Analyzing Alkaline Proteins in Human Colon Crypt ... - ACS Publications

Analyzing Alkaline Proteins in Human Colon Crypt Proteome Xin-Ming Li,† Bhavinkumar B. Patel,† Elena L. Blagoi,† Maketa D. Patterson,† Steven H. Seeholzer,† Tao Zhang,‡ Shirish Damle,‡ Zhenqiang Gao,‡ Bruce Boman,‡ and Anthony T. Yeung*,† Basic Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 19111, and Department of Medicine, Thomas Jefferson University, 1025 Walnut Street, Suite 1014, Philadelphia, Pennsylvania 19107 Received March 1, 2004

Normal human colon crypt protein extract was resolved by two-dimensional gel electrophoresis using pH 6-11 immobilized pH gradient strips in the first dimension. The optimized isoelectric focusing protocol includes cup-loading sample application at the anode and 1.2% hydroxyethyl disulfide (DeStreak), 15% 2-propanol and 5% glycerol in the rehydration buffer. Spots were well resolved across the entire pH range up to 11. A total of 311 protein spots were identified by mass spectrometry and peptide mass mapping. After combining isoforms, 231 nonredundant proteins were grouped into 16 categories according to their subcellular locations, and 17 categories according to their physiological functions. Histone proteins, ribosomal proteins and mitochondrial proteins were among the wellresolved highest pI proteins. Application of this protocol to the analysis of normal and neoplastic colon crypts will contribute to the proteomic study of colorectal tumorigenesis since a significant portion of the human proteins is in basic pH range. Keywords: 2D-electrophoresis • alkaline protein • colon crypt • colorectal cancer • proteomics

1. Introduction Proteomics aims to retrieve all of the information present at the protein level in a given biological system. It requires the ability to separate and resolve all proteins in the system, track changes in protein expression and subcellular location, identify and classify proteins into functional groups. At present, twodimensional electrophoresis (2-DE) gel based proteomics remains an important tool for achieving this goal. However, with all of the advancement in the field, such as immobilized pH gradient (IPG) gel strips, high-sensitivity fluorescent staining, sophisticated image analysis software, high-sensitivity mass spectrometers, and high-throughput automation, the separation and identification of all cellular proteins on 2-DE gel remains a challenge. Existing 2-DE technology is limited by the difficulties to resolve hydrophobic proteins, alkaline proteins and low abundance proteins.1 Most of the published results on 2-DE based proteomics are biased toward abundant proteins and proteins whose pIs fall within the range from 4 to 8. Efforts have been made to solve these problems since the beginning of 2-DE technology development. NEPHGE (nonequilibrium pH gradient electrophoresis) with carrier ampholytes can partially resolve the alkaline proteins, although the reproducibility was compromised by stopping the focusing process before reaching * To whom correspondence should be addressed. Tel: +1-215-728-2488. Fax: +1-215-728-3647. E-mail: [email protected]. † Basic Science, Fox Chase Cancer Center. ‡ Department of Medicine, Thomas Jefferson University. 10.1021/pr049942j CCC: $27.50

 2004 American Chemical Society

equilibrium.2 Gorg et al. described a protocol employing homemade high pH range (up to pH 12) IPG strips, and subsequently resolved some very alkaline proteins (ribosomal proteins and nuclear proteins with pI higher than 10) with a modified protocol.3-5 The difficulty in resolving alkaline proteins is often attributed to the reverse electro-endoosmotic flow that occurs during IEF, and migration or even exhaustion of the charged reducing agent such as DTT in the high pH range of the IPG strip. Hoving et al. described a modification in a 2-DE protocol by adding glycerol, 2-propanol, and large amount of reducing agent in the focusing buffer, thus improving the resolution of alkaline proteins up to pH 9.5.6 Another major improvement in high pH 2-DE resolving power was achieved by applying hydroxyethyl disulfide (HED) during IEF to block the thiol groups in proteins as mixed dithiols, thus improving the resolution for alkaline proteins and making the protein spot pattern highly reproducible.7 A recent study by Pennington et al. combining the use of HED and anodic cuploading was able to detect 353 protein spots of human brain proteins by silver stain at 100 µg loading scale on a typical pH 6-9 2-DE gel.8 Although technically difficult, it is very important to have a complete proteomic characterization of alkaline proteins in any given biological system. Theoretical simulation of human proteome 2-DE map reveals that about 40% of the total proteins have pIs higher than 8.9 Several classes of alkaline proteins have important cellular functions, such as the ribosomal proteins, mitochondrial proteins, histones, and other DNA binding proteins. These proteins are involved in various fundamental Journal of Proteome Research 2004, 3, 821-833

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research articles cellular processes, such as DNA replication, transcription, translation, metabolism, and apoptosis. Many of these cellular functions are perturbed in tumorigenesis. Therefore, analysis of the alkaline proteome may shed valuable insight on normal and cancer biology. In the present study, we concentrate our effort to the alkaline proteins from human colonic crypts as part of our ongoing project to study the molecular mechanism of colorectal cancer (CRC) tumorigenesis. CRC is the second leading cause of cancer death in the United States. CRC tumorigenesis is a multistep process involving a series of mutations in tumor suppressor genes and oncogenes. Normal colonic crypts develop into malignant cancer via the adenomatous polyp, a benign tumor of epithelial origin.10,11 Mutations on both alleles of the tumor suppressor gene APC (adenomatous polyposis coli) account for at least 85% of all colon cancers.12 Mutations in APC can be inherited or sporadic, but the inherited mutation on one allele increases the carrier’s risk of CRC to nearly 100%. Therefore, it is very important to investigate the molecular events occurring between the inactivation of the first allele and the second allele. Because of the two-hit nature of CRC, prevention of the second hit provides an opportunity to stop CRC development.13 As part of our concerted effort to study the earliest changes in colon crypt proteome during the stepwise molecular and pathological transition of CRC tumorigenesis, we report here our improvement on 2-DE separation of alkaline proteins as well as the identification of 231 alkaline human colon crypt proteins and their isoforms which can serve as reference points in future analysis.

2. Materials and Methods 2.1. Colon Crypts Preparation and Protein Extraction. 2.1.1. Colon Crypt Preparation. All of the human colon mucosa specimens used in this study and subsequent proteomics analysis were approved by respective Institutional Review Board of Fox Chase Cancer Center (FCCC) and Thomas Jefferson University (TJU) (FCCC protocol number: 00-852; TJU protocol number: 00.0023). Colon crypt preparation was based on a chelation method14 with some modifications. Briefly, the colon mucosal layer was dissected from surgically removed noncancerous region of a colon specimen and washed three times with cold PBS (phosphate-buffered saline), then incubated in 0.4% sodium hypochlorite for 15 min at room temperature. The cleaned mucosal layer was incubated in 3 mM EDTA in PBS for 45-60 min at room temperature, and the sample was vigorously shaken until detached crypts could be seen in suspension. After removing the tissue, the crypt suspension was transferred to a new centrifuge tube and centrifuged at 500 rpm for 3 min at 4 °C to pellet the crypts. The supernatant was removed, and the pellet was washed again in PBS. The quality of the isolated crypts was checked microscopically. If not used immediately, the crypts were stored at -80 °C. 2.1.2. Protein Extraction. Protein extracts from colon crypts were prepared using one of two protein extraction buffers: (A) Base Solution: 7 M urea, 2 M thiourea, 65 mM CHAPS, 60 mM Tris/HCl (pH 8.8) and 0.3% SDS; additives: 8 mM PMSF, 52 mM DTT; (B) Base Solution: the same as in A; additives: 8 mM PMSF, 97.4 mM hydroxyethyl disulfide (HED). For 200 mg wet weight of crypts, 2 × 500 µL extraction buffer was used. Vortex or homogenization by pipetting was necessary to dissolve the crypt preparations. To remove chromosomal DNA, 1% v/v of 0.4 M spermine and 0.2% v/v of 5% polyethylenimine 822

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Li et al. Table 1. Optimized IEF Protocol for pH 6-11 IPG Strip IEF protocol (pH 6-11) start step T (°C) volt (V)

1 2 3 4

20 18 18 18

0 0 150 300

end volt (V)

duration (h)

note

0 >12 passive rehydration 150 0.5 300 2.5-5 depends on sample vol. 10 000 60 000 Vh

were added to the solution, and incubated at room temperature for about 30 min, with vortexing every 10 min, to aggregate the polynucleotides. A clear protein extract was obtained after 45 min of centrifugation at 21 000 × g. Salt and SDS were removed by acetone precipitation. Protein extract was mixed with cold acetone at the ratio of 1:3 (v/v), and the mixture was stored at -20 °C overnight or longer. After 45 min of centrifugation at 21 000 × g, the supernatant was discarded and the pellet was washed with 80% cold acetone. After a brief centrifugation, the pellet was dried in a Speedvac for 5 min and redissolved in 2-DE sample buffer: 7 M urea, 2 M thiourea, 4% CHAPS. Any residue was removed by centrifugation for 45 min at 21 000 × g. Protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA), using a standard curve based on BSA in the same 2-DE sample buffer as for the preparation of colon crypt 2-DE sample. 2.2. Two-dimensional Electrophoresis and Protein Detection. 2.2.1. IEF and SDS-PAGE. IEF was performed using PROTEAN IEF Cell (Bio-Rad, Hercules, CA) with the cup-loading tray for anodic sample application (Bio-Rad, Hercules, CA). Linear gradient 18 cm (pH 6-11) Immobiline DryStrip (Amersham Biosciences, NJ) and ReadyStrip (pH 7-10) (Bio-Rad, Hercules, CA) were used according to the manufacturers’ instructions. The IPG strip was rehydrated overnight at 20 °C in 340 µL rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 5% glycerol, 15% 2-propanol, 1.2% DeStreak reagent, 2% IPG Buffer 6-11). For each IPG strip, 100 µg of protein samples in a volume less than 100 µL with 1.2% DeStreak reagent and 2% IPG buffer 6-11 was applied by cup loading at the anodic end. Electrode wicks immersed in rehydration buffer were applied at both ends of the strips, which were covered with mineral oil. IEF protocol as described in Table 1 was used. Total Volthours was about 65 kVh. The IEF parameters were recorded electronically to ensure reproducibility. After IEF, the IPG strip was immediately reduced and alkylated with a 2-step equilibration procedure. In the first equilibration solution, 2.6% (w/v) DTT was included in addition to 40 mM Tris-HCl, pH 8.8, 7 M urea, 10% glycerol and 2% SDS. The strip was incubated with continuous shaking at 100 rpm for 5 min on an orbital shaker. The second step was the same except that DTT was replaced by 21.6% (w/v) iodoacetamide. SDS/PAGE was carried out on a 12% SDS/polyacrylamide gels (20 cm × 20 cm × 1.0 mm) with PROTEAN II xi system (Bio-Rad, Hercules, CA). The strip was transferred to the top of the gel and held in place with 0.5% low-melt agarose dissolved in SDS-Tris-Glycine running buffer (Bio-Rad, Hercules, CA). Electrophoresis was performed with constant current at 40 mA/gel at 15 °C for approximately 4 h until the tracking dye was about 1 cm from the end of the gel. 2.2.2. Gel Staining and Image Acquisition. After electrophoresis, gels were placed in Fixation solution (40% methanol and 7.5% acetic acid) for at least 1 h, then transferred to Sypro

Alkaline Proteins in Human Colon Crypt

Ruby protein stain, 250 mL/gel (Bio-Rad Hercules, CA), and stained for at least 10 h, with 50 rpm orbital shaking in the dark at room temperature. Before imaging with a ProEXPRESS scanner (PerkinElmer, IL), the gel was destained with 250 mL Destain solution (10% methanol and 7.5% acetic acid) for 30 min. Two gel images were taken with exposure time of 200 ms and 2 s, respectively. The gel was destained for another 2 h and two more gel images were taken in the same manner. 2.3. Protein Spot-Cutting, Tryptic In-Gel Digestion, and Protein Identification by MALDI-TOF Analysis. 2.3.1. Spot-Cutting and Tryptic In-Gel Digestion. Protein spots were selected through image analysis with Progenesis Discovery v2003.02 software (Nonlinear Dynamics, UK), excised with a ProPic Robot (Genomic Solutions, MI) as 1-mm gel plugs, and deposited in a 96-well microplates. Liquid was removed and the gel plugs were dehydrated with 100% acetonitrile. Reduction was performed with 20 mM DTT in 50 mM NH4HCO3 for 30 min, followed by alkylation with 50 mM iodoacetamide in 20 mM NH4HCO3 solution for 30 min in the dark. Liquid was removed and the pH of the gel plugs was adjusted by 10 mM NH4HCO3 for 10 min. After dehydration with 100% acetonitrile, the gel plugs were rehydrated at 4 °C for 30 min in 7 µL Trypsin solution (10 ng/µL porcine modified sequencing grade trypsin, (Promega, Madison, WI), 10 mM NH4HCO3, 5% acetonitrile). Digestion was achieved by incubating the gel plug at 34 °C for about 12 h. 2.3.2. Protein Identification by MALDI-TOF Analysis. Tryptic digested peptides were analyzed by MALDI-TOF MS with a Reflex IV mass spectrometer (Bruker Daltonics, Billerica, MA). Digested samples (1 µL) were spotted onto a 400-µm spot size, 384-position Anchorchip (Bruker Daltonics, MA), then mixed with 2 µL matrix (alpha-cyano-4-hydroxycinnamic acid as 10 mg/mL in ethyl alcohol:ethyl alcohol:acetone:25%(w/v) TFA, volume wise as 18:400:200:5) and allowed to dry. Mass spectra were acquired between 500 and 3500 m/z in reflectron mode. Trypsin autolysis products were used for internal mass calibration. Peak assignment and internal calibration were performed using the software package XMASS (Bruker Daltonics, MA). Proteins were identified through database search (Swiss-Prot, MSDB, and NCBI), by using BioTool software (Bruker Daltonics, MA) and MASCOT (www.matrixscience.com). All of the identified spots shown in this publication were confirmed in duplicate using corresponding gel plugs cut from separate gels. 2.4. Reproducibility and Quality Control. Each sample used in this study were analyzed by more than two gels, with about 90% reproducibility in terms of total spot volume and spot matching result. Quality control for mass spectrometry peptide mapping was based on the following: (1) matched spots from replicate gels gave the same results; (2) pI and molecular weight of the excised protein spot were compatible with the assigned sequences; (3) mass error for matched masses were very tightly and evenly distributed around zero ppm; (4) masses matched to specific amino acid sequences accounted for most of the nonbackground spectral intensity.

3. Results 3.1. Cup-Loading Vs In-Sample Rehydration. In-sample rehydration was developed for IPG strips to enable higher sample loading and better resolution of the 2-DE gels. Through a prolonged low-voltage in-sample rehydration step, proteins, especially the larger ones, have more time to migrate into the IPG gel strip. However, our results showed that this protocol was not suitable for high-pH range IPG strips. Poor resolution

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Figure 1. Progressive improvement of high pH range 2-DE gel resolution by various strategies. Human colon crypt protein extract was resolved on IPG strip pH 7-10 (A, B, C, and D) or pH 6-11 (E and F). Second dimension gel: 12% acrylamide. (A) insample rehydration; (B) cup-loading; (C) 1.2% hydroxyethyl disulfide (DeStreak) was included in the IPG strip rehydration buffer; (D) extended 300 V protein entry step in IEF; (E) expansion of the IEF pH range from 7-10 to 6-11; (F) 15% instead of 10% 2-propanol, and 5% glycerol were included in the IPG strip rehydration buffer.

and heavy horizontal streaking were seen on gels prepared by in-sample rehydration (Figure 1A). In contrast, cup-loading at the anode end of the IPG strip significantly improved the 2-DE gel resolution, as shown in Figure 1B. 3.2. Applying Hydroxyethyl Disulfide in the Sample. DeStreak is the trade name for hydroxyethyl disulfide (HED), which was first shown by Olsson et al. to reduce the horizontal streakings on 2-DE gel, especially in high pH range gels.7 It forms mixed disulfides, thus protecting the active cysteine thiolgroups from uncontrolled modifications. We have used the HED in two ways: (1) the protein extract was prepared in the presence of low concentration of DTT (8 mM), and then a high concentration of HED (∼100 mM) was added to react with the thiol groups; (2) the cell extract was prepared in the presence of HED, thus eliminating the DTT step. Both procedures worked equally well, and streaking was significantly reduced (Figure 1C). 3.3. Increasing the Protein Entry Time. Although the cuploading and HED improved the gel resolution, the total protein presented on the 2-DE gel was less when compared with the Journal of Proteome Research • Vol. 3, No. 4, 2004 823

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Figure 2. Chart of a typical IEF process from the recorded parameters during an IEF run.

in-sample rehydration approach (Figure 1A-C), potentially compromising detection of low abundance proteins. Prolonging the total Vh did not improve the result (data not shown), but prolonging the initial protein entry step at 300 V enabled more protein to appear on the gel, as evidenced by both visual evaluation of the gel image and total spot intensity detected by image analysis software (Figure 1D). 3.4. IPG Strips of pH 6-11 vs pH 7-10. After comparing IPG strips of different pH ranges, we found pH 7-10 IPG strips caused some proteins to show an over-stretched pattern (Figure 1D), while other proteins gave tight spots. This phenomenon was observed also when very narrow pH range IPG strips were used. The focusing results were improved when the pH 7-10 IPG strip was replaced with pH 6-11 strip. In general, with the latter, there were more proteins being resolved, and spot shape also improved remarkably (Figure 1E). 3.5 Including 15% of 2-Propanol in Rehydration Buffer. Reverse electro-endoosmotic flow makes charged water molecules move from cathode to anode during IEF in high pH gradient gel, which can lead to drying or burning of the IPG gel strip. Sorbitol, 2-propanol, or methylcellulose have been used to circumvent this problem.3 We found that the addition of 15% 2-propanol into the IPG buffer achieved better-resolved protein spot pattern on our 2-DE gels, compared to using lower percentage 2-propanol in the buffer (Figure 1F). 3.6. Optimizing the IEF Protocol. Our operating protocol (Table 1) consists of a separate passive rehydration step for at least 12 h, and a sample-loading step with 300 V for 2.5 h for a loading volume of 50 µL (with an increment of 30 min for every additional 10 µL up to 100 µL total volume), and a final focusing step of 60 000 Vh. The quickest protocol can be completed in 22 h. However, the rehydration step may be extended or, after IEF, the IPG gel strip may be kept at -80 °C to accommodate a routine working schedule. A typical IEF record chart is shown in Figure 2. Note that the time necessary to reach the highest setting, 10 000 V, is long, and the final focusing time at 10 000 V is short. Sometimes the highest voltage may not reach 10 000 V during the run. 3.7. Protein Identification. Following the optimized protocol, from several selected gel images, a reference gel was established that contains about 800 protein spots (Figure 3). Most of the spots were excised in duplicate from replicate gels for protein identification. A total of 311 protein spots were identified as various modified forms of 231 nonredundant proteins. Specifically, 1 protein has 6 isoforms, 1 has 5 isoforms, 2 have 4 isoforms, 12 have 3 isoforms, 41 have 2 isoforms and 174 were identified as single spots in the pH 6-11 region. Table 824


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2 summarizes the details of the identified proteins. Although the isoforms of a protein can arise from several possibilities, such as genetic polymorphism, protein truncation, degradation, post-translational modification or artificial modification during 2-DE process, at this stage, we did not attempt to identify the causes nor the consequences of the protein isoforms. 3.7.1. Distribution of Identified Proteins as a Function of Molecular Weight and pI. Histograms showing the distribution of the identified proteins according to their theoretical molecular weights and pIs are shown in Figure 4. It is in good agreement with the pattern seen on the 2-DE gel (Figure 3). The majority of the proteins, more than 90%, were resolved in the 10-100 kDa region. About 40% of identified proteins had pI lower than pH 8.0, and about 60% higher than pH 8.0. There were 12 identified proteins with pI higher than 10, including histones, ribosomal proteins, and mitochondrial proteins. 3.7.2. Subcellular Location of the Identified Proteins. The subcellular locations of the identified protein showed that they belong to 16 categories, with 26 proteins in the unknown group (Figure 5). The largest group is the mitochondrial protein group, consisting of 71 members, representing almost 30% of the total identified proteins. 3.7.3. Functional Classification of the Identified Proteins. The distribution of the identified proteins based on functional categories in the Gene Ontology database (www. geneontology.org) and the Human Protein Reference Database15 (www.hprd.org) is shown in Figure 6. The 231 proteins identified were grouped into 17 categories. The group of 86 proteins involved in intermediary metabolism represents the largest group of the identified proteins. There were 17 proteins with no known functions assigned.

4. Discussion Analysis of the human genome sequence predicted approximately 35 000 coding genes.16 The proteome is further complicated by alternate splice junction variants and posttranslational modifications.16 Human proteome may have 40% proteins with pI higher than 8,9 and yeast proteome consists as high as 50% of the proteins having pI higher than 8.17 Therefore, it is necessary for any comprehensive proteome database to include information on alkaline proteins. In our current protocol, we use cup-loading sample application instead of the in-sample rehydration method, although in-sample rehydration has been used widely to resolve neutral or acidic proteins. Taking advantage of low-voltage and prolonged sample entry step, in-sample rehydration has the benefits of accommodating heavy loading and resolving high molecular weight proteins. However, we found that in-sample rehydration is not suitable for resolving alkaline proteins (Figure 1A,B). With cup-loading and a prolonged 300 V protein entry step, we were able to produce well-resolved and reproducible 2-DE gels in the range of pH 6-11 (Figure 1, Table 1). IPG matrix formulated with very basic (pK 10.3) acrylamido buffer exhibits a strong positive charge, developing a “reverseendoosmotic flow” of ionized water toward the anode during IEF. This results in disintegrated IPG matrix, disturbing the steady-state locations of the proteins in the IPG strip. To overcome this effect, it is necessary to include 15% 2-propanol and 5% glycerol in the rehydration buffer (Figure 1). So far, there are only a limited number of proteins related to CRC identified by proteomics, and most of them are around neutral pI. The first application of 2-DE to compare protein


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Figure 3. IPG pH 6-11 2-DE gel map. Proteins (100 µg) from human colon crypt whole cell extract were separated on pH 6-11 IPG strip in the first dimension and 12% acrylamide SDS-PAGE in the second dimension. The resulting gel was stained with Sypro Ruby. The protein spots identified by MALDI MS analysis are designated with spot numbers. The protein names for the annotated spots are listed in Table 2.

expression patterns of normal and transformed colorectal cells was published more than 20 years ago.18 Ji et al. studied the differences of normal and cancerous colon crypt based on 2-DE gel analysis in the range of pH 4-7.19 Cole et al. examined protein expression profiles from crypt, polyps and stroma from normal, APC mutated (multiple intestinal neoplasia, MIN) or p53 knock-out mice using two pH range IEF strips, pH 3-10 and pH 6-11, and one protein had been identified with pI above 8.20 The MIN mouse was studied using pH 3-10 2-DE gels to elucidate the role of APC in the development of familial and sporadic colorectal cancer. Several changes in protein expression were found among those samples, including two proteins with pI value higher than 8.21 Other proteomics studies on colon tissue identified tumor specific proteins, such as calgranulin A, calgranulin B and calgizzarin.22,23 To compare the protein expression profile more precisely, laser-capture microdissection (LCM) was performed on normal and cancer colonic epithelial cells, and the resulting samples were analyzed by 2-DE gels.24 A targeted proteomics analysis on rat intestinal epithelial cells using a tetracycline-regulated beta-catenin expression system found that 22 protein spots were either upregulated or down-regulated upon induction of β-catenin.25 In all these proteomics studies concerning colon crypt or CRC,

proteins resolved on 2-DE gels were limited to the neutral pH range. The only attempt to resolve proteins in pH 6-11 was suffered from poor resolution and inconsistency of the protein expression pattern above pH 8.20 In the present study, we have tested and modified several techniques (Figure 1) to resolve the alkaline proteins on 2-DE gels, and optimized the IEF protocol (Table 1) to get high resolution and reproducible gels. Applying this technology to human colon crypt samples, we resolved about 800 alkaline protein spots and identify 311 of them. Among the identified proteins, 18 are ribosomal proteins, and all have pI values above 9. It has been shown that ribosomal proteins are involved in extra-ribosomal activity independent of protein biosynthesis.26,27 Kasai et al. suggested that the extra-ribosomal activity is relevant to CRC by comparing the expression profile of 12 ribosomal proteins (Sa, S8, S11, S12, S18, S24, L7, L13a, L18, L28, L32, and L35a) in normal epithelium and cancer epithelium of human colorectal mucosa using immunohistochemistry. In normal mucosal epithelium, S11 and L7 ribosomal proteins appear to be barely expressed, in contrast to highlevel expression in immature mucosal cells and carcinoma cells.28 Furthermore, L7 ribosomal protein was expressed in secretory granules of the enterochromaffin cells in normal Journal of Proteome Research • Vol. 3, No. 4, 2004 825

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Table 2. List of Identified Proteins from Human Colon Crypt Whole Cell Extract Resolved on pH 6-11 2-DE Gels spot no.a

25 48 66 96 148 195 212 218 227 8 10 12 38 39 43 52 53 63 91 92 95 104 107 145 153 154 161 162 164 165 168 169 170 171

173 174 185 202 205 206 210 214 217 249

826

acc no.b

protein name

SW acc no.c

MW (Dal)d

structural proteins ARP2/3 complex 34 kDa subunit AR34_HUMAN 34425.53 (P34-ARC) P02545 Lamin A/C (70 kDa lamin) LAMA_HUMAN 74380 O15145 ARP2/3 complex 21 kDa subunit AR21_HUMAN 20761.46 (P21-ARC) P23528 Cofilin, nonmuscle isoform (18 COF1_HUMAN 18718.74 kDa phosphoprotein) O00151 PDZ and LIM domain protein 1 PDL1_HUMAN 36505 P07737 Profilin I PRO1_HUMAN 15000 O15509 ARP2/3 complex 20 kDa subunit AR20_HUMAN 19667.01 (P20-ARC) P18282 Destrin (Actin-depolymerizing DEST_HUMAN 18505.72 factor) P17661 Desmin DESM_HUMAN 53404.6 transcription and translation P13639 Elongation factor 2 (EF-2) EF2_HUMAN 96115.27 P21399 Iron-responsive element binding IRE1_HUMAN 98849.77 protein P23246 Splicing factor, proline-and SFPQ_HUMAN 76215.67 glutamine-rich P26599 Polypyrimidine tract-binding PTB_HUMAN 57356.7 protein 1 Q15233 54 kDa nuclear RNA- and DNANR54_HUMAN 54180.28 binding protein P47897 Glutaminyl-tRNA synthetase SYQ_HUMAN 88655.21 P41091 Eukaryotic translation initiation IF2G_HUMAN 51516.39 factor 2 subunit 3 P04720 Elongation factor 1-alpha 1 EF11_HUMAN 50451.24 Q14103 Heterogeneous nuclear ROD_HUMAN 38581.37 ribonucleoprotein D0 P22626 Heterogeneous nuclear ROA2_HUMAN 37463.75 ribonucleoproteins A2/B1 Q96IR1 Similar to RIKEN cDNA 1110033J19 Q96IR1 22269 gene -Homo sapiens (Human) Q9NVS2 28S ribosomal protein S18a, R18A_HUMAN 22511.92 mitochondrial P49241 40S ribosomal protein S3A RS3A_HUMAN 30023 P49411 Elongation factor Tu, EFTU_HUMAN 49852.31 mitochondrial Q15365 Poly(rC)-binding protein 1 PCB1_HUMAN 38015 (Alpha-CP1) Q15097 Polyadenylate-binding protein 2 PAB2_HUMAN 58709 Q15428 Splicing factor 3A subunit 2 S3A2_HUMAN 49338 P23396 40S ribosomal protein S3 RS3_HUMAN 26842 P09012 U1 small nuclear ribonucleoprotein RU1A_HUMAN 31259 A Q9Y2R9 mitochondrial ribosomal protein S7 Q9Y2R9 28258 small chain P46782 40S ribosomal protein S5 RS5_HUMAN 23033 P46783 40S ribosomal protein S10 RS10_HUMAN 18886 P08708 40S ribosomal protein S17 RS17_HUMAN 15466 P30050 60S ribosomal protein L12 RL12_HUMAN 17979 Q9NWU5 Hypothetical protein FLJ20594 Q9NWU5 23797 (Similar to mitochondrial ribosomal protein L22) P17075 40S ribosomal protein S20 RS20_HUMAN 13478 Q9UGG2 CB1E7.2 (Ribosomal protein S17 Q9UGG2 15763 - like 4) P39026 60S ribosomal protein L11 RL11_HUMAN 20300 Q16540 L23-related protein (Mitochondrial Q16540 17770 ribosomal protein L23) P04643 40S ribosomal protein S11 RS11_HUMAN 18590 P11940 Polyadenylate-binding protein 1 PAB1_HUMAN 70854 I38968 100 kDa coactivator - human Q13122 99690.37 P49406 60S ribosomal protein L19, RM19_HUMAN 28823.43 mitochondrial P07910 Heterogeneous nuclear ROC_HUMAN 33688.04 ribonucleoproteins C1/C2 P22090 40S ribosomal protein S4, Y RS4Y_HUMAN 29324.43 isoform O15144


pId

scoree

cov. (%)f

8.64

100

31

cytoskeletal

6.57 8.78

96 62

27 19

nuclear cytoskeletal

8.22

87

43

nuclear and cytoplasmic

6.56 8.48 8.53

60 100 90

27 64 48

cytoplasmic cytoskeletal cytoskeletal

8.06

60

27

cytoskeletal

5.21

60

11

cytoskeletal

6.42 6.23

265 60

43 13

cytoplasmic cytoplasmic

9.45

64

19

nuclear

9.22

62

16

nuclear

9.01

93

37

nuclear

6.71 8.66

222 78

41 30


9.1 7.62

70 60

34 19

8.97

118

43

9.93

70

25

cytoplasmic nuclear; component of ribonucleosomes nuclear; component of ribonucleosomes unknown

10.35

55

33

mitochondrial

9.75 7.26

55 213

19 48

ribosome mitochondrial

6.66

60

25

nucleus and cytoplasm

9.31 9.65 9.68 9.83

192 56 251 60

45 19 75 26

cytoplasmic nuclear ribosome nuclear

10

65

26

ribosome

9.73 10.15 9.85 9.48 9.95

62 57 70 77 72

20 30 44 54 25

ribosome ribosome ribosome ribosome unknown

9.95 9.64

71 70

38 45

ribosome ribosome

9.64 9.69

66 60

29 36

10.31 9.52 6.62 9.35

60 76 264 161

12 13 33 45

ribosome mitochondrial large ribosomal subunit ribosome cytoplasmic and nuclear nucleus mitochondrial

4.95

64

23

10.25

60

9

subcellular location

nuclear; copmponent of ribonucleosomes ribosome

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Alkaline Proteins in Human Colon Crypt Table 2 (Continued) spot no.a acc no.b

192

1 17 69 71 97 99 116 117 120

141 155 159 180 247

2 3 6 7 9 14 15 16 18 19 20 21 24 26 28 40 41 42 44 47 49

50 51 55 58

protein name

Q04837 Single-stranded DNA-binding protein, mitochondrial

SW acc no.c

MW (Dal)d

replication SSB_HUMAN 17200

pId scoree cov. (%)f

9.59 111

signaling and apoptosis proteins Q9BZE4 Nucleolar GTP-binding protein 1 NOG1_HUMAN 74316.97 9.52 O95831 Programmed cell death protein 8, PCD8_HUMAN 67144.01 9.04 mitochondrial P28482 Mitogen-activated protein kinase 1 MK01_HUMAN 41762.37 6.5 P50995 Annexin A11 (Annexin XI) ANXB_HUMAN 54697.2 7.53 P30086 PhosphatidylethanolaminePEBP_HUMAN 21026.67 7.42 binding protein P21796 Voltage-dependent anionPOR1_HUMAN 30736.57 8.63 selective channel P07355 Annexin II (Lipocortin II) ANX2_HUMAN 38676.86 7.56 P45880 Voltage-dependent anionPOR2_HUMAN 38638.88 6.32 selective channel P25388 Guanine nucleotide-binding GBLP_HUMAN 35510.73 7.6 protein beta subunit-like protein 12.3 Q9UIJ7 GTP:AMP phosphotransferase KAD3_HUMAN 25419 9.16 mitochondrial P20172 Clathrin coat assembly protein A2M1_HUMAN 49965 9.57 AP50 O35129 repressor of estrogen receptor O35129 33276 9.83 activity(BAP) Q12904 Multisynthetase complex auxiliary MCA1_HUMAN 39900 9.37 component p43 P19138 Casein kinase II, alpha chain KC21_HUMAN 45143 7.29 (CK II) intermediary metabolism P53396 ATP-citrate (pro-S-)-lyase ACLY_HUMAN 121660.21 6.95 P11586 C-1-tetrahydrofolate synthase, C1TC_HUMAN 102048.52 6.94 cytoplasmic P51659 Estradiol 17 beta-dehydrogenase DHB4_HUMAN 80092.47 8.96 4 P40939 Trifunctional enzyme alpha subunit, ECHA_HUMAN 83688.15 9.16 mitochondrial P11216 Glycogen phosphorylase, brain form PHS3_HUMAN 97318.78 6.4 P29401 Transketolase TKT_HUMAN 68518.98 7.58 P49748 Acyl-CoA dehydrogenase, ACDV_HUMAN 70744.58 8.92 very-long-chain P21397 Amine oxidase [flavin-containing] AOFA_HUMAN 60156.73 7.94 A P23786 Carnitine O-palmitoyltransferase CPT2_HUMAN 74243.59 8.38 II, mitochondrial P00915 Carbonic anhydrase I CAH1_HUMAN 28778.37 6.63 P18669 Phosphoglycerate mutase 1 PMG1_HUMAN 28768.84 6.75 P00918 Carbonic anhydrase II CAH2_HUMAN 29153.9 6.86 Q16762 Thiosulfate sulfurtransferase THTR_HUMAN 33504.88 6.83 P14618 Pyruvate kinase, M1 isozyme KPY1_HUMAN 58339.2 7.95 Q16851 UTP- -glucose-1-phosphate UDP2_HUMAN 55813.14 7.69 uridylyltransferase 2 P06744 Glucose-6-phosphate isomerase G6PI_HUMAN 63335.33 8.43 Q01518 Adenylyl cyclase-associated CAP1_HUMAN 51925.75 8.07 protein 1 P25705 ATP synthase alpha chain, ATPA_HUMAN 59827.63 9.16 mitochondrial Q99798 Aconitate hydratase, ACON_HUMAN 86113.2 7.36 mitochondrial P43304 Glycerol-3-phosphate GPDM_HUMAN 81200 6.98 dehydrogenase, mitochondrial Q06210 Glucosaminesfructose-6GFA1_HUMAN 77365.49 6.4 phosphate aminotransferase [isomerizing] 1 P55084 Trifunctional enzyme beta subunit, ECHB_HUMAN 51546.63 9.45 mitochondrial Q9Y6N5 Sulfide:quinone oxidoreductase, SQRD_HUMAN 50213.96 9.18 mitochondrial O60701 UDP-glucose 6-dehydrogenase UGDH_HUMAN 55673.5 6.73 P00966 Argininosuccinate synthase ASSY_HUMAN 46786 8.08


58

mitochondrial

55 175

23 35

92 129 61

26 30 38

nuclear; nucleolar mitochondrial intermembrane space nuclear & cytoplasmic cytoplasmic and possibly nuclear cytoplasmic

129

56

271 60

57 21

mitochondrial outer membrane and plasma membrane plasma membrane outer mitochondrial membrane

94

26

nuclear & cytoplasmic

96

36

mitochondrial matrix

78

27

plasma membrane

249

76

unknown

60

15

unknown

60

13

nuclear and plasma membrane

57 229

13 32


139

31

unknown

183

28

mitochondrial matrix.

153 147 102

19 28 20

endoplasmic reticulum unknown mitochondrial inner membrane

158

32

mitochondrial outer membrane

178

34

mitochondrial inner membrane

106 94 86 218 220 103

56 61 31 60 50 38

cytoplasmic cytoplasmic cytoplasmic mitochondrial matrix cytoplasmic cytoplasmic

121 113

28 40

cytoplasmic cell membrane

245

53


144

23

mitochondrial

55

13

mitochondrial

74

22

unknown

100

19


127

45

mitochondrial

230 88

47 24

unknown cytoplasmic

Journal of Proteome Research • Vol. 3, No. 4, 2004 827

research articles

Li et al.

Table 2 (Continued) spot no.a

59 60

P04075 P48735

61

P00558

62

P24752

70 72 73 74 75 76 77 78 79 82 84 85 87 88 89 90

94 98 101 103 106 108 109 110 114 115 118 119 121 122 123 125 127 131 132

828

acc no.b

protein name

Fructose-bisphosphate aldolase A Isocitrate dehydrogenase [NADP], mitochondrial Phosphoglycerate kinase 1

Acetyl-CoA acetyltransferase, mitochondrial Q9Y285 Phenylalanyl-tRNA synthetase alpha chain P00326 Alcohol dehydrogenase gamma chain P00505 Aspartate aminotransferase, mitochondrial P46439 Glutathione S-transferase Mu 5 P09622 Dihydrolipoamide dehydrogenase, mitochondrial P23368 NAD-dependent malic enzyme, mitochondrial Q9HCC0 Methylcrotonyl-CoA carboxylase beta chain P31939 Bifunctional purine biosynthesis protein PURH P12532 Creatine kinase, ubiquitous mitochondrial P48047 ATP synthase oligomycin sensitivity conferral protein, mitochondrial P00938 Triosephosphate isomerase P17080 GTP-binding nuclear protein RAN (TC4) P54868 Hydroxymethylglutaryl-CoA synthase, mitochondrial P06733 Alpha enolase P07954 Fumarate hydratase, mitochondrial P11310 Acyl-CoA dehydrogenase, medium-chain specific, mitochondrial Q92947 Glutaryl-CoA dehydrogenase, mitochondrial P00338 L-lactate dehydrogenase A chain P24539 ATP synthase B chain, mitochondrial P04406 Glyceraldehyde 3-phosphate dehydrogenase O75874 Isocitrate dehydrogenase [NADP] cytoplasmic P00352 Aldehyde dehydrogenase 1A1 P12268 Inosine-5′-monophosphate dehydrogenase 2 P40926 Malate dehydrogenase, mitochondrial P17174 Aspartate aminotransferase, cytoplasmic O60547 GDP-mannose 4,6 dehydratase Q16836 Short chain 3-hydroxyacyl-CoA dehydrogenase,mitochondrial Q02338 D-beta-hydroxybutyrate dehydrogenase, mitochondrial O95154 Aflatoxin B1 aldehyde reductase 2 P40925 Malate dehydrogenase, cytoplasmic Q9NR45 Sialic acid synthase O60218 Aldo-keto reductase family 1 member B10 P54819 Adenylate kinase isoenzyme 2, mitochondrial Q16822 Phosphoenolpyruvate carboxykinase, mitochondrial P00367 Glutamate dehydrogenase 1, mitochondrial


SW acc no.c

MW (Dal)d

pId

scoree cov. (%)f


ALFA_HUMAN IDHP_HUMAN

39720.44 8.39 51333.03 8.88

167 128

30 35

cytoplasmic mitochondrial

PGK1_HUMAN

44967.32 8.3

152

35

THIL_HUMAN

45455.75 8.98

79

22

mitochondrial & sarcoplasmic reticulum mitochondrial

SYFA_HUMAN

57584.53 7.31

75

25

cytoplasmic

ADHG_HUMAN

40565.95 8.63

203

46

cytoplasmic

AATM_HUMAN

47844.42 9.14

139

29


GTM5_HUMAN DLDH_HUMAN

25716.21 7.3 54686.14 7.59

60 129

28 32

cytoplasmic mitochondrial matrix

MAOM_HUMAN

66029.02 7.53

139

29


MCCB_HUMAN

61807.6

7.57

234

51


PUR9_HUMAN

65088.53 6.27

126

35

unknown

KCRU_HUMAN

47406.36 8.6

81

21

ATPO_HUMAN

23376.7

9.97

164

55

mitochondrial inner membrane mitochondrial matrix

TPIS_HUMAN RAN_HUMAN

26806.81 6.51 24578.68 7.01

192 107

85 45

cytoplasmic nuclear

HMCM_HUMAN

57112.61 8.4

119

26

mitochondrial

ENOA_HUMAN FUMH_HUMAN

47350.41 6.99 54773.23 8.85

220 116

49 28

ACDM_HUMAN

47014.75 8.61

95

32

cytoplasmic mitochondrial and cytoplasmic mitochondrial matrix

GCDH_HUMAN

48609.63 8.31

60

24


LDHA_HUMAN ATPF_HUMAN G3P2_HUMAN

36819.43 8.46 28947.36 9.37 36070.42 8.58

109 68 117

23 27 35

cytoplasmic mitochondrial cytoplasmic

IDHC_HUMAN

46914.62 6.53

177

52

DHA1_HUMAN IMD2_HUMAN

55323.14 6.29 56225.82 6.44

168 92

36 24

cytoplasmic and peroxisomal cytoplasmic unknown

MDHM_HUMAN 35964.93 8.92

186

52


AATC_HUMAN

46315.57 6.57

189

48

cytoplasmic

GMDS_HUMAN HCDH_HUMAN

42265.38 6.87 34312.93 8.88

165 67

41 16

unknown mitochondrial matrix

BDH_HUMAN

38531.61 9.11

61

18


AR73_HUMAN

37581.67 6.67

78

42

cytoplasmic

MDHC_HUMAN

36500.06 6.89

106

30

cytoplasmic

SIAS_HUMAN AKBA_HUMAN

40737.62 6.29 36225.91 7.12

153 88

41 42


KAD2_HUMAN

26557.82 7.85

60

31

PPCM_HUMAN

71446.93 7.56

158

34

mitochondrial intermembrane space mitochondrial

DHE3_HUMAN

61701.32 7.66

171

41


research articles

Alkaline Proteins in Human Colon Crypt Table 2 (Continued) spot no.a

133 135 136 140

144 146 147 149 152 177 182 186 189 191 197 209 213 226 233 234 244 248

acc no.b

protein name

P36871 P42765

Phosphoglucomutase 3-ketoacyl-CoA thiolase, mitochondrial P50416 Carnitine O-palmitoyltransferase I, mitochondrial P21912 Succinate dehydrogenase [ubiquinone] iron-sulfur protein, mitochondrial P12277 Creatine kinase, B chain Q92931 3-hydroxyisobutyryl-coenzyme A hydrolase P14550 Alcohol dehydrogenase [NADP+] P26440 Isovaleryl-CoA dehydrogenase, mitochondrial Q08426 Peroxisomal bifunctional enzyme (PBE) Q02127 Dihydroorotate dehydrogenase, mitochondrial P22234 Multifunctional protein ADE2 P18283 Glutathione peroxidasegastrointestinal Q9Y2Q3 Glutathione S-transferase, mitochondrial Q99714 3-hydroxyacyl-CoA dehydrogenase type II P36542 ATP synthase gamma chain, mitochondrial P07814 Bifunctional aminoacyl-tRNA synthetase P53007 Tricarboxylate transport protein, mitochondrial P30043 Flavin reductase O43837 Isocitrate dehydrogenase [NAD] subunit P37837 Transaldolase Q8WTW8 Hypothetical protein Q02252 Methylmalonate-semialdehyde dehydrogenase

27 64

Q99832 P23284

124

P11142

194

Q04984

231

P50991

34 86 93 113 128 183 184 236 241

P20618 P25787 P49721 O14818 P25789 P28062 P25774 Q9BSD7 Q92524

193

T-complex protein 1, eta subunit Peptidyl-prolyl cis-trans isomerase B Heat shock cognate 71 kDa protein 10 kDa heat shock protein, mitochondrial T-complex protein 1, delta subunit

SW acc no.c

MW (Dal)d

pId

scoree cov. (%)f


PGMU_HUMAN THIM_HUMAN

61565 42469

6.32 72 8.51 124

29 41


CPT1_HUMAN

89056

8.85

64

11

mitochondrial outer membrane

DHSB_HUMAN

32407

9.03

76

25


KCRB_HUMAN Q92931

42902 43279

5.34 8.34

78 64

28 18

cytoplasmic unknown

AKA1_HUMAN IVD_HUMAN

36761 46803

6.34 8.45

55 55

20 10

unknown mitochondrial matrix

ECHP_HUMAN

79843

9.22 164

35

peroxisomal

PYRD_HUMAN

42900

9.67

78

22


PUR6_HUMAN GPX2_HUMAN

47700 22100

6.95 55 7.6 116

18 51

de novo purine biosynthesis cytoplasmic mainly

GTK1_HUMAN

25400

8.53 106

60

HCD2_HUMAN

27100

7.66 165

75

mitochondrial matrix (by similarity) unknown

ATPG_HUMAN

33000

9.23

60

27

mitochondrial

SYEP_HUMAN

163026.31 7.77

60

9

TXTP_HUMAN

32738.14 9.81

80

22


FLRE_HUMAN IDHB_HUMAN

21988.16 7.31 42469.76 7.82

55 70

42 29


TAL1_HUMAN Q8WTW8 MMSA_HUMAN

37687.52 6.36 25474 9.4 58259 8.72

76 60 60

19 38 14

cytoplasmic unknown mitochondrial

chaperones TCPH_HUMAN 59842.12 7.55 121 PPIB_HUMAN 22785.03 9.33 141

32 50

cytoplasmic endoplasmic reticulum lumen

HS7C_HUMAN

71082.31 5.37

72

16

cytoplasmic

CH10_HUMAN

10700

8.91

79

52


7.52

cytoplasmic and nuclear

TCPD_HUMAN 58315.89 protein turnover PSB1_HUMAN 26700.45 PSA2_HUMAN 25865.27 PSB2_HUMAN 22992.74 O14818 28040.68 PSA4_HUMAN 29700 PSB8_HUMAN 30600 CATS_HUMAN 38100 Q9BSD7 20928 PRSX_HUMAN 44430.21

66

19

cytoplasmic

8.27 69 7.12 55 6.51 106 8.6 121 7.57 55 7.63 73 8.61 88 9.61 117 7.09 60

46 42 41 49 24 33 35 66 14

cytoplasmic and nuclear cytoplasmic and nuclear cytoplasmic and nuclear cytoplasmic and nuclear cytoplasmic and nuclear cytoplasmic and nuclear lysosomal unknown cytoplasmic and nuclear

Q9UBQ0 Vacuolar protein sorting 29

organelle trafficking VP29_HUMAN 20600

6.29 117

47

cytoplasmic

23

P13804

antioxidant proteins ETFA_HUMAN 35399.71 8.62 100

36


80

P22695

UCR2_HUMAN

48583.95 8.74 114

35

126

P38117

ETFB_HUMAN

28054.24 8.24 102

50

mitochondrial inner membrane; matrix side mitochondrial matrix

130 198

P16152 P04179

DHCA_HUMAN SODM_HUMAN

30500 24800

35 27

cytoplasmic mitochondrial matrix

Proteasome subunit beta type 1 Proteasome subunit alpha type 2 Proteasome subunit beta type 2 Proteasome subunit alpha type 7 Proteasome subunit alpha type 4 Proteasome subunit beta type 8 Cathepsin S precursor Hypothetical protein 26S protease regulatory subunit S10B

Electron-transfer flavoprotein alpha-subunit Ubiquinol-cytochrome C reductase complex Electron-transfer flavoprotein beta-subunit Carbonyl reductase [NADPH] 1 Superoxide dismutase [Mn], mitochondrial

8.55 8.35

66 82


research articles

Li et al.

Table 2 (Continued) spot no.a

219

32 33 57 129 137

160 166 181 187 200 207 211 216 220 221

235 237 11 46 163

67

222

158 230 176 215 54 56 229 232 151 178 35 36 13 4

830

acc no.b

P15559

protein name

NAD(P)H dehydrogenase [quinone] 1

SW acc no.c

NQO1_HUMAN

MW (Dal)d

30867.65

pId

8.91

molecular function unknown proteins JE0350 Anterior gradient-2 O95994 20000 9.03 O60844 Homologue of rat zymogen O60844 (AAC08708) 18336 9.43 AAC08708 granule membrane protein Q9BT58 Similar to RIKEN cDNA Q9BT58 37400 5.83 2610207I16 gene Q9NX63 Hypothetical protein FLJ20420.Q9NX63 26400 8.48 Homo sapiens (Human). Q9BTT5 Similar to NADH dehydrogenase Q9BTT5 38475 9.68 (Ubiqionone) 1 alpha subcomplex, 9 Q8NAT0 Hypothetical protein FLJ34830 Q8NAT0 39497 8.45 Q8N458 Similar to RIKEN cDNA Q8N458 24273 9.37 C030006K11 gene Q9H245 C1orf28 Q9H245 60693 9.63 Q8TD06 Anterior gradient protein 3 Q8TD06 19200 7.77 AAH04534 Similar to splicing factor Q9BSV4 69016 8.98 proline/glutamine rich Q9BTH4 RIKEN cDNA 1110037F02 gene.Q9BTH4 122486 6.01 Homo sapiens (Human). Q96AG0 Hypothetical protein.Q96AG0 101996.99 6.74 Homo sapiens (Human). P37802 Transgelin 2 (SM22-alpha TAG2_HUMAN 22391.45 8.41 homolog) P30042 ES1 protein homolog, ES1_HUMAN 24016.57 6.63 mitochondrial CAC69721 Sequence 48 from Patent CAC69721 23515 7 WO0162932 (Fragment).Homo sapiens (Human). Q9Y376 MO25 protein (CGI-66) MO25_HUMAN 40014.8 6.43 Q9H9B4 Sideroflexin 1 SFX1_HUMAN 35881.45 9.22 transporter, intracellular transporter Q9UJS0 Calcium-binding mitochondrial CMC2_HUMAN 74527.79 8.79 carrier protein P46459 Vesicle-fusing ATPase NSF_HUMAN 83115.25 6.38 P05141 ADP,ATP carrier protein, ADT2_HUMAN 33102 9.76 fibroblast isoform ion channel Q9Y277 Voltage-dependent anionPOR3_HUMAN 30981.41 8.85 selective channel extracellular matrix P02452 Collagen alpha 1(I) chain CA11_HUMAN 94738.01 9.29 precursor DNA repair P13051 Uracil-DNA glycosylase, UNG_HUMAN 34188 9.56 mitochondrial P09874 Poly [ADP-ribose] polymerase-1 PPOL_HUMAN 112952.6 8.99 cell proliferation Q14011 Cold-inducible RNA-binding CIRP_HUMAN 18637 9.51 protein P00568 Adenylate kinase isoenzyme 1 KAD1_HUMAN 21634.84 8.73 miscellaneous P04040 Catalase CATA_HUMAN 59815.8 6.95 P43490 Pre-B cell enhancing factor PBEF_HUMAN 55771.7 6.69 precursor Q14126 Desmoglein 2 precursor (HDGC) DSG2_HUMAN 116761.3 4.93 Q16181 Septin 7 (CDC10 protein SEP7_HUMAN 49041.23 8.85 homolog) Q99959 Plakophilin 2 PKP2_HUMAN 97852 9.39 P38159 Heterogeneous nuclear ROG_HUMAN 42306 10.06 ribonucleoprotein G Q9UFN0 NipSnap4 protein (HSPC299) NPS4_HUMAN 28562.58 9.21 Q9UNF6 Calcium-activated chloride channel Q9UNF6 100881 5.97 protein 1 O95340 Bifunctional 3′-phosphoadenosine PPS2_HUMAN 70026.81 8.18 5′-phosphosulfate synthethase 2 P12750 40S ribosomal protein S4, X RS4_HUMAN 29676.1 10.16 isoform


scoree cov. (%)f


55

18

cytoplasmic

62 86

36 71

cell membrane unknown

103

31

unknown

68

40

unknown

89

29

unknown

60 73

21 38

unknown unknown

94 76 98

24 45 35

unknown unknown unknown

55

12

unknown

264

32

unknown

102

48

unknown

64

18

mitochondrial

74

40

unknown

69 55

21 19

unknown mitochondrial

90

23

88 60

23 18

mitochondrial inner membrane cytoplasmic mitochondrial inner membrane

87

31

outer mitochondrial membrane

59

14

extracellular matrix

60

21

60

12

nuclear and mitochondrial nuclear

84

53

nuclear; nucleoplasm

91

28

cytoplasmic

187 84

42 22

peroxisomal secreted

55 75

11 25

cell membrane cytoskeletal

71 72

19 21

62 142

40 17

nuclear nuclear; ribonucleosomes unknown cell membrane

125

20

cytoplasmic

55

19

ribosome

research articles

Alkaline Proteins in Human Colon Crypt Table 2 (Continued) spot no.a

31 102 37 138

172 203 204 239 139 68 111 156 167 30 243

81

83 105 134 142 143 150 157

175 188 190 196 199 201

acc no.b

protein name

SW acc no.c

P22392 P42330

MW (Dal)d

pId

Nucleoside diphosphate kinase B NDKB_HUMAN 17401 8.52 Aldo-keto reductase family 1 AKC3_HUMAN 37219.99 8.05 member C3 Q06830 Peroxiredoxin 1 PDX1_HUMAN 22324.36 8.27 Q99623 B-cell receptor associated protein Q99623 33276 9.83 (D-prohibitin) (B-cell associated protein).- Homo sapiens (Human) P02278 Histone H2B.a/g/k H2BA_HUMAN 13767 10.32 P16106 Histone H3.1 H31_HUMAN 15377 11.13 P57053 Histone H2B.s H2BS_HUMAN 13805 10.37 O95644 Nuclear factor of activated TNFC1_HUMAN 102376.94 6.52 cells, cytoplasmic P12236 ADP,ATP carrier protein, ADT3_HUMAN 33073 9.76 liver isoform P17931 Galectin-3 (Galactose-specific LEG3_HUMAN 26098.04 8.61 lectin 3 P56470 Galectin-4 (Lactose-binding LEG4_HUMAN 36032.23 9.21 lectin 4) O00182 Galectin-9 (HOM-HD-21) LEG9_HUMAN 39835 9.34 (Ecalectin) O75251 NADH-ubiquinone oxidoreductase NUKM_HUMAN 23849 10.02 20 kDa subunit P05092 Peptidyl-prolyl cis-trans PPIA_HUMAN 18097.93 7.82 isomerase A P43034 Platelet-activating factor LIS1_HUMAN 47047.33 7.03 acetylhydrol intermediary metabolism & antioxidant proteins P49821 NADH-ubiquinone NUBM_HUMAN 51469.05 8.51 oxidoreductase 51 kDa subunit, mitochondrial P56556 NADH-ubiquinone NB4M_HUMAN 14900 10 oxidoreductase B14 subunit Q16698 2,4-dienoyl-CoA reductase, DECR_HUMAN 36329.91 9.35 mitochondrial P00390 Glutathione reductase, GSHR_HUMAN 56791 8.74 mitochondrial O96000 NADH-ubiquinone oxidoreductase NIDM_HUMAN 20917 8.77 PDSW subunit P30044 Peroxiredoxin 5, PDX5_HUMAN 22298 8.85 mitochondrial O95299 NADH-ubiquinone NUDM_HUMAN 41067 8.67 oxidoreductase 42 kDa Q16795 NADH-ubiquinone NUEM_HUMAN 42654 9.81 oxidoreductase 39 kDa subunit, mitochondrial O95139 NADH-ubiquinone NB7M_HUMAN 15348 9.63 oxidoreductase B17 subunit P14927 Ubiquinol-cytochrome C UCR6_HUMAN 13400 8.75 reductase complex O95168 NADH-ubiquinone NB5M_HUMAN 15100 9.85 oxidoreductase B15 subunit Q9Y6M9 NADH-ubiquinone NI2M_HUMAN 21900 8.59 oxidoreductase B22 subunit Q9P0J0 NADH-ubiquinone NB6M_HUMAN 16500 8.23 oxidoreductase B16.6 subunit CAA41703 STEROID 21Q16746 42438 8.85 MONOOXYGENASE (FRAGMENT)

scoree cov. (%)f


55 157

26 52

nuclear and cytoplasmic cytoplasmic

199 161

58 57

cytoplasmic unknown

60 55 60 55

29 28 29 12

nuclear nucleus nucleus cytoplasmic and nuclear

66

31

138

36

mitochondrial inner membrane nuclear

83

32

110

29

cytoplasmic & plasma membrane unknown

55

18

unknown

118

67

cytoplasmic

55

25

cytoplasmic

85

34

mitochondrial inner membrane, matrix side

62

37

75

29

mitochondrial inner membrane; matrix side mitochondrial

66

27

55

30

57

25

mitochondrial and cytoplasmic mitochondrial inner membrane, matrix side mitochondrial

87

26


60

20


80

55

66

49

63

39

64

56

114

48

60

17

mitochondrial inner membrane; matrix side mitochondrial inner membrane mitochondrial inner membrane; matrix side mitochondrial inner membrane; matrix side mitochondrial inner membrane; matrix side mitochondrial

The proteins are grouped based on their physiological functions. a Spot numbers refer to the number assigned on 2-DE gel shown in Figure 3. b NCBI protein accession numbers. c Protein entry name according to SWISS-PROT database. d Theoretical value. e Score from Mascot database search results. f Sequence coverage.

colon mucosa, but not in carcinoma cells expressing chromogranin A, implicating an extra-ribosomal function for this protein.28 In our study, five ribosomal proteins, among a total of 18 identified, were previously implicated in extra-ribosomal functions and tumorigenesis. S11 was shown to be specifically down-regulated in breast carcinoma MCF-7 cells undergoing staurosporin-induced apoptosis, suggesting an anti-apoptotic

function.29 Ribosomal protein S3 was identified as an ultraviolet endonuclease III DNA-repair enzyme, which cleaves DNA in response to ultraviolet irradiation.30 Enhancement of ribosomal protein S3a expression was shown to induce transformation of NIH 3T3 cells and to induce tumors in nude mice by up regulating anti-apoptotic proteins.31 Ribosomal protein L7 was proposed to be involved in apoptotic induction by specifJournal of Proteome Research • Vol. 3, No. 4, 2004 831

research articles

Li et al.

Figure 6. Functional classification of 231 identified proteins from human colon crypt whole cells. The 17 functional groups are the same as annotated in Gene Ontology database and Human Protein Reference Database. The total number of proteins in each category is shown on top of each bar.

Figure 4. Distribution of the 231 identified proteins from human colon crypt whole cell extract in relation to their theoretical isoelectric point (pI) (A), and molecular weight (B). The total number of proteins in each interval, pI or molecular weight respectively, is shown on top of each bar.

Figure 5. Subcellular locations of 231 identified proteins from human colon crypt whole cell extract. The 16 sub-cellular location categories are the same as annotated in the Swiss-Prot database and Human Protein Reference Database. The total number of proteins in each category is shown on top of each bar.

ically inhibiting the synthesis of anti-apoptotic factors, further implicating a role for some ribosomal proteins in apoptosis.32 We have also identified 71 mitochondrial proteins, consisting of 30.7% of total identified proteins in our present study. More than 70 years ago, Otto Warburg proposed that cancer cells may have impaired mitochondrial function resulting from elevated glycolysis and decreased respiration, which is a common feature of most tumors.33 Although the presumed impairment of mitochondrial function in cancer biology was never explained, in recent years it has been shown that 832


mitochondria play an essential role as sensors and executioners of apoptosis. The mitochondrial proteins ATP synthase R-, β-, and γ-chains were among the proteins identified in our study with pI values higher than 9. Cuezva et al. showed that the expression of beta-F1-ATPase and beta catalytic subunit of H+ATP synthase are selectively repressed in colon and kidney carcinomas, concurrent with increase in the expression of glyceraldehyde-3-phosphate dehydrogenase.34 In summary, through improvements of keeping proteins dissolved and focused during IEF, the most alkaline proteins of the colon crypt were well-resolved and characterized in this study. This established protocol provides the most complete view to date of the high pI colon crypt proteome and will allow a better characterization of the proteomic differences between normal and neoplastic crypts in the future. A better definition of the human colon crypt proteome will assist further understanding of CRC tumorigenesis, development, early detection and prevention. Abbreviations: 2-DE, 2-dimensional electrophoresis; APC, adenomatous polyposis coli; CRC, colorectal cancer; DTT, dithiothreitol; HED, hydroxyethyl disulfide; IEF, iso-electric focusing; IPG, immobilized pH gradient; MALDI-TOF MS, matrix assisted laser desorption-ionization time-of-flight mass spectrometry.

Acknowledgment. The Authors are grateful to Dr. L. H. Cohen, Fox Chase Cancer Center, for providing the purified histone proteins to test our protocol; to Dr. B. Bjellqvist, Amersham Biosciences, for communication concerning DeStreak (hydroxyethyl disulfide) application. We thank Drs. A. Bellacosa and A. G. Knudson for critical reading of the manuscript. This study was supported by National Cancer Institute grant NO1-CN-15103-MAO (Workstatement #40) to A. G. Knudson, DK063014 to B. Boman; We acknowledge FCCC Institutional Core Grant P30CA06927-41, The Pew Charitable Trust, The Sho¨ller Foundation and an appropriation from the State of Pennsylvania, for the purchase of the proteomics equipment. Note Added after Print Publication: This manuscript was published on the Web (05/06/2004) and in Print (08/09/2004) with a misspelled author name. The correct spelling is Steven H. Seeholzer. The correct electronic version of the manuscript

research articles


was published on 10/19/2004, and an Addition and Correction appears in the November/December 2004 issue (Vol. 3, No. 6).

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