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Park, Lee, Kim, Joo, Kwon, Youn, Song, Kang, Lee, Baik, Lee, Cho, and Rhee: Analysis of Low Molecular Weight Proteome from H. pylori Cell Extract Using the High Performance Liquid Chromatography
Original Article

J Bacteriol Virol 2010;40(2):67-75.

Published online: 18 January 2010

DOI: https://doi.org/10.4167/jbv.2010.40.2.67

Analysis of Low Molecular Weight Proteome from H. pylori Cell Extract Using the High Performance Liquid Chromatography

Jung Won Park1, Kyung Ja Lee2, Kyung Mi Kim2, Jung Soo Joo2, Yung Chul Kwon2, Hee Shang Youn3, Jae Young Song2, Hyung Lyun Kang2, Kon Ho Lee2, Seung Chul Baik2, Woo Kon Lee2, Myung Je Cho2, Kwang Ho Rhee2, ∗

1Research Institute for Subtropical Horticulture, Jeju National University, Jeju, Korea

2Department of Microbiology, Jinju, Korea

3Deaprtment of Pediatrics, School of Medicine, Gyeongsang National University, Jinju, Korea

∗Corresponding author: Kwang Ho Rhee. Department of Microbiology, School of Medicine, Gyeongsang National University Chiram-dong 92, Jinju, Gyeongnam 660-752, Korea. Phone: +82-55-751-8781, Fax: +82-55-759-1588 e-mail: khrhee1@gnu.ac.kr

      Revised 26 November 200914 April 2010       Accepted 12 May 2010

Copyright © 2010 Korean Society for Legal Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Low molecular proteins (LMPs) which are smaller than 20 kDa are difficult to visible on a standard two-dimensional SDS-polyacrylamide gel electrophoresis (2-D SDS-PAGE) map. LMPs must be enriched appropriately to be analyzed. We isolated LMPs of Helicobacter pylori 26695 from 1-D polyacrylamide gel and digested by pepsin. Pepsin-digested LMPs were separated by HPLC and each fraction was analyzed by hybrid tandem mass spectrometer. Seventy nine peptides, representing 27 genes, including copper ion binding protein (CopP, 7 kDa), thioredoxin (TrxA, 11.9 kDa) and ribosomal protein L23 (Rpl23, 10.5 kDa) were identified. Some proteins larger than 40 kDa including Omp2, Omp21, Omp27, Omp30, Omp32, catalase and HP1083 were also identified. This work may give researchers a useful way to analyse the expressed LMPs which could not be identified on the conventional 2-D SDS-PAGE.

Keywords: Helicobacter pylori, Hybrid tandem mass spectrometer, Low molecular protein

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Figure 1.
Extraction of the H. pylori LMW proteins. In lanes: M, molecular weight marker; 1, whole cell proteins of H. pylori 26695; 2, extracted low molecular proteins.
jbv-40-67f1.tif
Figure 2.
Profile of the H. pylori low molecular proteins separated by reverse phased HPLC.
jbv-40-67f2.tif
Figure 3.
2-DE map of the low molecular proteins of H. pylori that were extracted from 1-DE gel.
jbv-40-67f3.tif
Table 1.
Protein identification from reverse phased HPLC fractions of H. pylori 26695 low molecular proteins using the hybrid tandem mass spectrometry
Fraction No.§ MW (kDa) Protein name Match seq. TIGR locus name
23~24 22.2 Alkyl hydroperoxide reductase (ahpC) 34-NGAILFFWPK-43 HP1563
29~30 11.9 Thioredoxin (trxA) 55-VNTDEQEELSAK-67 HP0824
34~35 17.5 flavodoxin (fldA) 40-EQFNSFTK-47 HP1161
34~35 17.5 flavodoxin (fldA) 112-VVGQTPTDGYHFEASK-127 HP1161
34~35 17.5 flavodoxin (fldA) 25-AIGNAEVVDVAK-36 HP1161
34~35 16.9 Neutrophil activating protein (napA) 120-VTVTYADDQLAK-131 HP0243
34~35 19.1 Membrane-associated lipoprotein 69-AEDLITNNDVDYSTNQATAK-88 HP1456
36~37 27.8 Hypothetical protein HP0369 9-IQMTFYKL-16 HP0369
36~37 13.0 Co-chaperone (groES) 21-TSSGIIIPDNAK-32 HP0011
38~39 16.9 Neutrophil activating protein (napA) 30-GTDFFNVHK-38 HP0243
38~39 10.5 Ribosomal protein L23 (rpl23) 23-GVLVVQTAQNVTK-35 HP1317
38~39 14.5 Ribosomal protein S9 (rps9) 64-AVVFGGGYSAQAEALR-79 HP0083
38~39 20.4 Hypothetical protein (HP0305) 104-NVIEGDHAGSLTAYVR-119 HP0305
38~39 17.6 Hypothetical protein (HP0721) 34-MAGI(V)VAPQDIVDYTK-48 HP0721
40~41 16.9 Neutrophil activating protein (napA) 94-EILEDYKYLEK-104 HP0243
40~41 18.3 Adhesin thiol peroxidase (tagD) 28-LVNGDLQEVNLLK-41 HP0390
40~41 13.3 Ribosomal protein L7/L12 (rpl7/l12) 31-FGVSATPTVVAGAAVAGGAAAESEEK-56 HP1199
40~41 14.0 Ribosomal protein S11 (rps11) 59-STPYAAQQAVESALSK-74 HP1295
42~43 7.2 Copper ion binding protein (copP) 40-SVVVEFDAPATQDLIK-55 HP1093
42~43 17.5 Flavodoxin (fldA) 134-FVGLVIDEDNQDDLTDER-151 HP1161
42~43∗ 53.1 Hypothetical protein HP1083 322-AGTAGQSLLIR-332 HP1083
42~43 17.5 Flavodoxin (fldA) 4-IGIFFGTDSGNAEAIAEK-21 HP1161
42~43 69.7 Outer membrane protein (omp27) 610-IPTINTNYYSFLDTK-624 HP1177
44~45∗ 19.1 Membrane-associated lipoprotein 152-VFVLVGLDK-160 HP1456
44~45 11.9 Thioredoxin (trxA) 72-SIPTLLFTK-80 HP0824
44~45 26.5 Urease, alpha subunit (ureA) 195-IFGFNALVDR-204 HP0073
44~45 18.3 Adhesin thiol peroxidase (tagD) 147-EIVQNILEEPNYEALLK-163 HP0390
44~45∗ 26.8 Outer membrane protein (omp30) 90-LKNPNYNNEVVQLAGQVLGK-108 HP1395
44~45 18.3 ubiquinol cytochrome c oxidoreductase, Rieske 2Fe-2S subunit (fbcF) 123-GRFTSDGVNIAGTPPPRPFDIPPFK-145 HP1540
44~45 20.6 Hypothetical protein (HP1173) 159-ISLVGNFDGTGFLTEYK-175 HP1173
46~47∗ 21.9 lipoprotein, putative (Hypothetical protein) 127-LGIYINPNNQEVFALVR-143 HP0596
46~47 58.6 Catalase 29-GPVLLQSTWFLEK-41 HP0875
46~47 77.7 Outer membrane protein (omp2) 681-IPTINTNYYSLLGTTLQYR HP0025
46~47 43.0 Outer membrane protein (omp32) 331-IPTLPNYFFK-340 HP1501
46~47 22.2 Alkyl hydroperoxide reductase (ahpC) 112-DYDVLFEEAIALR-124 HP1563
46~47 20.4 Hypothetical protein (HP0305) 87-EAFFALFK-94 HP0305
46~47 16.9 Neutrophil activating protein (napA) 90-DIFKEILEDYK-100 HP0243
46~47 20.4 Hypothetical protein (HP0305) 87-EAFFALFK-94 HP0305
48~49∗ 16.1 Ribosomal protein L13 (rpl13) 27-LITEIAVLLR-36 HP0084
48~49 57.1 Outer membrane protein (omp21) 465-FQFLFDVGLR-474 HP0913
50~52 22.2 Alkyl hydroperoxide reductase (ahpC) 112-DYDVLFEEAIALR-124 HP1563
42~43∗ 53.1 Hypothetical protein HP1083 322-AGTAGQSLLIR-332 HP1083
42~43 17.5 Flavodoxin (fldA) 4-IGIFFGTDSGNAEAIAEK-21 HP1161
42~43 69.7 Outer membrane protein (omp27) 610-IPTINTNYYSFLDTK-624 HP1177
44~45∗ 19.1 Membrane-associated lipoprotein 152-VFVLVGLDK-160 HP1456
44~45 11.9 Thioredoxin (trxA) 72-SIPTLLFTK-80 HP0824
44~45 26.5 Urease, alpha subunit (ureA) 195-IFGFNALVDR-204 HP0073
44~45 18,.3 Adhesin thiol peroxidase (tagD) 147-EIVQNILEEPNYEALLK-163 HP0390
44~45∗ 26.8 Outer membrane protein (omp30) 90-LKNPNYNNEVVQLAGQVLGK-108 HP1395
44~45 18.3 ubiquinol cytochrome c oxidoreductase, Rieske 2Fe-2S subunit (fbcF) 123-GRFTSDGVNIAGTPPPRPFDIPPFK-145 HP1540
44~45 20.6 Hypothetical protein (HP1173) 159-ISLVGNFDGTGFLTEYK-175 HP1173
46~47∗ 21.9 lipoprotein, putative (Hypothetical protein) 127-LGIYINPNNQEVFALVR-143 HP0596
46~47 58.6 Catalase 29-GPVLLQSTWFLEK-41 HP0875
46~47 77.7 Outer membrane protein (omp2) 681-IPTINTNYYSLLGTTLQYR HP0025
46~47 43.0 Outer membrane protein (omp32) 331-IPTLPNYFFK-340 HP1501
46~47 22.2 Alkyl hydroperoxide reductase (ahpC) 112-DYDVLFEEAIALR-124 HP1563

§ HPLC fraction numbers from figure 2

∗ Unidentified in references

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