Nano-flow multidimensional liquid chromatography with electrospray ionization time-of-flight mass spectrometry for proteome analysis of hepatocellular carcinoma

Analytica Chimica Acta 530 (2005) 227–235

Nano-flow multidimensional liquid chromatography with electrospray ionization time-of-flight mass spectrometry for proteome analysis of hepatocellular carcinoma Yan Wang, Jie Zhang, Chun-Li Liu, Xue Gu, Xiang-Min Zhang∗ Department of Chemistry, Fudan University, Shanghai 200433, China Research Center of Proteome, Fudan University, Shanghai 200032, China Received 1 July 2004; received in revised form 10 September 2004; accepted 10 September 2004 Available online 8 December 2004

Abstract Hepatocellular carcinoma (HCC) is one of the top five cancers with the highest incident of a disease worldwide. To understand the mechanisms of hepatocarcinogenesis, proteomics analysis provides a powerful tool to identify proteins that associate with HCC. We developed a two-step procedure for mapping of HCC proteomics. In the first step, in order to simplify the complexity of proteomics of HCC, the subfractionation of complex protein mixtures in HCC into “subproteomes” is presented based on the solubility of protein. While in the second step an automate comprehensive two-dimensional (2D) separation system, coupling strong cation-exchange (SCX) in the first dimension with capillary reversed-phase chromatography (cRPLC) in the second dimension is developed further to separate and analyze proteins associated with HCC. By using this system, complex sample can be injected, desalted, separated and analyzed in complete automatization. The procedure for proteomics analysis was found to be applied for proteins with great molecular mass (>100 000), small molecular mass (<20 000), highly basic (pI > 9.5) and hydrophobicity, which are not well resolved in 2D-gel electrophoresis. In total 229 proteins were identified by using the described proteomics platform. Among them, several proteins related to the process of carcinogenesis were investigated further. © 2004 Elsevier B.V. All rights reserved. Keywords: Human hepatocellular carcinoma; Two-dimensional liquid chromatography; Electrospray ionization mass spectrometer; Proteomics; Nano-flow

1. Introduction Proteomics, the systematic study of the proteins expressed in cell or tissue, is now the focus of many fields of scientists [1–3]. Two-dimensional gel electrophoresis (2D-PAGE) [4] followed by protein identifications using mass spectrometry (MS) is the most widely used tool in proteomics studies [5,6]. However, the limitations in sensitivity, sample loading capacity, and amenability to automation restrict its use as a comprehensive proteome analysis platform. Column-based multidimensional chromatography is the most prospective approach to achieve high resolution as high as 2DE [7,8]. Giddings

∗

Corresponding author. Tel.: +86 21 6564 3983; fax: +86 21 6564 1740. E-mail address: [email protected] (X.-M. Zhang).

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.09.036

demonstrated that the overall peak capacity of multidimensional separations is the product of the peak capacities in each independent dimension provided that the separation mechanisms of each are orthogonal [9]. In addition to providing far greater resolving power, multidimensional chromatography approaches also offer versatility, automation, sensitivity, reproducibility and high throughput [10,11]. Jorgenson et al. have developed a number of coupled column separation schemes for performing comprehensive multidimensional analysis [12–14]. They differ in the separation modes and column sizes applied in the first dimension and the use of sample loops or on-column focusing for the transfer to the second dimension. Yates et al. describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT) [15,16]. MudPIT integrates a strong cation-exchange (SCX) resin and reversed-phase resin

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in a biphasic column and has been successfully applied for the analysis of protein expression profiles. To further identify proteins, the peptides eluted from multidimensional chromatography can be introduced into MS directly. At the present time, ESI source constructed for operation at flow rate in the nL/min (low flow ESI, also named nano-electrospray) attract particular attention for analyze low quantity of sample. So on-line coupling LC to MS has driven the development of nano-flow capillary column LC. When the chromatography is down to nano-scale, the chromatographic sensitivity and relative peak intensities that nanoflow capillary HPLC provides increases. Enhanced sensitivity or relative peak intensities also makes it possible to detect low abundance components by resolving them from high abundance components. In addition, by decreasing the mobile phase flow rate of the LC column, available for the acquisition of MS/MS spectra from the peptides increases by a corresponding amount, leading to improved proteome coverage. HCC is one of the leading causes of cancer deaths worldwide. Each year, 100 000 cancer deaths caused by HCC in China, account for 45% of all HCC cancer deaths worldwide. To attain a complete understanding of HCC, the proteomics analysis provides an important approach to study the proteins associate with HCC. Recently, proteomic characterization is also being applied to human HCC cell lines and tissues by 2D-gel and LC–ion trap MS [17–20]. Whole cell extracts of HCC contain a diverse set of protein, which vary from hydrophilic to hydrophobic (including integral membrane proteins), from highly acidic to neutral to highly basic, and from high abundance to low abundance. So in this paper, in order to simplify the complex of protein sample, the subfractionation was first carried out to classify the complex protein mixtures from HCC tissues by sequential extraction based on their solubility. Secondly, a nano-flow 2D-LC–ESI–MS–MS for the proteome analysis of HCC was established. This approach couples the principal advantages of nanaoscale LC with the advantages of nano-spray MS/MS, and provides an automation, high sensitivity, high throughput proteome analysis platform. By using these analysis procedures, we identified 229 proteins in HCC tissues, in which some proteins related to HCC.

2. Experimental 2.1. Chemicals Acteonitrile (ACN) was HPLC grade from Fisher Scientific (Fairlawn, NJ). The water used was MilliQ grade (Millipore, Bedford, MA, USA). Formic acid (FA) was HPLC grade obtained form Fluka. Urea, thiol urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), phynylmethylsulfonyl fluoride (PMSF), sequencing grade trypsin, dithiothreitol (DTT) were obtained from Sigma (St. Louis, MO). All chemicals used in making buffer solutions were analytical grade reagents.

2.2. Sample preparation The liver cancer tissue of D20, which is a metastatic model of human hepatocellular carcinoma in nude mice with high metastatic potential, was obtained from Liver Cancer Institute in Zhongshan Hospital, Fudan University. The liver tissue was diced and washed with cold physiological saline solution (0.9% NaCl solution) to remove blood and other possible contaminants. The sequential extraction of proteins from the liver tissue was carried out to prefractionate proteins on the basis of their relative solubility. First, the sample was homogenized in water contained 1 mM PMSF using glass homogenization vessel in ice bath. After 20 min swirl, the homogenate was centrifuged at 15000 × g for 10 min. The supernatant labeled as extract A, was divided into aliquot and stored at −80 ◦ C freezer. The insoluble pellet was resuspended in lysis buffer (7 M urea, 2 M thiol urea, 2% (w/v) CHAPS, 50 mM DTT, 2% (w/v) SB3-10 and 1 mM PMSF). Then, the lysate was centrifuged, and the supernatant was labeled as extract B. Protein concentration of sample was measured by the Bradford assay using bovine serum albumin (BSA) as standard [21]. The extract A was digested with trypsin (protein/trypsin 35:1, w/w) overnight at 37 ◦ C. The extract B was diluted to 2 M urea containing 50 mM ammonium hydrogencarbonate, then follow the same trypsin digest as extract A. 2.3. Comprehensive nano-SCX–RPLC–MS/MS system Peptide separations were performed with an UltiMateTM nano-scale LC system combined with a FAMOS microautosampler and a SWITCHOS valves from LC Packings (Amsterdam, The Netherlands). A FAMOS micro-autosampler with additional built-in sixport valves was used for sample injection. Elution of strong cation-exchange chromatography (SCX), sample preconcentration and desalting on the trapcolumn was performed with an auxiliary LC quaternary pump building in SWITCHOS that was operated isocratically at a flow rate of 15 ␮L/min. In the first dimension of 2D LC, 20 ␮L trypsin digested extract A or extract B was injected to SCX column (Bio-SCX, 300 ␮m i.d., 150 mm, LC packings) equilibrated with buffer consisted of 2% acetonitrile (v/v) and 0.1% formic acid (v/v). The peptides were eluted with a step gradient of ammonium acetate (0, 10, 50, 75, 100, 150, 200, 300, 400, 500, and 1000 mM) and captured on a cartridge type trapcolumn (C18 PepMapTM , 300 ␮m i.d., 1 mm, LC Packings) for preconcentration and desalt. Nanoscale RPLC column (C18 PepMapTM , 75 ␮m i.d., 15 cm, LC packings) was used for second dimension LC. Flow rate was 200 nL/min. Mobile phase A consisted of 0.1% formic acid in acetonitrile/water (2:98, v/v) and mobile phase B of 0.1% formic acid in acetonitrile/water (98:2, v/v). Following a 20 min isocratic elution with 5%B the column was developed using gradient conditions as follows: 5% B linearly increased to 50% B in 40 min, then increased up to 95% B in 10 min, further maintained at 95% B for 10 min

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for washing the column, and then ramp down to 5% B for equilibrium. The nano-RP column was linked directly to a nano-LC electrospray device. This device holds a PicoTipTM EMITTER Silica TipTM needle (FS360-75-15-D-5, New Objective, Inc., Woburn, MA, USA), which is a nano-electrospray needle with a 15 ␮m tip.

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selected as protein cleavage specificity. Both b-ions and y-ions were included in the database search. Carboxymethyl (C) was selected as a variable modification and no static modification was selected. The taxonomy was homo sapiens. Peptide tolerance is ±0.8 Da, and MS/MS is ±0.5 Da.

3. Results and discussion 2.4. Mass spectrometer 2D-LC–MS/MS analysis was performed on a QSTARXL ESI MS equipped with an orthogonal time-of-flight (TOF) mass analyzer (Applied Biosystems, USA). The instrument was operated in positive ion mode. The instrument was set to perform an MS survey scan of 1 s with m/z range of 400–2000. Information dependent acquisition (IDA) will be generated using the survey experiments conditions as a template. The collision gas was argon and the collision energy was kept at 51.2 V. Any peak with a threshold of 20 counts/s was automatically detected, and the top three precursors from the each MS survey scan were selected by the quadrupole for fragmentation. The ions with two to four charges were selected for MS/MS analysis. A product ion was scanned for 2 s. The instrument was calibrated prior to analysis using horse myoglobin. 2.5. Data processing and database searching Peak list files were searched against a non-redundant protein database SWISS-PROT using the MASCOT search engine (http://www.matrixscience.com/) [22]. Trypsin was

3.1. Development of the 2D-LC mode Analysis of the extracts from the liver tissue of D20 was accomplished by on-line 2D-LC–ESI–MS–MS analysis. The chromatographic system developed was based upon separation of peptides in two dimensions LC. To achieve maximal orthogonality the first dimension was based on electrostatic interactions while the second was based on hydrophobic interactions. The complete valve set up of the 2D-LC is shown in Fig. 1. The valve switching process consisted of the following sequential steps. First, the sample was loaded into a loop by a syringe pump, illustrated as Fig. 1a, loading mode. The SCX column was connected to the trapcolumn. After loading into the sample loop, the sample was pumped into SCX column by the auxiliary pump at 15 ␮L/min. The peptides, which do not bind to the SCX column, were trapped on the top of the trapcolumn. The loading solvent was an aqueous 2% acetonitrile/0.1% formic acid solution. Loading time used was 10 min. After 10 min, the valve B was switched to clean-up and desalting mode, shown as Fig. 1b. An aqueous 2% acetonitrile/0.1% formic acid solution delivered at 15 ␮L/min

Fig. 1. Flow diagram of system valves of nano-2D-LC.

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by the auxiliary pump to clean the trapcolumn and desalting. The desalting time used was 10 min also. During these two modes, the RP nano-scale analytical column was equilibrated with buffer A, an aqueous 2% acetonitrile/0.1% formic acid solution at 200 nL/min by nano-pump. After clean-up and desalting, the valve A was switched and the trapcolumn placed in-line with the RPLC nano-scale analytical column, shown as Fig. 1c. The bound peptides were blackflushed from the trapcolumn and separated on RPLC nano-scale column with gradient elution. The flow rate through the analytical column during this stage of the separation process was maintained at 200 nL/min. The column effluent was prayed into the nano-electrospray ionization MS–MS system. After the first part of the analysis was completed, the valve B was switched back and the trapcolumn was connected with the SCX column again. At the same time, the valve A was switched back to equilibrate RP nano-scale column with buffer A. Peptides, which retained on the SCX column, were separated with a step-elution by 20 ␮L ammonium acetate in different concentrations, respectively. The effluent from each step was loaded onto the trapcolumn to desalting. The analysis was followed by RPLC and ESI–MS–MS. The procedure was repeated until all peptide fragments were eluted and analyzed. 3.2. Choice and evaluation of the nano-2D-LC–MS/MS Although both anion and cation-exchange HPLC were viable options for protein ion-exchange separations, cation-exchange HPLC was selected for the first dimension primarily on the basis of buffer pH compatibility with the subsequent reversed-phase dimension, and the use of positive ion mode mass detection. In addition, it should be sufficient to use for the most biological samples. Since the norm linear gradient might cause the fraction splitting, the step gradient was applied for peptide analysis in SCX [23]. The tip of nano-electrospray needle is only 15 ␮m, nonvolatile components (such as urea and buffer salts) would lead to physical blockage of the nano-spray source, causing significant suppression of analyte ionization. So desalting is indispensable before the trapcolumn was connected with the analytical RP column. In this study, we used buffer A to wash the trapcolumn for 10 min at flow rate of 15 ␮L/min to remove the salt before subjected to MS system. Reversed-phase HPLC is a favorable technique for resolving proteins and peptides. However, for the traditional analytical bore RP columns of 4.6 mm internal diameter and flow rate of 1 mL/min, the concentration sensitivity of electrospray ionization mass spectrometry led to a poor. By using the smaller inner diameter chromatography columns, the limit of detection can be significantly improved. The increase in concentration sensitivity is proportional to the inverse square of the column diameter [24]; thus, a 75 ␮m i.d. capillary column should offer a 4-fold improvement in sensitivity over the 150 ␮m columns used, assuming the same amount of sample is injected. Dilution factor is much less in capillary RPLC.

Therefore, using nano-flow capillary RPLC prior to ESI, it would obtain high peak concentration for the low levels (ca. 100 pmol) of digested peptides. In addition, a discrete fraction of peptides is displaced from the SCX column directly onto a RP trapcolumn, which concentrated at the head of the trapcolumn to avoid for the band broading. Trifluoroacetic acid (TFA) is commonly used as an RPLC mobile phase modifier in peptide and protein separations [25], which provided good peak shape and recovery, and high volatility to facilitate easy removal. However, recent studies found TFA could result in spray instability and ionization suppression, while formic acid would significantly increase MS sensitivity for peptide and protein analyses [26,27]. So formic acid was selected as mobile phase modifier in our study. The choice of ESI–TOF MS as a detector for the on-line 2D separations was made to provide an additional effective dimension of separation (i.e. ions in m/z space). In addition, an unambiguous structural elucidation of a certain peptide would be obtained from MS/MS. The time of MS scan was set 1 s in quadrupole part, and the time required for MS/MS experiments was set to 2 s. The average peak width of RPLC is about 10–30 s, so each peak might be scanned about 3–6 times. This is sufficient to acquire enough data points for adequate resolution. The top three precursor were selected by the quadrupole from each MS survey scan for producing product ion. Former target ions were excluded for 30 s. Quadrupole resolution is low (4 amu) to ensure the isotope ions of all precursors and products pass through quadrupole. 3.3. 2D-LC–MS/MS analysis of tryptic peptides of D20 Tryptic peptides of D20 were eluted from SCX column by ammonium acetate solution with different concentration from 0 to 1000 mM. Fig. 2 shows the total ion currents (TIC) of a representative 2D-LC experiment of extract A at the concentration of 100 mM ammonium acetate. The chromatogram shows much strong signals, with the highest intensity of 6.1 × 105 . The average base peak width for resolved peaks in this separation is ∼20 s. Analysis of the SCX fraction, and 11 subsequent salt step fractions from the first dimension in the 2D system yielded a peak capacity ∼1800. Peptides eluted from the nano-HPLC were detected by ESI–MS with a full scan mode. Fig. 3 shows the representative mass spectrum corresponding to peaks 1–4 marked in Fig. 2. Since the limitation in RPLC separation, one peak in TIC may contain several co-eluted peptides. In other words, mass spectrometry provides a third dimension separation to the system (m/z space). The fragment peptide spectra can be examined for sequence information. As an illustration, Fig. 4 shows the MS/MS spectrum of the peak 3 in Fig. 2. This is the doubly charged precursor ion. Its sequence was confirmed from the labeled b- and y-ions in the spectrum. The sequence is PGGLLLGDVAPNFEANTTVGR, and ion score is 106. Fragments observed in the spectrum are bolded and underlined in

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Fig. 2. Full-scan total ion current (TIC) profile of tryptic digest of extraction A at the concentration of 100 mM ammonium acetate.

seen for ions which have lost ammonia (−17 Da) denoted b* and y* and water (−18 Da) denoted b0 and y0 . The MS/MS data were searched against SWISSPROT database by MASCOT search engine. Total ion scores,

Table 1. In low energy CID, 51.2 V applied for this experiment, generate predominantly b and y ions. In Fig. 4, we see that the peaks do not account for is very little. At the same time y series have very good coverage. In addition, peaks are

Table 1 Fragments of sequence PGGLLLGDVAPNFEANTTVGR observed in Fig. 4 which are bolded and underlined #

b

b++

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

98.06 155.08 212.10 325.19 438.27 551.26 608.38 723.40 822.47 893.51 990.56 1104.61 1251.67 1380.72 1451.75 1565.80 1666.84 1767.89 1866.96 1923.98

49.53 78.04 106.56 163.10 219.64 276.18 304.69 362.21 411.74 447.26 495.79 552.81 626.34 690.86 726.38 783.40 833.93 884.45 933.98 962.49

b*

1087.58 1234.65 1363.69 1434.73 1548.77 1649.82 1750.87 1849.93 1906.96

b*++

544.29 617.83 682.35 717.87 774.89 825.41 875.94 925.47 953.98

b0

705.39 804.46 875.50 972.55 1086.59 1233.66 1362.71 1433.74 1547.79 1648.83 1749.88 1848.95 1905.97

b0++

353.20 402.73 438.25 486.78 543.80 617.34 681.86 717.38 774.40 824.92 875.44 924.98 953.49

Seq.

y

y++

y*

y*++

y0

y0++

P G G L L L G D V A P N F E A N T T V G R

2001.04 1944.02 1887.00 1773.91 1660.83 1547.75 1490.72 1375.70 1276.63 1205.59 1108.54 944.50 847.43 718.38 647.35 533.30 432.26 331.21 232.14 175.12

1001.02 972.51 944.00 887.46 830.92 774.38 745.87 688.35 638.82 603.30 554.77 497.75 424.22 359.70 324.18 267.16 216.63 166.11 116.57 88.06

1984.01 1926.99 1869.97 1756.89 1643.80 1530.72 1473.70 1358.67 1259.60 1188.56 1091.51 977.47 830.40 701.36 630.32 516.28 415.23 314.18 215.11 158.09

992.51 964.00 935.49 878.95 822.41 765.86 737.35 679.84 630.30 594.79 546.26 489.24 415.70 351.18 315.66 258.64 208.12 157.60 108.06 79.55

1983.03 1926.01 1868.99 1755.90 1642.82 1529.73 1472.71 1357.69 1258.62 1187.58 1090.53 976.49 829.42 700.37 629.34 515.29 414.25

992.02 963.51. 935.00 878.46 821.91 765.37 736.86 679.35 629.81 594.29 545.77 488.75 415.21 350.69 315.17 258.15 207.63

# 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

b++ = (b + H)/2; b* = b − NH3 ; b*++ = (b − NH3 + H)/2; b0 = b − H2 O; b0++ = (b − H2 O + H)/2; y++ = (y + H)/2; y* = y − NH3 ; y*++ = (y − NH3 + H)/2; y0 = y − H2 O; y0++ = (y − H2 O + H)/2.

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Fig. 3. The representative mass spectrum corresponding to the peaks 1–4 marked in Fig. 2: (a) MS of peak 1; (b) MS of peak 2; (c) MS of peak 3; (d) MS of peak 4.

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Fig. 4. MS/MS spectrum for sequence PGGLLLGDVAPNFEANTTVGR.

protein coverage (was calculated by amino acid count), and the number of peptides matched from MS fragmentation data indicate the identity of the detected molecules. In total, 150 proteins were identified from extract A using the multidimensional platform, 113 were identified in extract B. Dynamic range and protein solubility issues complicate the detection and separation of low-abundance proteins. Sequential extraction has been shown to be a useful technique for resolving an increased proportion of a functional proteome [28]. In our study, the complex protein mixture was classified based on the solubility of protein, the hydrophilic protein might be extracted into the extract A, and the hydrophobic protein might be extracted into the extract B. Some proteins however were present in two extracts. For example, 78 kDa glucose-regulated protein precursor (P11021) was present in the two extracts. Total score of it from extract A

is 190, the peptide matched is 21, while the total score from extract B is 139, the peptide matched is 11. Some proteins were present only in one extract. 67 KD Laminin receptor (P08865), a protein bearing the membrane protein property and having a high binding affinity with laminin in HCC compared to normal hepatic cells [29], was only present in extract B. By using the prefractionation method, the complexity of the sample was simplified, so that more proteins would be identified. In the final reduction the redundant proteins identified presented in extract A and B, 229 proteins were identified. In Table 2, we summarize the proteins with the top 20 scores for all experiments. Among the identified protein, the largest isolated protein is Fibrillin 2 precursor (P35556), with an Mr of 314 131. The smallest is the protein Ubiquitin, with an Mr of 8560

Table 2 Top 20 score proteins identified in the LCI-D20 proteome sample Accession number

Protein name

Mr

pI

Total ion score

No. of peptides matched

Protein coverage

P14618 P00938 P23141 P07195 P06733 O43707 P52209 P04687 P15531 P11021 P07437 P04083 P55072 P07900 P00352 Q15084 P30041 P35579 P02546 P42655

Pyruvate kinase Triosephosphate isomerase Liver carboxylesterase precursor l-Lactate dehydrogenase B chain Alpha enolase Alpha-actinin 4 6-Phosphogluconate dehydrogenase, decarboxylating Tubulin alpha-1 chain Nucleoside diphosphate kinase A 78 kDa glucose-regulated protein precursor Tubulin beta-1 chain Annexin A1 (annexin I) Transitional endoplasmic reticulum ATPase Heat shock protein HSP 90-alpha Aldehyde dehydrogenase 1A1 Probable protein disulfide isomerase P5 precursor Peroxiredoxin 6 Myosin heavy chain, nonmuscle type A Lamin C 14-3-3 protein epsilon

57769 26522 62481 36484 47008 104788 52975 50126 17138 72288 49727 38559 89266 84490 54696 48091 24888 226461 65096 29155

7.95 6.51 6.06 5.72 6.99 5.27 6.88 4.94 5.83 5.01 4.75 6.64 5.14 4.94 6.29 4.95 6.02 5.50 6.57 4.63

426 336 271 265 265 236 216 208 197 190 186 173 165 164 163 161 160 159 154 148

49 29 30 61 26 13 24 37 24 21 14 8 24 24 20 7 15 33 23 22

36.9 44.8 22.0 32.0 18.0 6.2 14.9 21.1 25.0 17.1 6.3 13.9 14.2 14.5 26.3 15.2 18.3 9.5 18.0 19.2

Shown are SWISS-PROT accession number, total ions scores, protein coverage (was calculated by amino acid count), total number of matched peptides, isoelectric point (pI), and relative molecular mass (Mr ) of the respective proteins.

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(P02248). Among the identified protein, 74% has the mass between 20 000 and 100 000 Da, 15% has the mass below 20 000 Da, and 11% has the mass greater than 100 000 Da. Protein with a molecular mass of less than approx. 20 000 are generally not well resolved in 2D-PAGE. Under conditions where smaller protein can be observed, the separation of large protein with a molecular mass of larger than 100 000 is still also a problem in 2D-PAGE. The pI range of the identified protein is between 4.09 and 11.14. Among them, 8% were highly basic proteins with pI greater than 9. As usual, these highly basic proteins could not be well mapped by using 2D-gel separation methods. Using nano-2D-LC–ESI–MS–MS platform as described in this paper, we have been able to improve the separation of proteins in a total cell extract. High molecular weight, low molecular weight and high basic proteins are well separated, even if the separation conditions are the same. Remarkably, these proteins account for the 34% of the all identified protein. If these proteins cannot be well resolved and identified by using 2D-PAGE, which would lead to a potential loss of important biological information. Nano-2D-LC–ESI–MS–MS circumvents these drawbacks of the 2D-PAGE, and provides an unbiased platform for proteomics. 3.4. Cancer-related proteins Since the biological processes are directly executed by proteins, the state of an organism is essentially reflected in its proteome rather than genome, which is much more stable. Finding functional proteome is the main aim for mapping global expression patterns of proteins. The analysis indicates that some proteins are related to cancer. These proteins are annexin-I (P04083), Vimentin (P08670), Cytokeratins CK8 (P05787) and CK18 (P05783), pyruvate kinase M2 (P14786), carbonyl reductase (P16152), AXII (P07335), epoxide hydrolase (P07099), alpha-enolase (P06733), nucleoside diphosphate kinase A (P15531), Thioredoxin (P10599), histone H2B (P02278), aldehyde dehydrogenase (P05091), calreticulin precursor (P27797), manganese superoxide dismutase (P04179), Cu/Zn superoxide dismutase (P00441), Macrophage migration inhibitory factor (MIF) (P14174), annexin V (P08758), alcohol dehydrogenase (P14550), aldose reductase (P15121), glyceraldehyde-3phosphate dehydrogenase (GAPDH) (P04406), calmodulin (P02593), ubiquitin (P02248), and 67 KD Laminin receptor (P08865). Annexins (AX) is a family of structurally related proteins that have calcium- and phospholipid-binding domains. AXI plays an important role in the malignant transformation process leading to HCC and that it is closely related to the histological grade of HCC [30]. AXII and AXI may act together in the occurrence and development of HCC [31]. Thioredoxin (TRX) is known to contain an active site with a redox-active disulfide and has various biological activities. It has been found that patient with HCC have a significantly elevated serum level of TRX have been revealed, suggesting

that measurement of serum of TRX might be a useful clinical parameter when HCC is suspected [32]. An association between liver disease severity and common alleles of microsomal epoxide hydrolase (mEH), an enzyme involved in the metabolism of highly reactive epoxide intermediates, has been found. mEH gene polymorphisms were significantly associated with chronic hepatitis C virus (HCV)-related liver disease severity and HCC risk [33]. Other protein, such as macrophage migration inhibitory factor (MIF) may contribute to multiple aspects of tumor progression, including control of cell proliferation, differentiation, cell survival and angiogenesis [34], and overexpression of Vimentin (VIM) was significantly associated with HCC metastasis [35], and Cu/Zn superoxide dismutase could serve as a “signature” for diagnosis and prognosis of HCC [36].

4. Conclusion We have demonstrated the capability of nano-2DLC–MS–MS for the separation and identification of complex peptide samples. It highly automates about sample injection, desalting, analysis, and identification without further manual interference. Coupling SCX in the first dimension with RPLC in the second dimension, the total peak capacity of this system as a measure for chromatographic performance can be calculated to be as high as 1800. Prefractionation of proteomics was also applied based on the solubility of protein in HCC tissue. Some extremely large, small, highly basic and hydrophobic proteins were identified, that prove this system is an unbiased platform for proteomics. Therefore, this procedure could be proposed as a general approach to deal with current limits in proteomics analysis. Finally, by using this platform 229 protein of HCC tissue was identified, among them several are found to be related with the development of HCC.

Acknowledgement This paper was supported by the National Basic Research Priorities program, project: 2001CB5102, the National 863project: 2002AA2Z2042, and the National Natural Science Foundation of China, project 20075004.

References [1] P.A. Haynes, S.P. Gygi, D. Figeys, R. Aebersold, Electrophoresis 11 (1998) 1862. [2] E. Jung, M. Heller, J.C. Sanchez, D.F. Hochstrasser, Electrophoresis 21 (2000) 3369. [3] A. Pandey, A. Podtelejnikow, B. Blagoev, X. Bustelo, M. Mann, H. Lodish, Proc. Natl. Acad. Sci. USA 97 (2000) 179. [4] P.H. O’Farrell, J. Biol. Chem. 250 (1975) 4007. [5] R. Aebersold, D.R. Goodlett, Chem. Rev. 101 (2001) 269. [6] M. Mann, R.C. Hendrickson, A. Pandey, Annu. Rev. Biochem. 70 (2001) 437.

Y. Wang et al. / Analytica Chimica Acta 530 (2005) 227–235 [7] F.E. Regnier, G. Huang, J. Chromatogr. A 750 (1996) 3. [8] J.P. Lumann, A.V. Lemmo, A.W. Moore, J.W. Jorgenson, Electrophoresis 14 (1993) 439. [9] J.C. Giddings, Anal. Chem. 56 (1984) 1258. [10] A.W. Moore, J.W. Jorgenson, Anal. Chem. 67 (1995) 3448. [11] M.T. Davis, J. Beierle, E.T. Bures, M.D. McGinley, J. Mort, J.H. Robinson, C.S. Spahr, W. Yu, R. Luethy, S.D. Patterson, J. Chromatogr. B 752 (2001) 281. [12] M.M. Bushey, J.W. Jorgenson, Anal. Chem. 62 (1990) 978. [13] G.P. Opitek, J.W. Jorgenson, R.J. Anderegg, Anal. Chem. 69 (1997) 2283. [14] G.J. Opitek, K.C. Lewis, J.W. Jorgenson, Anal. Chem. 69 (1997) 1518. [15] D.A. Wolters, M.P. Washburn, J.R. Yates III, Anal. Chem. 73 (2001) 5683. [16] A.J. Link, J. Eng, D.M. Schieltz, E. Carmack, G.J. Mize, D.R. Morris, B.M. Garvik, J.R. Yates III, Nat. Biotechnol. 17 (1999) 676. [17] J. Kim, S.H. Kim, S.U. Lee, G.H. Ha, D.G. Kang, N.Y. Ha, J.S. Ahn, H.Y. Cho, S.J. Kang, Y.J. Lee, S.C. Hong, W.S. Ha, J.M. Bae, C.W. Lee, J.W. Kim, Electrophoresis 23 (2002) 4142. [18] S.O. Lim, S.J. Park, W. Kim, S.G. Park, H.J. Kim, Y. Kim, T.S. Sohn, J.H. Noh, G.H. Jung, Biochem. Biophys. Res. Commun. 291 (2002) 1031. [19] T.K. Seow, S.E. Ong, R.C.M.Y. Liang, E.C. Ren, L. Chan, K.L. Ou, M.C.M. Chung, Electrophoresis 21 (2000) 1787. [20] L.R. Yu, R. Zeng, X.X. Shao, N. Wang, Y.H. Xu, Q.C. Xia, Electrophoresis 21 (2000) 3058. [21] M.M. Bradford, Anal. Biochem. 72 (1976) 248. [22] D.N. Perkins, D.J.C. Pappin, D.M. Creasy, J.S. Cottrell, Electrophoresis 20 (1999) 3551. [23] H.J. Liu, S.J. Berger, A.B. Chakraborty, R.S. Plumb, S.A. Cohem, J. Chromatogr. B 782 (2002) 267.

235

[24] B. Mehlis, U. Kertscher, Anal. Chim. Acta 352 (1997) 71. [25] G. Winkler, P. Wolschann, P. Briza, F.X. Heinz, C.J. Kunz, J. Chromatogr. 347 (1985) 83. [26] A. Apffel, S. Fischer, G. Goldberg, P.C. Goodley, F.E. Kuhlmann, J. Chromatogr. A 712 (1995) 177. [27] C.G. Huber, A. Premstaller, J. Chromatogr. 443 (1988) 119. [28] M.P. Molloy, B.R. Herbert, B.J. Walsh, M.I. Tyler, M. Traini, J.C. Sanchez, D.F. Hochstrasser, K.L. Willians, A.A. Gooley, Electrophoresis 19 (1998) 837. [29] D.L. Zheng, B.W. Peng, Q.L. Huang, J.Y. Lin, Chin. J. Cancer 22 (2003) 248. [30] T. Masaki, M. Tokuda, M. Ohnishi, S. Watanabe, T. Fujimura, K. Miyamoto, T. Itano, H. Matsui, K. Arima, M. Shirai, T. Maeba, K. Sogawa, R. Konishi, K. Taniguchi, Y. Hatanaka, O. Hatase, M. Nishioka, Hepatology 24 (1996) 72. [31] T. Masaki, M. Tokuda, M. Ohnishi, Y. Tai, T. Itano, H. Matsui, S. Watanabe, K. Arima, K. Kohno, T. Maeba, Y. Ikeda, O. Hatase, M. Nishioka, Int. Hepatol. Commun. 4 (1995) 113. [32] K. Miyazaki, N. Noda, S. Okada, Y. Hagiwara, M. Miyata, I. Sakurabayashi, N. Yamaguchi, T. Sugimura, M. Terada, H. Wakasugi, Biotherapy 11 (1998) 277. [33] L. Sonzogni, L. Silvestri, A. De Silvestri, C. Gritti, L. Foti, C. Zavaglia, R. Bottelli, M.U. Mondelli, E. Civardi, E.M. Silini, Hepatology 36 (2002) 195. [34] Y. Ren, H.T. Tsui, R.T.P. Poon, I.O.L. Ng, Z. Li, Y.X. Chen, G.P. Jiang, C. Lau, W.C. Yu, M. Bacher, S.T. Fan, Int. J. Cancer 107 (2003) 22. [35] L. Hu, S.H. Lau, C.H. Tzang, J.M. Wen, W.S. Wang, D. Xie, M.H. Huang, Y. Wang, M.C. Wu, J.F. Huang, W.F. Zeng, J.S.T. Sham, M.S. Yang, X.Y. Guan, Oncogene 23 (2004) 298. [36] D. Goldenberg, S. Ayesh, T. Schneider, O. Pappo, O. Jurim, A. Eid, Y. Fellig, T. Dadon, I. Ariel, N. de Groot, A. Hochberg, E. Galun, Mol. Carcinogen 33 (2002) 113.