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Abstract
It has been reported that most of Helicobacter pylori proteome components appear so crowded in the region of pH 4.5∼8.0 that a lot of them were inseparable in 2-DE using the broad range IPG strip. Therefore, inseparable protein spots in 2-DE profiles have to be apart from each other for improving the protein identification. Here, we attempt to examine the usability of the narrow range IPG strips for separating close spots in the broad range IPG strip at proteomic analysis of H. pylori. The whole cell proteins of H. pylori strain 26695 were separated by narrow range IPG strips (pI 3.9∼5.1, 4.7∼5.9, 5.5∼6.7, and 6.3∼8.3, respectively), followed by SDS-PAGE, and visualized by silver staining, showing that the distances between spots were widened and the total number of detectable spots was increased. Resolved protein spots were identified by the peptide fingerprinting using MALDI-TOF-MS. As a result, 87 expressed proteins were identified by the peptide fingerprinting. Of them, 23 proteins, including hydrogenase expression/formation protein, purine-binding chemotaxis protein, and ribosomal protein S6, have not been reported in the previous proteome studies of H. pylori. Thus, these results demonstrate that the high complexity proteome components could be effectively separated using the narrow range IPG strips, which might be helpful to strengthen the contents of the master protein map of the H. pylori reference strain.
References
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Figure 1.
Two-dimensional-gel electrophoresis of whole cell proteins from H. pylori strain 26695. Whole cell proteins solution was separated on a narrow range IPG strips, followed by 12.5% SDS-PAGE, and visualized by silver staining. The original gel size was 20×18×0.15 cm. (A) pH 3.9∼5.1; (B) pH 4.7∼5.9; (C) pH 5.5∼6.7; (D) pH 6.3∼8.3.
Figure 2.
The composite profile of the whole cell proteins of H. pylori strain 26695 resolved in the 2-DE using the narrow pH range IPG strips. The whole cell proteins of H. pylori strain 26695 was subjected to 2-DE using IPG strips of 3.9∼5.1, 4.7∼5.9, 5.5∼6.7, and 6.3∼8.3 ranges, the gels were visualized with silver staining. The 4 kinds of images were taken up and analyzed with PDQUEST software, and excluded with the overlapping zones of each right and left termini, and then combined by PHOTOSHOP software to generate the composite one ranged from pI 3.9 to pI 8.3. The marked protein spots have been identified and listed in Table 1.
Figure 3.
Blow-up images of two different regions in 2-DE gels of H. pylori whole cell proteins resolved by broad (pH 3.0∼10.0), intermediate (pH 5.0∼8.0), narrow (pH 4.7∼5.9) pH strips. Here, the narrower is the pH range of the strip, the higher resolution does the spots profile show in which the resolution and the number of spots are improved. HtpG spot appears in the gel of narrow pH strips but did not in otherwise.
Table 1.
Protein identification of spots from narrow range 2-DE gels the whole cell proteins of H. pylori strain 26695
| TIGR locus§ | MW (Da) | pI | Annotation |
|---|---|---|---|
| HP 0010 | 58263.91 | 5.4772 | Chaperone and heat shock protein (groEL) |
| HP 0011 | 12990.87 | 6.5917 | Co-chaperone (groES) |
| HP 0014 | 31681.02 | 5.7841 | Hypothetical protein |
| HP 0026 | 48350.02 | 8.0209 | Citrate synthase (gltA) |
| HP 0027 | 47531.05 | 7.9527 | Isocitrate dehydrogenase (icd) |
| HP 0047∗ | 36405.28 | 4.8329 | Hydrogenase expression/formation protein (hypE) |
| HP 0059∗ | 32723.51 | 4.8431 | Hypothetical protein |
| HP 0060∗ | 93128.96 | 4.8450 | Hypothetical protein |
| HP 0061∗ | 21764.88 | 5.9064 | Hypothetical protein |
| HP 0067 | 29722.06 | 6.1629 | Urease accessory protein (ureH) |
| HP 0068 | 21955.25 | 4.7602 | Urease accessory protein (ureG) |
| HP 0069 | 28618.77 | 7.6498 | Urease accessory protein (ureF) |
| HP 0072 | 61683.52 | 5.9110 | Urease beta subunit (ureB) |
| HP 0073 | 26539.49 | 8.9593 | Urease, alpha subunit (ureA) |
| HP 0096 | 34796.18 | 7.0468 | Phosphoglycerate dehydrogenase |
| HP 0109 | 67051.91 | 4.7568 | Chaperone and heat shock protein70 (dnaK) |
| HP 0137∗ | 23610.48 | 7.6666 | Hypothetical protein |
| HP 0153∗ | 37686.28 | 5.5065 | Recombinase (recA) |
| HP 0166 | 25855.46 | 5.0781 | Response regulator (ompR) |
| HP 0176 | 33772.69 | 6.2517 | Fructose-bisphosphate aldolase (tsr) |
| HP 0191 | 27651.76 | 5.1972 | Fumarate reductase, iron-sulfur subunit (frdB) |
| HP 0192 | 80121.10 | 7.3217 | Fumarate reductase, flavoprotein subunit (frdA) |
| HP 0197 | 42362.06 | 6.4292 | S-adenosylmethionine synthetase 2 (metX) |
| HP 0201 | 36483.11 | 6.5193 | Fatty acid/phospholipid synthesis protein (plsX) |
| HP 0210 | 71274.03 | 5.2608 | Chaperone and heat shock protein C62.5 (htpG) |
| HP 0220 | 42403.38 | 6.1542 | Synthesis of [Fe-S] cluster (nifS) |
| HP 0243 | 16933.34 | 5.8309 | Neutrophil activating protein (napA) (bacterioferritin) |
| HP 0264 | 96683.36 | 6.2660 | ATP-dependent protease binding subunit (clpB) |
| HP 0389 | 24617.63 | 6.1683 | Superoxide dismutase (sodB) |
| HP 0391∗ | 18966.68 | 5.0808 | Purine-binding chemotaxis protein |
| HP 0399 | 62826.43 | 6.7727 | Ribosomal protein S1 (rps1) |
| HP 0452∗ | 60506.79 | 5.9621 | Conserved hypothetical protein |
| HP 0480 | 66676.31 | 5.0937 | GTP-binding protein, fusA-homolog (yihK) |
| HP 0512 | 54513.58 | 6.0379 | Glutamine synthetase (glnA) |
| HP 0557 | 34881.13 | 5.3110 | Acetyl-coenzyme A carboxylase (accA) |
| HP 0569 | 40574.45 | 5.5302 | GTP-binding protein (gtp1) |
| HP 0570 | 54433.07 | 7.1054 | Aminopeptidase a/i (pepA) |
| HP 0589 | 41508.81 | 6.3623 | Ferredoxin oxidoreductase, alpha subunit |
| HP 0617 | 65601.53 | 6.8834 | Aspartyl-tRNA synthetase (aspS) |
| HP 0618 | 21243.21 | 5.0039 | Adenylate kinase (adk) |
| HP 0620 | 19271.89 | 4.8028 | Inorganic pyrophosphatase (ppa) |
| HP 0646∗ | 30975.69 | 6.5192 | UDP-glucose pyrophosphorylase (galU) |
| HP 0649 | 51980.86 | 6.8341 | Aspartate ammonia-lyase (aspA) |
| HP 0658 | 53288.14 | 6.0094 | Glutamyl-tRNA(Gln) amidotransferase subunit B (gatB) |
| HP 0691 | 25362.24 | 5.8082 | 3-oxoadipate coA-transferase subunit A (yxjD) |
| HP 0692 | 22262.85 | 5.3450 | 3-oxoadipate coA-transferase subunit B (yxjE) |
| HP 0695 | 78532.79 | 6.9435 | Hydantoin utilization protein A (hyuA) |
| HP 0703 | 43414.34 | 6.7535 | Response regulator |
| HP 0799∗ | 19676.86 | 5.3363 | Molybdopterin biosynthesis protein (mog) |
| HP 0829 | 51801.80 | 8.2155 | Inosine-5′-monophosphate dehydrogenase (guaB) |
| HP 0852∗ | 35776.74 | 6.6806 | Hypothetical protein |
| HP 0857∗ | 21102.26 | 6.3967 | Phosphoheptose isomerase (gmhA) |
| HP 0859 | 37412.66 | 7.3588 | ADP-L-glycero-D-mannoheptose-6-epimerase (rfaD) |
| HP 0900 | 27310.33 | 5.2449 | Hydrogenase expression/formation protein (hypB) |
| HP 0902 | 11029.96 | 6.6745 | Hypothetical protein |
| HP 0975∗ | 10656.15 | 4.7389 | Glu-tRNA(Gln) amidotransferase, subunit C |
| HP 0978 | 54492.59 | 6.6324 | Cell division protein (ftsA) protein |
| HP 0979∗ | 40954.34 | 5.1659 | Cell division protein (ftsZ) |
| HP 1038 | 18483.25 | 4.9294 | 3-dehydroquinase type II (aroQ) |
| HP 1043∗ | 25468.16 | 4.9740 | Response regulator |
| HP 1059 | 37360.31 | 6.0818 | Holliday junction DNA helicase (ruvB) |
| HP 1079∗ | 42919.70 | 6.3237 | Hypothetical protein |
| HP 1102 | 25727.47 | 7.3447 | Glucose-6-phosphate 1-dehydrogenase (devB) |
| HP 1104 | 38645.51 | 7.4067 | Cinnamyl-alcohol dehydrogenase ELI3–2 (cad) |
| HP 1110 | 44743.62 | 5.8122 | Pyruvate ferredoxin oxidoreductase, alpha subunit |
| HP 1114 | 75916.20 | 6.9096 | Excinuclease ABC subunit B (uvrB) |
| HP 1132 | 51478.08 | 5.0767 | ATP synthase F1, subunit beta (atpD) |
| HP 1134 | 55143.11 | 5.0512 | ATP synthase F1, subunit alpha (atpA) |
| HP 1135∗ | 20368.81 | 8.4730 | ATP synthase F1, subunit delta (atpH) |
| HP 1161 | 17492.37 | 4.2040 | Flavodoxin (fldA) |
| HP 1164 | 35985.98 | 6.8739 | Thioredoxin reductase (trxB) |
| HP 1195 | 77020.88 | 5.0111 | Translation elongation factor EF-G (fusA) |
| HP 1199 | 13313.38 | 4.9125 | Ribosomal protein L7/L12 (rpl7/l12) |
| HP 1203 | 20261.24 | 7.8735 | Transcription termination factor NusG (nusG) |
| HP 1205 | 43647.70 | 4.9302 | Translation elongation factor EF-Tu (tufB) |
| HP 1226∗ | 40196.14 | 6.5108 | Oxygen-independent coproporphyrinogen III oxidase (hemN) |
| HP 1237∗ | 41635.41 | 7.1579 | Carbamoyl-phosphate synthetase (pyrAa) |
| HP 1246∗ | 16971.34 | 7.4983 | Ribosomal protein S6 (rps6) |
| HP 1293 | 38499.11 | 4.6887 | DNA-directed RNA polymerase, alpha subunit (rpoA) |
| HP 1299 | 27576.93 | 6.4208 | Methionine amino peptidase (map) |
| HP 1345∗ | 44771.64 | 6.7477 | Phosphoglycerate kinase |
| HP 1374∗ | 50353.20 | 6.4377 | ATP-dependent protease ATPase subunit (clpX) |
| HP 1507∗ | 43904.36 | 6.5698 | Conserved hypothetical ATP-binding protein |
| HP 1514 | 44649.63 | 5.5915 | Transcription termination factor NusA (nusA) |
| HP 1517 | 149715.58 | 7.5020 | Type IIS restriction enzyme R and M protein (ECO57IR) |
| HP 1563 | 22235.57 | 6.2482 | Alkyl hydroperoxide reductase (tsaA) |
| HP 1582 | 29720.44 | 8.0532 | Pyridoxal phosphate biosynthetic protein J (pdxJ) |
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