From the Research Center for Proteome Analysis, Key Lab of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; and
Eastern Hepatobiliary Surgery Hospital, Shanghai 200438, China
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ABSTRACT |
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In order to improve the diagnosis and prognosis of HCC, there is an urgent need to identify molecular markers to detect the disease. Using tissue samples from patients with HCC may be the most direct and persuasive way to find useful diagnostic and/or prognostic markers. Recently, proteomic analysis was applied to HCC tissues. Nineteen cases of HCC were analyzed by two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) by Paik et al. (46). Proteome alterations in normal, cirrhotic, and tumorous tissue were observed using 2DE-MALDI-TOF-MS assay by Jung et al. (7). Kim et al. analyzed 11 cases of HCC using 2DE and delayed extraction-MALDI-TOF-MS (8).
Nowadays, nonenzymatic sample preparation (NESP) is one of the regular techniques for tissue sample preparation and can be modified due to tissue type-specific properties (9). However, problems may be associated with heterogeneity and contaminating proteins, e.g. blood proteins. Several approaches have been developed to resolve those problems. Selection of cell types of interest by dissection has received a great deal of attention. Since 1996, a laser-assisted technique, laser capture microdissection (LCM), has emerged as a good choice. LCM under direct microscopic visualization permits rapid one-step procurement of selected cell populations from a section of complex, heterogeneous tissue (10, 11). LCM has been used to isolate specific types of cells for protein, DNA, and RNA analysis. In the age of proteomics, the proteins obtained by laser capture microdissected cells can be analyzed by 2DE (12, 13), immunoassay (14, 15), and surface-enhanced laser desorption/ionization time-of-flight (1621). The only shortcoming of LCM may be that it requires a long time to pick up sufficient cells for one experiment: 27 h for 20,00040,000 cells per immunoassay and 15 h for 250,000 cells per 2DE gel (22).
Our previous work had applied proteomic analysis to HCC cell lines (23, 24) and HCC metastatic cells (25). Furthermore, we extended our work to clinical tissues using LCM. However, at present the LCM assay only obtains about several hundred micrograms of proteins with dissection for at least several hours, which is hard to be analyzed by the traditional 2DE-MS proteomic route, especially for preparative 2DE gels followed by MS identification.
Since 1999, the isotope-coded affinity tag (ICAT) strategy has been a leading technology for relative protein quantification relying on post-harvest stable isotope labeling (26). Post-harvest labeling with stable isotopes can be used for protein quantification in cells and tissues from any organism, and the ICAT method as initially described has been shown capable of accurate quantification of proteins in complex mixtures (26). After the first generation 2H-ICAT reagents, the second generation cleavable 13C-ICAT (cICAT) reagents provided improved performance (2729). Also, the two-dimensional liquid chromatography coupled with tandem mass spectroscopy (2D-LC-MS/MS) method has been shown to be capable of identifying a large numbers of proteins, including proteins of low abundance (30, 31).
In this study, we used LCM to isolate HCC and non-HCC hepatocytes and combined LCM with cICAT labeling technology and 2D-LC-MS/MS to carry out accurate qualitative and quantitative analysis of HCC and non-HCC tissues. The flowchart used is outlined in Fig. 1. A total of 644 proteins in HCC hepatocytes were qualitatively determined, and 261 differential proteins between HCC and non-HCC hepatocytes were quantitated. To date, this is one of the largest qualitative and qualitative proteome studies for HCC and non-HCC tissues. Our strategy and method provided an accurate, fast, and sensitive approach for proteomic analysis of clinical tissues, which will facilitate the understanding of the mechanism of HCC or other diseases and mining of potential markers and drug targets for diagnosis and treatment.
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EXPERIMENTAL PROCEDURES |
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Tissue Specimen and Sample Preparation by Nonenzymatic Method (NESP)
The tissues used were obtained from a 50-year-old male patient with HCC in Edmondson grade III (HBV infected, -fetoprotein 7.3 µg/liter, size 15 x 13 x 10.5 cm). HCC tissues and their adjacent paired non-HCC tissues were isolated from freshly partial-hepatectized tissues of HCC. Tissues were washed several times with cold glutamine-free RPMI 1640 medium (glutamine-free, 5% fetal calf serum, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and antibiotics: 25 µg/ml oxacillin, 50 µg/ml gentamycin, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, 50 U/ml nistatin) and were homogenized in liquid nitrogen-cooled mortar and pestle. These obtained cells were dissolved in lysis buffer (8 mol/liter urea, 4% CHAPS, 40 mmol/liter Tris, 65 mmol/liter DTT). Samples were treated on ice for a while using an ultrasonic processor and centrifuged for 1 h at 15,000 rpm to remove DNA, RNA, and any particulate materials. The protein concentrations of samples were measured by Bio-Rad Protein Assay kit. All samples were stored at -80 °C until use.
Laser Capture Microdissection
Eight-micrometer sections of freshly prepared liver tissues were stained with toludine blue using standard manufacturers protocols with minor modifications. The sections were air-dried and microdissected with a Leica AS LMD Laser Capture Microdissection System using laser pulses of 7.5 µm diameter, 70 mW, and with 23 ms duration. Approximately 50,000100,000 cells of HCC and non-HCC hepatocytes were microdissected and stored on microdissection caps at -80 °C until lysed. Each cell population was determined to be 95% homogeneous by microscopic visualization of the captured cells. Laser capture microdissected HCC and non-HCC hepatocytes were dissolved in lysis buffer (8 mol/liter urea, 4% CHAPS, 40 mmol/liter Tris, 65 mmol/liter DTT). Samples were treated on ice for a while using an ultrasonic processor and centrifuged for 1 h at 15,000 rpm to remove DNA, RNA, and any particulate materials. The protein concentrations of samples were measured by Bio-Rad Protein Assay kit. All samples were stored at -80 °C until use.
Removal of Toludine Blue and Digestion of Protein Mixture for Qualitative Analysis
Samples prepared by NESP or LCM technology were deposited in precipitation solution (50% acetone/50% ethanol/0.1% acetic acid, sample volume:precipitation solution volume = 1:5) for at least 12 h at -20 °C. The pellets were washed by 100% acetone and 70% ethanol, then redissolved in 6 mol/liter guanidine·HCl/100 mmol/liter Tris (pH 8.3), and the concentrations were measured by Bio-Rad Protein Assay kit. Next, 200 µg of soluble proteins were reduced with DTT (final concentration 20 mmol/liter) and subsequently alkylated with IAA (final concentration 40 mmol/liter). After desalting and removal of toludine blue by ultrafiltration with Microcon-10, the protein mixture was incubated with trypsin (trypsin:protein mixture = 1:30 w/w; Promega) at 37 °C for 16 h.
cICAT Labeling of Proteins
A total of 100 µg of HCC and 100 µg of non-HCC soluble proteins prepared by LCM technology were reduced with tri-n-butylphosphate (final concentration 5 mmol/liter). Reduced HCC- and non-HCC-soluble proteins were transferred into the vial containing cICAT light or heavy reagent (Applied Biosystems, Framingham, MA), respectively, and mixed. After a brief centrifugation, proteins were incubated for 2 h at 37 °C in the dark. The labeled proteins were then combined into one tube. After desalting by ultrafiltration, the protein mixture was incubated with trypsin (trypsin:protein mixture = 1:30 w/w; Promega) at 37 °C for 16 h. Avidin cartridge (Applied Biosystems) was used to purify the ICAT-labeled peptides from the tryptic digests according to the manufacturers protocol. The elution from the avidin cartridge was dried through lyophilization. The dried cICAT-labeled peptides were dissolved in cleaving reagents and cleaved for 2 h at 37 °C. The cICAT-labeled peptides were condensed through lyophilization.
1D- and 2D-LC-MS/MS
All the two-dimensional high-performance LC separations were performed on a ProteomeX work station (Thermo Finnigan) equipped with two capillary LC pumps. The flow rate of both salt and analytical pumps was at 200 µl/min and was about 2 µl/min after split. Nine different salt concentration ranges from 0, 25, 50, 75, 100, 150, 200, 400, and 800 mM ammonium chloride were used for step gradient. The mobile phases used for reverse phase were: A, 0.1% formic acid in water, pH 3.0; B, 0.1% formic acid in acetonitrile. The one-dimensional high-performance LC separation is performed using same system but without a strong cation exchange column.
An electrospray ionization ion trap mass spectrometer (LCQ Deca XP; Thermo Finnigan) was used for peptide detection. The positive ion mode was employed and the spray voltage was set at 3.2 kV. The spray temperature was set at 150 °C for peptides. Collision energy is automatically set by LCQ Deca XP. After acquisition of a full-scan mass spectra, three MS/MS scans were acquired for the next three most intense ions using dynamic exclusion.
Peptides and proteins were identified using TurboSequest software (Thermo Finnigan), which uses the MS and MS/MS spectra of peptide ions to search against the publicly available NCBI nonredundant protein database (May 16, 2003; www.ncbi.nlm.nih.gov). The protein identification criteria that we used were based on Delta CN (0.1) and Xcorr (one charge
1.8, two charges
2.2, three charges
3.7).
Bioinformatics Analysis
The pI and Mr of the proteins were analyzed using ExPASy proteomics tools accessed from cn.expasy.org/tools/#proteome. The general average hydropathicity or GRAVY score is calculated as the arithmetic mean of the sum of the hydropathic indices of each amino acid (32). We conducted the transmembrane prediction using the program TMHMM 2.0, which can be accessed from the Center for Biological Sequence Analysis (www.cbs.dtu.dk/services/TMHMM/). All identified proteins were classified by their molecular function, cellular component, and biological process with the tools on www.geneontology.org.
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RESULTS |
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Comparison of 1D-LC-MS/MS and 2D-LC-MS/MS for Qualitative Analysis
To compare 1D-LC-MS/MS and 2D-LC-MS/MS for qualitative analysis, 200 µg of solubilized proteins from HCC tissues were reduced, alkylated, and digested with trypsin, and the resulting peptides were separated on a capillary C-18 column and then detected by ion trap mass spectrometry (1D-LC-MS/MS). A total of 208 proteins were unambiguously identified in a single analysis. Another 200 µg of HCC-solubilized proteins were analyzed by two-dimensional chromatography separation prior to MS/MS (strong cation exchange chromatography and reversed-phase chromatography) and then detected by ion trap mass spectrometry (2D-LC-MS/MS). A total of 626 solubilized proteins were unambiguously identified. It was clear that 2D-LC-MS/MS could achieve better qualitative analysis than 1D-LC-MS/MS. Thus, we combined 2D-LC-MS/MS with the LCM method in order to perform large-scale qualitative analysis. 2D-LC-MS/MS analyzed 200 of µg HCC-solubilized proteins prepared by LCM, and a total of 644 solubilized proteins were unambiguously identified. The data from all three tests are summarized in Table II. By bioinformatic analysis, 25 (12.0%), 64 (10.2%), and 80 (12.4%) hydrophobic proteins were found in HCC-NESP-1D-LC-MS/MS, HCC-NESP-2D-LC-MS/MS, and HCC-LCM-2D-LC-MS/MS, respectively. We conducted the transmembrane mapping prediction using the program TMHMM 2.0 and found 8 (3.9%), 30 (4.8%), and 54 (8.4%) transmembrane proteins in HCC-NESP-1D-LC-MS/MS, HCC-NESP-2D-LC-MS/MS, and HCC-LCM-2D-LC-MS/MS, respectively. The pI and Mr of the proteins were analyzed using ExPASy proteomics tools. There were 19 (9.1%), 77 (12.3%), and 75 (11.6%) proteins with Mr beyond routine 2DE (Mr <10 kDa, Mr >100 kDa) and 21 (10.1%), 78 (12.5%), and 126 (19.6%) with pI beyond traditional 2DE (pI > 9) in HCC-NESP-1D-LC-MS/MS, HCC-NESP-2D-LC-MS/MS, and HCC-LCM-2D-LC-MS/MS, respectively.
Quantitative Analysis with cICAT Technology and 2D-LC-MS/MS
Furthermore, we combined LCM technology with 2D-LC-MS/MS coupled with cICAT technology in order to perform large-scale and accurate quantitative analysis. One hundred micrograms of HCC- and 100 µg of non-HCC-solubilized proteins prepared by LCM technology were reduced, labeled with cICAT light or heavy reagent, respectively, and then combined and digested with trypsin. The affinity-purified ICAT-labeled peptides were cleaved with cleaving reagents and then analyzed by two-dimensional chromatography MS/MS.
After database searching and quantitation by Xpress followed by manual check, a total of 261 proteins had quantitative information, and 149 (57.1%) of all identified proteins displayed more than 2-fold expression differences. Among these 149 proteins, 94 proteins were found to be down-regulated in HCC hepatocytes, while 55 proteins were found to be up-regulated in HCC hepatocytes (Table III).
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DISCUSSION |
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Both 1D-LC-MS/MS and 2D-LC-MS/MS can rapidly analyze mixtures and identify a large numbers of proteins. In this study, we compared the capabilities of 1D-LC-MS/MS and 2D-LC-MS/MS for qualitative analysis. As the data shown, the number of proteins found with 1D-LC-MS/MS (HCC-NESP-1D-LC-MS/MS, 208 proteins) is about one-third (33%) of the number of proteins found with 2D-LC-MS/MS (HCC-NESP-2D-LC-MS/MS, 626 proteins). Therefore, 2D-LC-MS/MS could achieve better qualitative analysis than 1D-LC-MS/MS. Thus, we combined the LCM method with 2D-LC-MS/MS alone or coupled with ICAT technology in order to perform large-scale qualitative and quantitative analyses. A total of 644 proteins in HCC hepatocytes were qualitatively determined and 261 differential proteins between HCC and non-HCC hepatocytes were quantitated. To date, this is one of the largest qualitative and qualitative proteome studies for HCC and non-HCC tissues.
To our knowledge, compared with previous work (2629), our work is the first report that applies the ICAT method to clinical tissues. Schmidt et al. reported the complementary analysis of 2DE and ICAT (34). Compared with 2DE, which could only offer a qualitative and quantitative relationship based on individual spots and frequently the same protein was detected in different spots or different spots contained more than one protein (34), our LCM-ICAT-2D-LC-MS/MS assay provides alteration of overall expression of a protein instead of individual spots resolved by 2DE. Moreover, protein qualitative and quantitative analysis in 2DE mostly depends on protein recovery during isoelectric focusing and SDS-PAGE processes. Especially for tissue samples, it can be difficult to achieve good recovery of basic proteins, membrane proteins, or proteins with large molecular mass through isoelectric focusing. In contrast, 2DE is more powerful in resolution of protein isoforms resulting from post-translational modifications. Thus, LCM-ICAT-2D-LC-MS/MS and traditional 2DE-MS methods have certain complements and the combination of two assays may offer more overall understanding of HCC or other biological systems studied.
Our previous work reported several differentially expressed proteins based on HCC cell lines (23, 24) and HCC metastatic cells (25) with the 2DE-MS strategy. It is difficult to obtain large-scale differentially expressed proteins through 2DE based on cell lines, similar to LCM-ICAT-2D-LC-MS/MS based on tissues in this work, resulting in few overlaps of the differential expressed proteins. Recently, Kim et al. reported that fructose-bisphosphate aldolase B (P05062), argininosuccinate synthetase (NP_446464), and cathepsin B preproprotein (NP_001899, APP secretase; cathepsin B1) have been down-regulated in HCC tissues (35). Our data further showed down-regulation of these three proteins in LCM-purified HCC hepatocytes. Two members of the chlordecone reductase family, aldo-keto reductase family 1 members C2 (NP_001345) and C3 (NP_003730), are up-regulated in HCC tissue based on our data, while genes of the chlordecone reductase family have been found up-regulated (36) at the transcriptomic level. Proteomic and transcriptomic results indicate that the up-regulated expression of the chlordecone reductase family in HCC may start at the transcript level.
We found four kinases in differentially expressed proteins between HCC and non-HCC hepatocytes, two up-regulated proteins (XP_222218.1, similar to STE20-like kinase; AAD03058.1, Eph-family protein) and two down-regulated proteins (AAC33435.1, mitotic checkpoint protein kinase Bub1A; AAD02030.1, Eph-like receptor tyrosine kinase hEphB1). We noticed that the Eph family of receptor tyrosine kinases and their cell-presented ligands, the ephrins, play diverse roles in carcinogenesis (37). Moreover, one of down-regulated proteins in HCC cells is Rho guanine nucleotide exchange factor 7 isoform b (NP_663788.1), which belongs to a family of cytoplasmic proteins that activate the Ras-like family of Rho proteins by exchanging bound GDP for GTP and may form a complex with G proteins and stimulate Rho-dependent signals.
DEAD (Asp-Glu-Ala-Asp) box polypeptide 1 (DDX-1, NP_004930.1) is one of down-regulated proteins in HCC hepatocytes. Interestingly, DDX1 can interact with heterogeneous nuclear ribonucleoprotein K (hnRNP K), and their interaction is disrupted by the addition of poly (C), poly (A), and poly (U) RNA substrates (38). Another member of the DEAD protein family, HLA-B-associated transcript 1 (NP_004631.1), is also down-regulated in HCC hepatocytes. This protein is a negative regulator of inflammation and a translation initiation factor, whose gene has multiple alternatively spliced transcript variants and has been found to have multiple polyadenylation sites. More interestingly, three members of splicesome (NP_006793.1, splicing factor 3a, subunit 3, 60 kDa; NP_003085.1, small nuclear ribonucleoprotein polypeptide E; NP_079555.1, heterogeneous nuclear ribonucleoprotein K) were found in up-regulated proteins in HCC hepatocytes. In the LCM-ICAT-2D-LC-MS/MS assay, hnRNP K had the same up-regulated trend in HCC hepatocytes. Moreover, up-regulation of hnRNP K and a nuclear-translocated phenomenon of hnRNP K in tumor areas are found with immunohistochemistry assay.2 Recently, Ostrowski and Bomsztyk demonstrated that in several states of enhanced cell proliferation, there are increased hnRNP K protein levels in the nucleus (39). It is likely that the increased hnRNP K protein levels seen in the nuclei of the proliferating cells serve to support nuclear process that not only composes inducible expression of a very large number of genes but also maintains conducive chromatin topology in growing cells (39). Therefore, hnRNP K may be an important tumor-related protein, and the significance and mechanism of the DEAD protein family and splicesome members in HCC need further investigation.
Interesting, we found 16 unknown or hypothetical up-regulated proteins and 14 unknown or hypothetical down-regulated proteins in HCC hepatocytes (Table III). These maybe indicated that the LCM-ICAT-2D-LC-MS/MS method could obtain lower-abundant proteins than routine two-dimensional gel methods and extend our abilities to get information about complicated HCC-involved multi-aspects and multi-genes. Roles of the unknown or hypothetical proteins we found in HCC will be the subjects of future studies.
In this report, LCM coupled with 2D-LC-MS/MS and cICAT labeling technology can achieve accurate qualitative and quantitative clinical proteomic analysis. The LCM technology can significantly purify specific cells, and the 2D-LC-MS/MS coupled with cICAT quantification provide rapid and accurate qualitative and quantitative information on the proteomes of HCC and non-HCC tissues. In contrast, ICAT methods and traditional 2DE methods are complimentary in protein identification and quantification. In summary, this technical advance may contribute to proteomic research on HCC, including looking for tumor-interrelated proteins and finding potential biomarkers and drug targets. This method also provides accurate qualitative and quantitative analysis for proteomes in a variety of types of clinical tissue samples. Further studies will focus on increasing the capability of identification of low-abundant proteins using prefractionation by ion exchange chromatography or by SDS-PAGE (27) after ICAT labeling. Moreover, the function of the differential proteins will be trailed in more clinical tissues to ascertain their roles in the diagnosis of HCC.
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FOOTNOTES |
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Published, MCP Papers in Press, January 15, 2004, DOI 10.1074/mcp.M300133-MCP200
1 The abbreviations used are: HCC, hepatocellular carcinoma; LCM, laser capture microdissection; ICAT, isotope-coded affinity tag; cICAT, cleavable ICAT; 1D-LC-MS/MS, one-dimensional liquid chromatography coupled with tandem mass spectrometry; 2D-LC-MS/MS, two-dimensional liquid chromatography coupled with tandem mass spectrometry; NESP, nonenzymatic sample preparation; 2DE, two-dimensional electrophoresis; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; DTT, dithiothreitol; IAA, iodoacetamide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; GO, Gene Ontology.
2 C. Li et al., manuscript in preparation.
* This work was supported by National High-Technology Project (2001AA233031, 2002BA711A11) and Basic Research Foundation (2001CB210501). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Hong-Yang Wang, Eastern Hepatobiliary Surgery Hospital, No. 225, Changhai Road, Shanghai 200438, China. Tel.: 86-21-25070856; Fax: 86-21-65566851; E-mail: hywangk{at}online.sh.cn; Rong Zeng, Research Center for Proteome Analysis, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 YueYang Road, Shanghai 200031, China. Tel.: 86-21-54920170; Fax: 86-21-54920171; E-mail: zr{at}sibs.ac.cn
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REFERENCES |
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