Quantitative Protein Expression Analysis Of CLL B Cells from Mutated

Synopsis. Protein expression levels were compared in two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (...
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Quantitative Protein Expression Analysis Of CLL B Cells from Mutated and Unmutated IgVH Subgroups Using Acid-Cleavable Isotope-Coded Affinity Tag Reagents David R. Barnidge,*.†,‡ Diane F. Jelinek,§ David C. Muddiman,†,‡ and Neil E. Kay| Mayo Proteomics Research Center, Department of Biochemistry and Molecular Biology, Department of Immunology, and Department of Internal Medicine, Division of Hematology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905 Received February 11, 2005

Relative protein expression levels were compared in leukemic B cells from two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy chain genes (IgVH). Cells were separated into cytosol and membrane protein fractions then labeled with acid-cleavable ICAT reagents (cICAT). Labeled proteins were digested with trypsin then subjected to SCX and affinity chromatography followed by LC-ESI-MS/MS analysis on a linear ion trap mass spectrometer. A total of 9 proteins from the cytosol fraction and 4 from the membrane fraction showed a 3-fold or greater difference between M-CLL and UM-CLL and a subset of these were examined by Western blot where results concurred with cICAT abundance ratios. The abundance of one of the proteins in particular, the mitochondrial membrane protein cytochrome c oxidase subunit COX G was examined in 6 M-CLL and 6 UM-CLL patients using western blot and results showed significantly greater levels (P < 0.001) in M-CLL patients vs UM-CLL patients. These results demonstrate that stable isotope labeling and mass spectrometry can complement 2D gel electrophoresis and gene microarray technologies for identifying putative and perhaps unique prognostic markers in CLL. Keywords: CLL • B cell • quantitative comparison • cICAT • UM-CLL • M-CLL • linear ion trap mass spectrometer

Introduction Chronic lymphocytic leukemia is an incurable adult B cell malignancy that occurs in both men and women and is the most frequently observed leukemia in Western countries.1 While the cause of CLL is not yet known, it is known that a subset of individuals with CLL have more aggressive disease that requires treatment while others have an indolent form of the disease that is stable over many years. While clinical diagnosis of CLL is routinely performed by examining blood lymphocytes using flow cytometry,2 a prognosis for each patient is difficult to determine using routinely available clinical analyses and laboratory tests. Therefore, efforts have focused on finding new prognostic tests that can assist physicians in determining efficient treatment options for patients. Presently, the most widely accepted predictor that can distinguish between aggressive and nonaggressive forms of CLL is the mutational status of the genes encoding the variable region for the heavy chain of the B cell receptor (IgVH). Studies * To whom correspondence should be addressed. Mayo Proteomics Research Center, Mayo Clinic College of Medicine, 200 1st Street SW, Rochester, MN 55905. Phone: (507) 266-4777. Fax: (507) 284-9261. E-mail: [email protected]. † Mayo Proteomics Research Center. ‡ Department of Biochemistry and Molecular Biology. § Department of Immunology. | Department of Internal Medicine.

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have demonstrated that patients with unmutated IgVH genes (UM-CLL) are likely to have a more aggressive form of the disease as compared to patients with mutated genes (IgVH) (MCLL).3,4 Due to the cost and time associated with sequencing of these genes, surrogate prognostic indicators that can distinguish between patients with M-CLL and UM-CLL have been sought after using DNA microarray technology. An example of a prognostic protein identified on the transcript level using this approach is the tyrosine kinase ZAP-70. Subsequent studies have shown that ZAP-70 can indeed distinguish between M-CLL and UM-CLL in a majority of CLL patients using protein based immunological techniques such as western blot and flow cytometry.5 We have recently reviewed several of the novel prognostic indicators, including ZAP-70 and IgVH mutational status, in relation to their clinical prognostic value6 and have found that although progress has been made, there is still the need for a prognostic indicator that can be readily and easily quantified. In this study, we use a proteomic approach as opposed to a DNA microarray technology approach to begin identifying the relative levels of proteins expressed in M-CLL versus UM-CLL. Others groups have examined CLL B-cells using a proteomic approach both on a qualitative level using 1DE7 or LC-MS/ MS,8 and on a quantitative level using 2DE.9,10 We show here the combined use of cell fractionation, acid cleavable isotope coded affinity tags (cICAT) and LC-ESI-MS/MS,11-13 as a 10.1021/pr050028f CCC: $30.25

 2005 American Chemical Society

Protein Expression Analysis of M-CLL vs UM-CLL

means of quantifying proteins that could be used to distinguish between a patient with M-CLL versus a patient with UM-CLL. The comparison of two patients using a shortened protocol with a single SCX cleanup step in combination with an extended reverse-phase LC gradient resulted in the identification of 13 proteins that were expressed at a 3-fold level or greater between M-CLL versus UM-CLL. Western blots were performed on proteins with equal ratios as well as proteins exhibiting a 3-fold or greater difference to validate the labeling experiments at an intact protein level.

Materials and Methods CLL B-Cell Isolation. Peripheral blood was obtained from two untreated patients diagnosed with classical B-cell CLL2 using protocols approved by the Mayo Clinic Institutional Review Board under IRB 1827-00. Approximately 0.5-1 × 109 peripheral blood mononuclear cells were isolated by FicollPaque gradient centrifugation from 10 mL of blood, washed once with cold phosphate buffered saline (PBS) then frozen at -80 °C. Using two color flow cytometry, g95% of the mononuclear cells were positive for both CD5 and CD19. Residual erythrocytes were lysed when the cells were thawed by adding RBC lysis buffer for 10 min followed by two washings with cold PBS prior to cytosol and membrane fractionation. The two CLL patients were determined to have either mutated (M-CLL) IgVH genes or unmutated (UM-CLL) IgVH genes based on our previously published techniques.14 Cytosol and Membrane Fractionation. Isolated cells were kept on ice and disrupted using sonication for 20 s in PBS. Crude membranes were separated from cytosol by ultracentrifugation at 100 000 × g for 45 min. The supernatant was saved and used as the cytosol fraction. The crude membrane pellet was then resuspended in PBS containing 1 M NaCl using sonication. The suspended crude membranes were subjected to ultracentrifugation again at 100 000 × g for 45 min then solubilized in 100 mM NH4HCO3 buffer pH 8.0 containing 6 M urea and 1% octylglucoside using sonication followed by stirring at 4 °C for 30 min. Total protein concentrations were determined in triplicate for cytosol and crude membrane fractions using the BCA colorimetric protein assay with bovine serum albumin as the standard (Pierce, Rockford, IL). cICAT Labeling of Samples. Equal amounts of protein from each patient were subjected to labeling. Membrane fractions from each patient had similar total protein concentrations; however, the total protein concentration in the cytosol fraction from the M-CLL patient was lower than the UM-CLL patient. Rather than labeling a larger volume of cytosol from the M-CLL patient and a smaller volume of cytosol from the UM-CLL patient (equal amounts of protein in each), the M-CLL cytosol fraction was concentrated using a 10 kDa cutoff Microcon concentrator (Millipore Corp., Bedford, MA). To keep sample handling procedures equal, the UM-CLL cytosol fraction was diluted to equal the volume of the M-CLL cytosol fraction then both samples were concentrated to an equal volume. After concentrating fractions were diluted 1:1 with 6M Gdn‚HCl in 50 mM NH4HCO3, pH 8.3 then reduced with TBP at a concentration of 5 mM for 1 h at room temperature with mixing and intermittent sonication. After reduction, each sample was alkylated with a 2-fold molar excess of cICAT reagent (ABI, Framingham, MA) for 2 h at 37 °C according to the manufacturer’s instructions. The patient with a mutated IgVH gene status (M-CLL) was labeled with the lightscICAT reagent (13C0) while the patient with an unmutated IgVH gene status (UM-CLL) was

research articles labeled with heavyscICAT reagent (13C9). After labeling samples were combined and desalted using a 10 mL Fast Desalting column on a Bio-Rad PathFinder FPLC system into 50 mM NH4HCO3, pH 8.0 to a final volume of 1.5 mL. The combined sample was boiled for 10 min then allowed to cool prior to digestion using 60 µg of trypsin (Promega, Madison, WI) and a proteolysis time of 12 h at 37 °C. The sample was boiled for 10 min to stop the digestion then acidified to pH 3.0 using TFA. The sample was then subjected to SCX using the cartridge column provided by the manufacturer and following the manufacturer’s instructions (ABI, Framingham, MA). Eluate from the SCX column was brought to neutral pH using buffers supplied by the manufacturer then cICAT labeled peptides were purified using an avidin cartridge provided by the manufacturer per the manufacturer’s instructions (ABI, Framingham, MA). ICAT labeled peptides eluted from the avidin affinity cartridge had the acid cleavable portion of the ICAT label removed per the manufacturer’s instructions and lyophilized to dryness. The sample was hydrated into 50 µL of 98% v/v water, 1% v/v n-propanol, 1% v/v acetonitrile, 0.5% v/v formic acid then centrifuged at 20 000 × g for 5 min prior to analysis. Capillary Reverse Phase LC. Labeled peptides were subjected to capillary reverse-phase LC using a LC Packings Ultimate system (Dionex Corp., Sunnyvale, CA). A volume of 5 µL was injected onto a Pepmap C18 cartridge trap column (300 µm × 0.5 cm; LC Packings, Amsterdam, NL) via a FAMOS autosampler. Trapped peptides were washed for 2 min at 20 µL/minute with mobile phase A (98% v/v water, 1% v/v n-propanol, 1% v/v acetonitrile, 0.2% v/v formic acid). Using a 10-port valve, the Pepmap column was put in-line with the gradient and the reverse-phase column. Peptides were separated using a 160 min gradient starting with 2% mobile phase B (96% v/v acetonitrile, 2% v/v n-propanol, 2% v/v water, 0.2% formic acid) for 5 min then ramping from 2 to 45% B over 80 min, stopping at 45% B for 10 min, then ramping from 45 to 95% B over 110 minutes stopping for 10 min then ramping back to 2% B. Empty IntegraFrit capillary columns (New Objective. Woburn, MA) were packed in-house for the analysis with TARGA C18 packing material, 5 µm bead size and 120 Å pore size, (Higgins Analytical, Mountain View, CA) in a 75 µm i.d. × 360 µm o.d. fused silica capillary with a length of 5 cm. The flow rate for the reversed-phase analysis was set by splitting the LC pump flow from 25 µL/minute to approximately 300 nL/minute. A 50 µm i.d. × 15 µm tip ES emitter (New Objective. Woburn, MA) was connected to the other end of the union and a positive electrospray voltage of approximately 1 kV was applied via the union. MS/MS Data. All MS and MS/MS spectra were collected on a linear ion-trap mass spectrometer (LTQ, Thermo Finnigan, San Jose, CA.). The instrument was set up to operate in a ‘triple play’ mode where the three most intense precursor ions found in a scan range from 500 to 1500 m/z were selected for MS/ MS. Each precursor ion was subjected to a broad range zoom scan (( 10 Da scan window) followed by collision-induced dissociation (CID) using normalized collision energy of 35%. The MS precursor scan was acquired in 1 µscan while zoom scans were acquired using 4 µscans and CID spectra were acquired using 3 µscans. Raw MS/MS files were searched using SEQUEST against the human subset of Swiss-Prot/TREMBL protein database where cysteine residues were modified to include the addition of the acid-cleavable ICAT labels (227.13 Da for the light label and 236.16 Da for the heavy label) along with oxidation of methionines. Protein identifications were Journal of Proteome Research • Vol. 4, No. 4, 2005 1311

research articles sorted in Bioworks browser by filtering data according to Xcorr values. The following Xcorr values were used to accept peptide assignments in SEQUEST for precursor ions; Xcorr g 2.0 for [MH+]+1, Xcorr g 2.0 for [MH2+]+2, and Xcorr g 2.8 for [MH3+]+3.15 The use of these Xcorr value criteria serves as a filter reducing the number of peptides reported in BioWorks from a SEQUEST search that are likely false positives. All protein abbreviations listed are taken from the Swiss-Prot protein database located at http://us.expasy.org/cgi-bin/sprot-search-de and can be found using the suffix, _HUMAN (i.e., CD5_HUMAN). All ICAT ratios found by the XPRESS software option in Bioworks were manually confirmed, as were all MS/MS spectra. Protein abundance ratios were calculated using the area obtained for the average mass of each labeled peptide pair with the mean abundance used for proteins having multiple peptide identifications. Western Blot Analysis. Western blot analysis was performed according to the method of Towbin.16 Briefly, samples were diluted to a concentration of 0.5 µg/mL using Laemmli buffer and separated by 1DE using precast Criterion gels (Bio-Rad Corp., Hercules, CA). Gels were equilibrated in transfer buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol, pH 8.3 then transferred to nitrocellulose blotting material under constant voltage (100 V for 30 min). Membranes were blocked with casein in TBS, pH 7.4 (Blocker Casein, Bio-Rad Corp., Hercules, CA) for >1 h prior to the addition of the primary antibody and all primary and secondary antibodies were diluted in 10% blocker casein and 0.1% Tween 20 in TBS, pH 7.4. Primary antibodies were allowed to equilibrate for 2 h then the blots were washed 5 times, 5 min each, with TBS, pH 7.4 containing 0.05% Tween 20. The primary antibodies used include the following; anti-actin, mouse mAb 1:5000 dilution (Novus Biologicals, Littleton, CO, Cat# NB 600-501), anti-A32A, rabbit polyclonal Ab 1:2,500 dilution (Novus Biologicals, Littleton, CO, Cat# ab5989), anti-tubulin, mouse mAb 1:5,000 dilution (Novus Biologicals, Littleton, CO, Cat# ab7291), antiCOXVIb, mouse mAb 1:5,000 dilution (Molecular Probes, Inc., Eugene, OR, Cat# A21366). Secondary antibodies were either goat anti-mouse or goat anti-rabbit HRP conjugated monoclonal antibodies (Jackson Immuno Research Laboratories, West Grove, PA) and were used at a dilution of 1:40 000. Proteins were visualized on imaging film (Biomax, Eastman Kodak Company, Rochester, NY) by chemiluminescence using SuperSignal West Femto substrates (Pierce Biotechnology, Inc., Rockford, IL). Densitometry measurements were performed on a GS800 densitometer with Quantiy One software (Bio-Rad Corp., Hercules, CA). Isolation of Mitochondria. Peripheral blood mononuclear cells isolated by Ficoll-Paque gradient and frozen at -80 °C were thawed and rinsed with PBS. Mitochondria were then isolated from CLL B cells using a commercially available reagent-based mitochondria isolation kit (Pierce, Rockford, IL). The pellet containing mitochondria was solubilized in 100 mM NH4HCO3 buffer pH 8.0 containing 6M urea and 1% octylglucoside then total protein concentrations were determined in triplicate for cytosol and crude membrane fractions using the BCA colorimetric protein assay with bovine serum albumin as the standard (Pierce, Rockford, IL).

Results and Discussion Control Labeling Using the Same Patient Fraction. Control labeling experiments were performed on cytosol and membrane fractions to determine the error associated with the 1312

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labeling of CLL B cell fractions using the cICAT reagents. Equal amounts of protein from the same patient (in this case the UMCLL patient) were labeled with light and heavy isotope reagents. Figure 1 shows the histograms generated from these experiments with all ratios presented as Light:Heavy along with representative zoom scans from chromobox protein homologue 5 (CBX5) and the membrane marker protein CD5. The bin size was set at 0.1 resulting in 20 different bins ranging in values from 0.1 to 10. ICAT ratios were set into each bin using the histogram function in Excel. These results illustrate that the variability associated with the labeling process for this particular sample preparation was negligible, suggesting that when comparing different patients any ratios observed outside those observed for controls (Light/Heavy ratio ) 1.06 ( 0.1 for cytosol and 1.03 ( 0.12 for membranes) are estimated to be due to biological variability and/or sample preparation variability (vide infra). The protein identifications listed in the cytosol (N ) 79) and membrane (N ) 71) control labeling histograms in Figure 1 are a fraction of the number of proteins that were found to meet the Xcorr criteria. For example, the cytosol control labeling experiment had a total of 326 protein assignments meeting the Xcorr criteria while the membrane control labeling experiment had a total of 212 protein assignments meeting the Xcorr criteria. The difference observed between the number of proteins identified with acceptable Xcorr criteria and the number of proteins used to create the histograms in Figure 1 was due in part to the presence of non-ICAT labeled peptides that resulted in a protein ID. Others have reported the presence of nonbiotin containing peptides binding nonspecifically to the avidin cartridge.11 Also, peptides were found that met the Xcorr criteria but lacked insufficient abundance (S/N < 3) to determine the relative amounts of each label. Additionally, some MS/MS spectra assigned an acceptable Xcorr value by SEQUEST were dropped since they were found to have high background allowing SEQUEST to assign b- and y-ion series for ions that were visibly noise, often in the presence of high abundance peaks that were not assigned to the sequence. While a more rigorous separation of tryptic peptides using a gradient elution SCX step with fractionation would help increase the number of protein identifications by reducing the influence of the three factors mentioned above, such an approach increases the sample handling and analysis time 10-fold or more depending on the number of SCX fractions collected. Nonetheless, the histograms in Figure 1 effectively define the variation expected for a control labeling experiment for cytosol and membrane fractions. The question of sample preparation variability was examined by splitting a cell pellet from a M-CLL patient into three equal portions then preparing cytosol and membrane fractions from each portion in parallel. Figure 2 shows a silver stained gel of these replicate fractions along with a Western blot probing the cytosol and membrane fractions using an anti-tubulin mouse mAb. The similarities in the staining patterns and the blot intensities are readily observed indicating that no dramatic differences in protein yield were detected for the replicate preparations. Optical density values obtained for replicates 1, 2, and 3 from the anti-tubulin Western blot intensities for cytosol were 0.95, 0.92, and 0.92, CV ) 1.3, while the membrane replicates had O. D. values of 0.40, 0.37, and 0.44, CV ) 8.0. These results indicated that, barring any major discrepancies in cell isolation procedures from patient to patient prior to fractionation; large differences observed using cICAT reagents

Protein Expression Analysis of M-CLL vs UM-CLL

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Figure 1. Histograms shown in the figure were generated using relative abundance ratios found for cICAT labeling controls where an equal amount of the M-CLL patient sample was split and labeled. The frequency represents the number of proteins identified and each bin represents a difference of 0.1. Ratios were averaged for proteins with multiple peptide assignments. Next to each histogram is a representative example of a precursor ion zoom scan showing the relative abundance ratios for the light and heavy ICAT label. The histograms demonstrate that the ICAT labeling process does not introduce appreciable error.

Figure 2. M-CLL patient sample cell pellet was split into three equal parts and processed in parallel as described in Material and Methods then subjected to 1D SDS-PAGE. The same amount of protein (2 µg) as determined by BCA protein analysis was added to each lane of a 4-15% precast gradient gel then either silver stained or probed with an anti-tubulin mouse mAb (1° dilution 1:5000) after being transferred to nitrocellulose. The reproducibility of the replicate preparations suggests that there is no appreciable error associated with the cell fractionation procedure.

would likely be due to biological differences and not sample preparation variability. Comparison of Patients with Mutated and Unmutated IgVH Genes using cICAT Reagents. The M-CLL sample was obtained from an untreated female patient with low Rai risk, a 4.2% mutation level in the IgVH region, and a 13q-x1/13q-x2

fluorescence in situ hybridization (FISH) karyotype while the UM-CLL sample was obtained from an untreated male patient with low Rai risk, a 0% mutation level in the IgVH region, and a 13q-x1 FISH karyotype. Equal amounts of protein from cytosol and membrane fractions were cICAT labeled as described in the Materials and Methods and the resulting tryptic Journal of Proteome Research • Vol. 4, No. 4, 2005 1313

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Figure 3. Histograms showing the distribution of ICAT ratios observed for labeling experiments performed on the cytosol and membrane fractions; M-CLL (Light): UM-CLL (Heavy). Western blot results for four of the proteins identified are presented to the right of the fraction where identified. The histograms illustrate the broad distribution of abundance ratios observed for the experiments suggesting extensive biological variability between the two patients.

peptides isolated by the combination of SCX and avidin affinity chromatography were subjected to LC-MS/MS. Figure 3 shows the two histograms produced by placing each ICAT ratio into a bin for the protein identifications made from labeling of the cytosol and membrane fractions from the M-CLL and UM-CLL patient comparison. Also included in Figure 3 are the bin locations for two proteins from cytosol, actin and A32A, and two proteins from membranes, tubulin and COXG, along with their corresponding Western blots. The histograms produced from the cytosol and membrane fraction labeling experiments show two distinct distributions, both of which extend over a broader range of ratios than the control histograms. However, the cytosol histogram exhibits a distribution closer to that observed for the control as opposed to the membrane histogram. The Western blots shown in Figure 3 serve to confirm the observed ICAT ratios found for the four proteins with the intact protein used as a measure of abundance rather than a labeled tryptic peptide. Examples of the abundances observed for the ICAT labeled tryptic peptides from the four proteins probed by Western blot are shown in Figure 4. The striking differences in the abundances of the ICAT labeled peptides from COX G and A32A from the two patients are clearly observed in the spectra on the bottom of the figure. In addition, the abundances of the ICAT labeled tryptic peptides originating from the two structural proteins actin and tubulin, often used as Western blot loading controls, were not significantly different between the M-CLL patient and the UM-CLL patient. The figure also lists an observed and normalized ratio for the two proteins, actin and A32A, originating from the cytosol fraction. An observed value of 1:0.62 Light/Heavy is listed for the ICAT labeled actin peptides in the spectrum along with a normalized value of 0.99: 1. After all the ICAT labeled peptide abundance ratios were compiled, the histogram produced had the most abun1314

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dant bin value at 1:0.6 (data not shown). Subsequent BCA analysis of the cytosol fractions that were concentrated but had not been labeled, confirmed that a change in total protein had taken place between the two samples during the concentrating step. Therefore, all ICAT ratios for labeled peptides identified from cytosol were adjusted to compensate for this difference. However, as can be observed for A32A in Figure 4, the abundance of the labeled tryptic peptide from the UM-CLL patient was already greater than the M-CLL patient, consequently after normalizing to total protein the difference was even more pronounced. Also, as Figure 3 demonstrates, western data derived from cytosol fractions that were not concentrated, concur with the normalized ICAT values from the cytosol fractions that were concentrated. Table 1 gives a list of proteins that were found to have a 3-fold or greater difference in abundance between the M-CLL patient and the UM-CLL patient. A cutoff of 3-fold was chosen since these proteins were clearly outside the control ratios (Control: Light/Heavy ratio ) 1.06 SD ) 0.10 for cytosol and 1.03 SD ) 0.12 for membranes) and were the most likely to distinguish between M-CLL and UM-CLL. A complete list of the proteins that were identified in cytosol and membrane fractions and their respective Light/Heavy ratios (M-CLL ) Light, UM-CLL ) Heavy) are provided in the Supporting Information. Table 1 shows a cluster of proteins from the cytosol fraction with a 3-fold difference in abundance between the M-CLL patient versus the UM-CLL patient. However, on closer examination it is clear that three of the four proteins are not related to mutational status. This is evident from the identification of ICAT labeled peptides from beta- and deltachain hemoglobin that were identified in the M-CLL patient but not the UM-CLL patient. The presence of hemoglobin is probably related to residual erythrocytes that were not completely lysed prior to fractionation in the M-CLL patient

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Figure 4. Zoom scan ion abundance mass spectra for ICAT labeled tryptic peptides from actin and A32A (cytosol) and tubulin and COXG (membranes). The spectra demonstrate the similarities in ion abundances found for the tryptic peptides using cICAT labeling as compared to the blot intensities for intact proteins observed in Figure 3. Table 1. Proteins Identified with at Least a 3-Fold Difference in Expression between M-CLL and UM-CLL

a

cytosol

Swiss-Prot ID

Acidic nuclear phosphoprotein pp32a Splicing factor, arginine/serine-rich 1 Calgranulin A Heterogeneous nuclear ribonucleoproteins Glutathione peroxidase 1

A32A_HUMAN SFR1_HUMAN S108_HUMAN ROA2_HUMAN GPX1_HUMAN

Thioredoxin domain containing protein 5 HLA class I histocompatibility antigen, B-4 Hemoglobin beta chain Hemoglobin delta chain

TXN5_HUMAN 1B46_HUMAN HBB_HUMAN HBD_HUMAN

membrane

Swiss-Prot ID

Poly [ADP-ribose]polymerase-1 (PARP-1)

PPOL_HUMAN

Cytochrome c oxidase polypeptide VIb* Cytochrome c oxidase polypeptide Vb Vimentin

COXG_HUMAN COXB_HUMAN VIME_HUMAN

fold difference

Mb < UMc 7 4 4 3 3 M > UM 3 3 3 3 fold difference

M < UM 3 M > UM 6 4 3

Examined by western blot. b M ) Mutated CLL labeled with Light cICAT reagent. c UM ) Unmutated CLL labeled with Heavy cICAT reagent.

preparation. This finding is an indication of the challenges associated with having consistent starting material from patientto-patient prior to fractionation. In addition, the labeled peptide identified from the HLA class I histocompatibility antigen is likely a result of HLA typing differences between the

two patients. We are currently investigating HLA typing as an internal control for ICAT labeling differences observed between membrane preparations from different patients. However, thioredoxin domain containing protein 5 is apparently a putative discriminator between M-CLL and UM-CLL as are the Journal of Proteome Research • Vol. 4, No. 4, 2005 1315

research articles other proteins identified in Table 1, with emphasis on the two proteins found with the greatest difference in abundance between M-CLL and UM-CLL, A32A in cytosol and COXG in membrane. The protein A32A, also referred to as tumor suppressor putative HLA-DR-associated protein (PHAP I), is an inhibitor of protein phosphatase 2A and is involved in promoting apoptosis by accelerating caspase-9 activation.17 In addition, another member of the PHAP family of proteins was also identified, A32E, at a 2-fold greater level in the UM-CLL patient as compared to the M-CLL patient. Similar findings were observed in the case of COXG where another subunit from cytochrome c oxidase, COXB, was found to have nearly the same ICAT ratio as COXG further supporting the finding that subunits of cytochrome c oxidase may be differentially expressed between M-CLL and UM-CLL (data not shown). In the case of the membrane fraction it is important to note that a number of peptides from mitochrondrial proteins were identified and found-to-have ratios near 1:1 including the electron transporter cytochrome c-1, R and γ-chains of ATP synthase, import inner membrane translocase (IM9A), ADP/ATP carrier protein, ubiquinol-cytochrome-c reductase complex core protein I, and the voltage-dependent anion-selective channel proteins 1 and 2 (VDAC-1,2), along with others. It is unlikely that the cytochrome c oxidase subunits COXG and COXB would be preferentially isolated in one patient membrane fraction over another considering the number of other mitochondrial peptides that were found to be at or near 1:1 ratios. Levels of COX G and A32A were analyzed by western blot in a set of patients including 6 patients with M-CLL and 6 patients with UM-CLL. Prior to checking patient samples CLL B cell mitochondria were isolated to enrich levels of COX G rather than using crude membranes derived from sonication and ultracentrifugation. This approach not only enriched the levels of COX G in preparations, but it also allowed for more patient samples to be processed in a shorter amount of time. In addition, the cytosol fraction obtained from the mitochondria isolation procedure was used for testing the levels of A32A. Figure 5 shows the results for Western blots of mitochondria and cytosol fractions probing for COX G and A32A. The figure clearly demonstrates that COX G is found in the mitochondria fraction while A32A is found in the cytosol fraction. Reproducibility of the mitochondria preparations was established by the density of the 4 bands for COX G which were found to have a CV ) 7.9 while the bands for A32A were more variable having a CV ) 31. The variation that was observed was most likely due to preparation variability for COX G and A32A not in-gel variability since equal amounts of Replicate 1 were placed into 3 separate lanes in the same gel as an internal control (data not shown) and the CV for the density of the COX G bands were found to be 4.6 while the CV for the density of the A32A bands were found to be 2.2. All 12 patient samples checked for levels of COX G and A32A were randomly selected using only the patient’s IgVH gene status as a parameter for sorting into mutational subgroups. Mitochondria and cytosol were isolated as described in the Materials and Methods section then analyzed by Western blot. Figure 6 shows the results for M-CLL (labeled M1-M6) and UM-CLL (labeled UM1-UM6) patient samples probed for COX G and A32A. The band density values for M-CLL patients were compared to the density values for UM-CLL patients using a two-tailed t-test assuming equal variances and an alpha value of 0.05. A P-value of 0.0008 was calculated for the COX G 1316

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Figure 5. Control Western blot analysis of replicate mitochondrial and cytosol fraction isolated from a patient with M-CLL and processed as described in Material and Methods. A total of 5 µg of protein was added to each lane of a 15% precast 1DE gel that was transferred to nitrocellulose and probed using an anti-COX G mouse mAb. The blot was then stripped and re-probed with an anti-A32A rabbit polyclonal Ab. The blot clearly shows the presence of COX G in the mitochondrial fraction and the presence of A32A in the cytosol fraction. Reproducibility of the replicate preparations was measured by quantifying the abundance of the bands using densitometry; CV for COX G ) 7.9 and CV for A32A ) 31.

Figure 6. Multiple patient western blot analysis of 12 patient samples, 6 with M-CLL (labeled M1-M6) and 6 with UM-CLL (labeled UM1-UM6). Mitochondrial and cytosol fractions were isolated from each patient using the same process as described for the patient control samples in Figure 5. For the COX G western, a total of 5 µg of protein was added to each lane of a 15% precast 1DE gel that was transferred to nitrocellulose and probed using an anti-COX G mouse mAb. For the A32A Western, a total of 5 µg of protein was added to each lane of a 4-15% precast 1DE gradient gel that was transferred to nitrocellulose and probed using an anti-A32A rabbit polyclonal Ab. Quantification of each band was performed using densitometry and sample abundances were compared using standard t-statistics (see text).

comparison while a P-value of 0.265 was calculated for the A32A comparison. Thus, for the M-CLL and UM-CLL patient set compared, COX G was found at a statistically greater level in M-CLL patients than UM-CLL patients, in agreement with the findings from the ICAT experiments. However, the findings for the comparison of A32A in M-CLL and UM-CLL patients suggest that levels of A32A in M-CLL patients vs UM-CLL are not statistically different when examined by Western blot indicating that A32A cannot be used to discriminate between M-CLL and UM-CLL. The findings presented here suggest that COX G, aka. COX 6B, may be expressed at different levels in M-CLL vs UM-CLL patients. While other groups have studied the levels of cyto-

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Protein Expression Analysis of M-CLL vs UM-CLL

chrome c oxidase subunits in prostate cancer,18,19 to the best of our knowledge there are no published reports describing the abundance of a subunit of cytochrome c oxidase in CLL B cells at the protein level. A number of recent studies have examined mitochondrial DNA mutations in CLL as a result of chemotherapy, including mutations in mtDNA for the cytochrome c oxidase subunit COX 2,20,21 but little is known about the changes in the mitochondrial proteome of CLL B cells as a result of disease progression. The results of this study demonstrate the utility of stable isotope labeling and mass spectrometry for comparing M-CLL and UM-CLL subgroups and point to the use of this technology to better understand the mitochondrial proteome of CLL B cells. Abbreviations: CLL, chronic lymphocytic leukemia; IgVH, immunoglobulin variable heavy chain; M-CLL, chronic lymphocytic leukemia with mutated immunoglobulin variable heavy chain gene; UM-CLL, chronic lymphocytic leukemia with unmutated immunoglobulin variable heavy chain gene; cICAT, acid-cleavable isotope coded affinity tags; CV, coefficient of variation.

Acknowledgment. The authors wish to acknowledge the efforts of Renee Tschumper and Cheryl Jankiewicz for their help in preparing patient samples prior to cell fractionation. Supporting Information Available: A complete list of proteins identified in cytosol and membrane fractions from the comparison of M-CLL and UM-CLL patients along with their respective Light/Heavy ICAT ratios. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Rozman, C.; Montserrat, E. N. Engl. J. Med. 1995, 333, 10521057. (2) Cheson, B. D.; Bennett, J. M.; Grever, M.; Kay, N.; Keating, M. J.; O’Brien, S.; Rai, K. R. Blood 1996, 87, 4990-4997. (3) Damle, R. N.; Wasil, T.; Fais, F.; Ghiotto, F.; Valetto, A.; Allen, S. L.; Buchbinder, A.; Budman, D.; Dittmar, K.; Kolitz, J.; Lichtman, S. M.; Schulman, P.; Vinciguerra, V. P.; Rai, K. R.; Ferrarini, M.; Chiorazzi, N. Blood 1999, 94, 1840-1847.

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