top candidate proteins, 10; peptide masses, M + H+; mass tolerances, 0.5 Da. 2. For the MS-Fit program: species, Methanococcus jannaschii; protein pI, 3–10; N terminus, hydrogen; C terminus, free acid; maximum reported hits, 5; search mode, identity; minimum number of matches with no amino acid substitutions, 1; peptide mass shift, 40.1 Da; possible modifications mode, default; user defined modification 1, phosphorylation of S, T, Y; minimum number of peptides required to match; 4 (or variable); Pfactor, 0.4; peptide mass tolerance, 0.5 Da.
The portion of the identified polypeptide's amino acid sequence covered by the matched fragments.
The propeptide sequence was located by comparison with the corresponding M. voltae homolog, for which experimental evidence is available (13).