Abstract The purpose of screening and identification of significant differentially expressed proteins, and to provide a theoretical basis for research on the pathogenesis and early diagnosis of human lung adenocarcinoma. Two-dimensional electrophoresis of immobilized pH gradient two-dimensional gel electrophoresis separation of 12 cases of stage Ⅰ lung adenocarcinoma and its matching cancer normal lung tissues of total soluble protein Ⅰ of lung adenocarcinoma and matched cancer adjacent normal lung tissue protein gel maps; PDQuest gel image analysis software comparative analysis of differentially expressed protein spots screened; matrix-assisted laser desorption ionization time-of-flight mass spectrometry peptide mass fingerprinting of the corresponding protein spots; Search protein database identified differentially expressed proteins. Results reproducible Stage I lung adenocarcinoma and matched cancer adjacent normal lung tissue proteins by two-dimensional electrophoresis gel maps; 26 filter out the presence of significantly differentially expressed protein spots, dig 9 protein spots mass spectrometry, nine proteins have been satisfied peptide mass fingerprinting; Search protein database identified four proteins, respectively: 60S ribosomal protein P2, cathepsin B1, apolipoprotein A  I precursors and La 4.1 proteins. Conclusion The two-dimensional electrophoresis gel Ⅰ of lung adenocarcinoma and matched cancer adjacent normal lung tissue protein maps identified in lung adenocarcinoma early the obvious protein expression, for further exploration of the human lung adenocarcinoma The pathogenesis of early and looking for specific molecular markers provides a theoretical basis.

Key words lung adenocarcinoma; proteomics; dimensional gel electrophoresis; matrix-assisted laser desorption ionization time-of-flight mass spectrometry

 Differential Analysis of Two  dimension Gel Electrophoresis Profiles of Human Early  stage Lung Adenocarcinoma and Tumor  adjacent Tissue

    LIU Gui  zhi1, WU Yi  ming2

    1.Department of Physical Examination, First Teaching Hospital, Zhengzhou University, Zhengzhou 450052, China; 2.Department of Toxicology, College of Public Health, Zhengzhou University

    Corresponding Author: WU Yi  ming, E  mail: wuym@zzu.edu.cnAbstract: Objective To screen and identify differential expression proteins, and to obtain theoretical data for studying human stage Ⅰ lung adenocarcinoma molecular mechanism and finding early biomarker.Methods The total proteins of 12 human stage Ⅰ lung adenocarcinoma tissue and normal tumor  adjacent tissue were separated, using 170mm long immobilized pH gradient (IPG) gel strips, pH3  10, for the first dimension and homogeneous SDS  polyacrylamide gel electrophoresis (SDS  PAGE ) (12% T) for the second dimension.The differential expression proteins were analyzed with PDQuest image analysis software, then identified using matrix  assisted laser desorption / ionization time of flight mass spectrometry (MALDI  TOF  MS) and database searching.Results The well  reproducible 2  DE gel patterns of human stage Ⅰ lung adenocarcinoma and normal tumor  adjacent tissue were established and 26 differential proteins were screened.Nine of 26 differential protein spots were cut out from the preparation gels and were identified with MALDI  TOF  MS.Comparing with the protein database, four candidate proteins were identified with MS. They were 60S acidic ribosomal protein P2, Cathepsin B precursor, Apolipoprotein A  I precursor and La 4.1 protein.Conclusion In this study, 2  DE gel images of human stage Ⅰ lung adenocarcinoma and paired normal tumor  adjacent tissue were established successfully, and identified 4 differential proteins.These data will be helpful for studying human lung adenocarcinoma molecular mechanism and screening early biomarker.

    Key words: Lung adenocarcinoma; Proteomics; Two  dimensional gel electrophoresis; Matrix  assisted laser desorption  ionization time of flight mass spectrometry

    Lung cancer is recognized worldwide cancer killer. Incidence of occult high mortality. Currently, early diagnosis is the key measures to improve the survival of lung cancer patients. Occurrence, development and prognosis of lung cancer is an extremely complex, multi-stage, multi-factor abnormal regulation of the process, if direct protein overall level from the start to study the different clinical stages of lung cancer, will be able to be more comprehensive, truly reveals lung cancer carcinogenesis, specific molecular markers for early diagnosis and prognosis of lung cancer. With the emergence of proteomics and its continuous development and improvement of analytical techniques for the display and analysis of proteins from the overall level to provide a strong technical support.

    This study using proteomics technology to people early lung adenocarcinoma and matched cancer adjacent normal lung tissue proteome research, the establishment of a two-dimensional gel electrophoresis pattern and differentially expressed protein spots were identified to the human lung adenocarcinoma pathogenesis and early diagnosis of the study provides a theoretical basis.

    1 Materials and methods

    1.1 specimens

    Thoracic Surgery, First Clinical Medical College of Zhengzhou University, this group of specimens taken from March 2005 to May 2006, a single line of surgical treatment of 12 cases of stage Ⅰ (T1N0M0, T2N0M0) lung adenocarcinoma patients. Cases drawn including cancer tissues and the corresponding adjacent normal lung tissue of each copy of 24 specimens, cancer tissue and matched normal lung tissue samples were confirmed by histopathological examination.

    1.2 Reagents

    Immobilized pH gradient (immobilized pH gradient, IPG) prefabricated strips (pH3 of  10,17 cm), 3 to 10 ampholytes bio  lyte, urea, thiourea were purchased from Bio  Rad; CHAPS (3  [3  cholamidopropyl] diethylamine  1  propane sulfonic acid), Aprotinin (aprotinin), PMSF (phenylmethylsulfonyl fluoride), DTT (dithiothreitol), iodoacetyl ammonium, acrylamide, methylene bisacrylamide, SDS (sodium dodecyl sulfate) of TEMED (N’N’N’N, ‘ tetramethyl ethylene ammonia) were purchased from Sigma Chemical Company.

    1.3 The main instrument

    Two-dimensional gel electrophoresis system  PROTEIN IEF CELL isoelectric focusing instrument PROTEAN xi cell electrophoresis apparatus, GS  800 image acquisition instrument, the PDQuest 7.1 gel image analysis software. American Bio  Rad products.

    1.4 Organization Preparation of total protein

    80 ~ 100mg tissue samples in liquid nitrogen, under fully ground to a powder, placed in 500μl lysis buffer (containing 7M urea, 2M thiourea, 40 mM Tris, 4% CHAPS, 1% Triton  X100, 100 mM DTT, 10.2% (W / v) Bio  Lyte, 0.5mM PMSF, 0.5mM EDTA, 1μg/ml Aprotinin), fully vortex mixing, and incubated at 37 ℃ for 1h fully vortex then remove 15 000r/min 4 ° C for 30 min supernatant is the organization of total protein. Using BradFord method [1] The determination of the protein concentration of the sample set  80 ° C low temperature refrigerator to save.

    1.5 immobilized pH gradient two-dimensional gel electrophoresis

    IPGphor isoelectric focusing system guide. Always isoelectric focusing electrophoresis procedures are as follows: 50V Active hydration, 16h (17 ℃); 250 V desalination, 30min; 1 000V desalination 1h; boost 5h to 10 000V focus to 80 000Vh. The dichroic SDS  polyacrylamide gel electrophoresis (polyacrylamide gel electrophoresis, PAGE) under the following conditions: the sample concentrator current 5mA/gel/17cm sample separating current 30mA/gel/17cm. The gel of the electrophoresis obtained silver staining or Coomassie blue staining (CBB staining).

    1.6 Two-dimensional electrophoresis pattern analysis

    Staining after SDS polyacrylamide gel, the PDQuest software internal support by the image scanning apparatus GS-800 scan imaging, digitized images analyzed using PDQuest 7.1 software. The image analysis process including protein spots detected (detect spots on gels), edit and matching of protein spots (edit spot and edit match), the molecular weight of the protein spots (Mr) and isoelectric point (pI) correction [2].

    1.7 In-gel digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (matrix  assisted laser desorption / ionization time of flight mass spectrometry, MALDI  TOP  MS) analysis

    Scan analysis after preparative CBB staining gel placed under the bright lights, turned their guns silylation cut protein spots with 2mg / L trypsin in-gel enzymatic protein samples by Shanghai GeneCore biotechnology limited assist with the identification of MALDI  TOP  MS, the sample is detected peptide mass fingerprinting.

    1.8 database retrieval

    Peptides m / z data, software with Mascot (http://www.matrixscience.com) and Profound (http://129.85.19.193/profound  bin / WebProFound.exe), set the appropriate search parameters, the NCBInr, Swissprot protein database search theoretically enzymatic peptides matched with proteins, according to the search results, combined with the isoelectric point and molecular weight of the protein on the gel ultimately determine a target protein.

    2 Results

    2.1 Stage I lung adenocarcinoma and matched cancer adjacent normal lung tissue proteome

    The amount of the sample from the control of the total protein of the same specimen, 0.15mg, two-dimensional gel silver staining, and repeatability of detection was performed three times, and very similar results show that 3 times of two-dimensional electrophoresis silver staining. GS  800 to obtain images PDQuest 7.1 software point detection, the average spots get three gel Ⅰ of lung adenocarcinoma and matched cancer adjacent normal lung tissue were 1873 ± 79 and 1715 ± 63. Background subtraction, which is a glue for the reference gel protein spots matching three plastic the average match points I lung adenocarcinoma and matching paracancerous of normal lung tissue were 1576 ± 62 and 1445 ± 48 matching rate of 87.1% and 85.0%, respectively. Reproducible Stage I lung adenocarcinoma and matched cancer adjacent normal lung tissue of two-dimensional gel electrophoresis, silver staining, as shown in Figure 1.

    2.2 Stage I lung adenocarcinoma and matched cancer adjacent normal lung tissue protein expression profiling analysis and differential display identification of proteins

    The amount of total protein control sample 0.15mg, two-dimensional gel electrophoresis of the proteins of normal lung tissue of 12 patients with Stage I lung adenocarcinoma and its matching paraneoplastic PDQuest 7.1 software analysis were established 12 glue average gel silver staining. Ⅰ lung adenocarcinoma tissue protein gel silver staining protein spots were detected in 1977, 1826 protein spots were detected in normal lung tissue protein gel Figure. Will establish the average gel match, after repeated comparison screened Ⅰ lung adenocarcinoma and normal lung tissues was significantly differentially expressed protein spots (think 26 more than five times the expression of difference and at least five pairs organization have the same change point for differentially expressed protein spots). By the above method, the total protein amount of control on the kind of 3mg, stained gel of the preparation of two-dimensional electrophoresis test to find differentially expressed protein spots corresponding to the silver-stained gel stained gel from the test, the first batch of digging experimental molecular weight less than 40kD 9 protein spots were identified by mass spectrometry. 1,6,4,7 No. protein spots in matched normal lung tissue expression in stage I lung adenocarcinoma was significantly diminished or disappeared, 2,3 and 5 protein only in lung adenocarcinoma tissues, 8 and No. 9 protein expression was significantly increased in the cancer tissue, as shown in Figure 1.9 protein spots were obtained satisfactory enzymatic fragment peptide mass fingerprinting. Search protein databases, combining the candidate protein isoelectric point, molecular weight and isoelectric point measured in the experiment, the molecular weight is consistent and other information, the final identification of a protein, 4, as shown in Table 1.

    3 Discussion

    Using proteomics technology, domestic and foreign scholars the respectively adjacent normal lung tissue in lung cancer cell lines, human lung cancer tissues and matched cancer research different lung cancer cell lines and normal bronchial epithelial cells [3  5], lung cancer and cancer next to two-dimensional electrophoresis gel maps of the normal lung tissue [6  9], which screened a variety of meaningful existence of significantly differentially expressed proteins. At present, the domestic yet to see the proteomics methods of lung adenocarcinoma do installments research literature. Foreign scholars have noted that the different stages of lung adenocarcinoma exist protein Table 14 differentially expressed protein spots by peptide mass fingerprint search SWISS  PROT database results expression differences between stage I lung adenocarcinoma and matched normal lung tissue, however which protein expression differences in stage I lung adenocarcinoma how two-dimensional electrophoresis gel maps have not been reported.

    In this study, using proteomics technology, successfully established Ⅰ of lung adenocarcinoma and matched normal lung tissue protein proteome, and to filter out the presence of significantly differentially expressed protein spots 26, select one of the nine proteins point for mass spectrometric analysis, the search protein databases, and ultimately only 4 is consistent with the experimental molecular weight and isoelectric point of the protein. Other five proteins have been good peptide mass pattern map, but did not find a match with which molecular weight and isoelectric point protein in the protein database, possible reasons: the same kind of protein that may exist multiple shear contain different subunits, subjected to different chemical modifications such as phosphorylation, acetylation; well as the protein database at this stage is not perfect, need to be further enriched. This part of the protein has yet to do further identification by tandem mass spectrometry or amino acid sequence analysis methods. Four proteins: 60S ribosomal protein P2, cathepsin B1, the precursor of the apolipoprotein A  I, La 4.1 protein.

    Identification of four proteins, 60S ribosomal protein P2 is an acidic ribosomal protein. Acidic ribosomal protein is a multifunctional protein, by P0, P1 and P2. Molecule of P0 and two molecules of a heterodimer the P1/P2 composed of the total of the handle of the large ribosomal subunit structure of the active center in the ribosome, mRNA, tRNA in protein synthesis and transcription factor interactions involved in the regulation of the translation process mechanism. In addition, the acidic ribosomal proteins, there are a lot of ribosome function in vitro, as involved in DNA repair, regulation of gene expression, participate in metabolism plays a role in tumorigenesis, development [10]. The ultimate synthesis of the protein product, the concentration of each component of the P0, P1, P2, play a crucial role in certain metabolic pathways may produce a very important influence [11]. In the present study, 60S phosphorylation of ribosomal protein P2 expression in stage I lung adenocarcinoma was significantly increased, which will lead to the concentration of each component of the ribosomal P0, P1, P2 imbalance, P0, P1, P2 each ingredient concentration is relatively stable on the ultimate synthesis of the protein product plays a key role in this change is bound to affect the synthesis of certain proteins, so that normal tissue dysfunction, whether high expression of P2 lung adenocarcinoma, pending further study. In the present study, cathepsin B1 expression in stage I lung adenocarcinoma, only the lack of protein spots in normal lung tissue. Protease B precursor known as tissue or tissue cathepsin B1 Protease B original synthesized and of the zymogen in the rough endoplasmic reticulum, glycosylation, glycosylated in the Golgi apparatus is further phosphorylation, which can be lysosomal on 6  phosphate mannose receptor recognition, then endocytosis into lysosomes by itself or other protease activation. Activated cathepsin B1 that cathepsin B, which can promote the proliferation of tumor blood vessels, and enhance the ability of the tumor cell movement, degradation or activation of plasminogen activator and matrix metalloproteinase indirect degradation many extracellular matrix components, and tumor growth and invasion and metastasis [12  14]. Research reports, lung cancer, breast cancer and other tumor cathepsin B expression was significantly increased [15], the the precursor protein content is likely along with it; another enzyme expression level of transcription and post-translational level regulation , Gong et al [16] found that the protease B gene in the tumor tissue, tissue outer exons 2 and 3, as well as activate the precursor protein peptide of 7 amino acid residue deletions, formed specific mRNA product, because of its missing the signal peptide, protein synthesis can not, along the endoplasmic reticulum into the lysosome and dispersed in the cytoplasm, organizations protease B1 activation barriers to content increases. The apolipoprotein A  Ⅰ precursor Another difference of this experiment is to sieve out significant protein expression in stage I lung adenocarcinoma silence. By the apolipoprotein A-I is the major protein component of high density lipoproteins. Apolipoprotein A  Ⅰ precursor to the mature apolipoprotein A  I the conversion process is conducted outside the cell, catalyze the reaction of the enzyme present in the plasma. However, studies have shown that in vitro hepatoma cell line Hep G2, both secretion of apolipoprotein A  Ⅰ precursor, while the secretion of apolipoprotein A  Ⅰ precursor into the apolipoprotein A  Ⅰ converting enzyme , the precursor to the the mature apolipoprotein A  Ⅰ [17]. Whether Ⅰ of lung adenocarcinoma similar to the process of transformation of the apolipoprotein A  Ⅰ precursor completely transformed into mature apolipoprotein A  Ⅰ, the exact mechanism has yet to be confirmed by further studies. La4.1 protein is one of the La protein, the La protein is a nuclear protein, but also early Sjogren’s syndrome major target antigen of autoantibodies in patient serum, the protein is unclear relationship between the tumor.

    In this study, the application of proteomics methods, the initial establishment stage I lung adenocarcinoma spectra of two-dimensional electrophoresis gels, and identified four kinds occurred early expression of abnormal proteins in lung adenocarcinoma, further study of these proteins for molecular markers for early diagnosis of lung adenocarcinoma possible.