Solution Isoelectric Focusing for Peptide Analysis: Comparative

Oct 5, 2005 - Solution Isoelectric Focusing for Peptide Analysis: Comparative Investigation of an Insoluble Nuclear Protein Fraction. Yanming An,Zongm...
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Solution Isoelectric Focusing for Peptide Analysis: Comparative Investigation of an Insoluble Nuclear Protein Fraction Yanming An,† Zongming Fu,† Peter Gutierrez,‡ and Catherine Fenselau*,†,‡ Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, and Greenebaum Cancer Center, University of Maryland Medical School, Baltimore, Maryland 21201 Received July 18, 2005

In this study, a solution isoelectric focusing apparatus was modified and built into a two-dimensional separation method for peptides. Newly commercialized isoelectric membranes, which carry immobilized ampholytes, were integrated to establish the pH boundaries in this apparatus. High-performance liquid chromatography was employed as the second dimension, interfaced with mass spectrometry. An insoluble nuclear protein fraction was used to evaluate and optimize this method. This two-dimensional separation method dramatically improves peptide detection and identification compared with a single dimension LC-MS analysis. Off-line reversed-phase HPLC was used to ascertain reproducibility. The two-dimensional separation method was combined with 18O labeling for comparative analysis of protein expression in two cell lines. Separation of peptides by solution isoelectric focusing (sIEF) offers the advantage that it can be accomplished after the 18O labels are introduced. The labeled peptides can be mixed with unlabeled ones before fractionation by sIEF. The relative abundances of nuclear proteins from a drug resistant MCF-7 cancer cell line were compared to those from the drug susceptible parent cell line using this combined strategy. The abundances of several heterogeneous nuclear ribonucleoproteins were found to be increased in the mitoxantrone-resistant line. Keywords: solution isoelectric focusing • shotgun proteomics • 18O labeling • mass spectrometry • drug resistance • nuclear proteins • reversed-phase liquid chromatography • MCF-7 cancer cells • heterogeneous nuclear ribonucleoproteins

Introduction In proteomic research, experimental and computational approaches are combined to provide global analysis of the proteomes of cells and tissues. The identification and quantification of multiple proteins, which constitute a specific biological system, are important for understanding complex problems in biology. The coupling of highly efficient separations and mass spectrometry instrumentation is evolving rapidly and these systems are widely applied to problems ranging from biological function to drug development. The primary separation method employed for protein mixtures is two-dimensional gel electrophoresis. Despite the high selectivity and sensitivity of 2-D PAGE, it is often not sufficient for the analysis of a given proteome. Limited dynamic range (max. 104) has hampered the ability to identify low-abundance proteins from complex biological samples.1,2 Two-dimensional PAGE also has difficulties in detecting proteins with extreme molecular masses and isoelectric points (outside the ranges of 20200 kDa and pH 3-10). The 2-D PAGE is also limited for more hydrophobic proteins, e.g., membrane proteins.3 An alternative strategysmultidimensional protein identification technologyshas been introduced for more automated and * To whom correspondence should be addressed. E-mail: fenselau@ umd.edu. † Department of Chemistry and Biochemistry, University of Maryland. ‡ Greenebaum Cancer Center, University of Maryland Medical School.

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more comprehensive proteome analysis.4,5 This technique involves multidimensional separations of peptides in proteome digestions and tandem mass spectrometry. Several methods, including cation-exchange chromatography,4,5 capillary isoelectric focusing,6 gel isoelectric focusing,7,8 and free flow electrophoresis9-11 have been proposed for the first dimension, followed by reversed-phase liquid chromatography coupled with mass spectrometry. The isoelectric focusing techniques are considered to have the advantage over ion-exchange chromatography that they provide pI-based separations. Thus, they provide another physical characteristic to assist database searching. However, these isoelectric focusing methods have limited loading capacity, and/or require complex instrumentation. Another alternative is preparative-scale solution isoelectric focusing (sIEF), which has been introduced for protein prefractionation by several prominent proteomics laboratories.12-14 This technique separates proteins based on their isoelectric points and is considered an orthogonal methodology to reversed phase HPLC or SDS-PAGE. Incorporation of this method into proteomic studies decreases the complexity of protein samples and improves protein detection and identification. The large sample capacity and the profound concentration effect of isoelectric focusing make relatively low abundance components in the protein mixture more detectable and provide a wider dynamic range. To keep the intact proteins in 10.1021/pr050221+ CCC: $30.25

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Solution Isoelectric Focusing in Shotgun Proteomics

solution during focusing, detergents are added to the sIEF fractions,15-17 which are not directly compatible with downstream LC-MS/MS analysis. In the present study, a twodimensional strategy has been established for peptide separation, which incorporates solution isoelectric focusing with reversed-phase LC-MS/MS. In this strategy, sIEF is adapted for use at the peptide level, and to avoid the use of detergents. The method has now been demonstrated to provide efficient and reproducible separations for both protein and peptides. Solution isoelectric focusing (sIEF) with the apparatus used here and free flow electrophoresis (FFE) share several strengths for peptide analysis, including high loading capacity, high sample recovery and good reproducibility. FFE has the advantage of narrow range pH peptide separations,10 while solution IEF has the advantage that the separated and concentrated peptide samples do not contain polymeric HMPC or detergent, and need only to be desalted for subsequent LC-MS/MS analysis. For evaluation, the sIEF-based peptide strategy has been applied to analysis of an insoluble nuclear protein fraction isolated from human MCF-7 breast cancer cells. Following partial solubilization with detergents, the proteins were digested into peptides. The peptide mixture was initially separated into six fractions using solution isoelectric focusing. To evaluate the reproducibility of the separation, fractions were analyzed by off-line reverse phase HPLC. For peptide identification, each fraction was collected and further analyzed by LC-MS/MS and bioinformatics. The two-dimensional separation method dramatically improves peptide detection and identification compared with a single dimension LC-MS analysis. A second objective was to investigate changes in the abundances of proteins in the insoluble nuclear fractions of a drug resistant human breast cancer cell line and its parental drug susceptible cell line. The 2-D separation strategy described above is coupled with 18O labeling. Drug susceptible MCF-7 cancer cells (parental control cells), have been widely used in laboratory studies since the line was established from the pleural effusion of a patient with metastatic mammary carcinoma in 1973.18 The drug resistant MCF-7/MX cell subline studied here is a stable line derived from the parental cell line by stepwise increases in the concentration exposures to mitoxantrone.19

Experimental Section Materials. L-lysine, L-arginine, and phosphoric acid were obtained from Sigma Co. (St. Louise, MO). IPG Buffer, pH 3-10 was purchased from Amersham Biosciences (Piscataway, NJ) Modified porcine trypsin (sequence grade) was purchased from Promega (Madison, WI). Dialysis membrane (500 Da cutoff) came from Millipore (Billerca, MA). MCE kit was purchased from Proteome Systems (Woburn, MA). Isotopically enriched H218O, > 95% 18O was purchased from Isotech, Inc. (Miamisburg, OH). Trypsin immobilized on Poros beads was purchased from Applied Biosystems (Foster City, CA). PepClean C-18 spin columns came from Pierce (Rockford, IL). Water was purified by a MilliQ system and filtered with 0.22 µm membrane (Millipore, MA). Cell Culture and Nuclear Protein Preparation. MCF-7 and MCF-7/MX cells were cultured in MEM (Sigma, St. Louis, MO) with 10% FBS and 1% penicillin streptomycin. Every 6 months, the MCF-7/MX cell line was subjected to a reselection cycle of three passages with culture medium containing increased concentrations of mitoxantrone. Cultured MCF-7 and MCR-

research articles 7/MX cells were harvested at 95% confluence, released with trypsin, centrifuged at 500 × g, and washed twice with PBS. A nuclei isolation kit (Sigma) was used to isolate and purify MCF-7 nuclei, according to the manufacturer’s instructions. The nuclei pellets were suspended in a buffer containing 0.42 M NaCl, 20 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 25%(v/v) Glycerol (pH 7) and centrifuged at 16 000 × g for 10 min. The supernatant was collected for another use.20 The pellets were washed with water and resuspended in the Sample Solubilizing Buffer (ProteomeSystems, Woburn, MA) with the detergent excluded, and held for 30 min on ice. This suspension was vortexed vigorously for 60 s every 10 min. Then the suspension was centrifuged at 16 000 × g for 10 min. The supernatant fraction was immediately transferred to new tubes and stored at -80 °C. Protein concentration was determined with the Bradford protein assay (BioRad). The proteins were reduced with tributylphosphine (TBP) (1:40, v/v) and alkylated with Acrylamide Alkylation Reagent (1:100, v/v) (ProteomeSystems) for 90 min at room temperature. Then the solution was diluted 10 times with 50mM NH4HCO3 pH 8.0. Proteins were digested with trypsin (Promega) (1:50, w/w) at 37 °C overnight. Solution Isoelectric Focusing Fractionation. A six-chamber solution isoelectric focusing device was assembled using the multichamber Teflon dialyzer system (Amika Corp., MD) and pH membranes from ProteomeSystems Inc. The pH values of the membranes were 3.0, 5.0, 6.5, 8, and 11 (ProteomeSystems, Woburn, MA). Two 500 Da dialysis membranes (Millipore, MA) were put at each end of the terminal chambers to protect the system from the running buffer outside. The sample was loaded in the chamber with pH 5-6.5. The two terminal chambers were filled with Electrode Buffer and the chambers without sample were filled with Chamber Buffer, both of which were purchased from ProteomeSystems. The running buffers were: 7 mM phosphoric acid (anode) and 20 mM lysine/20 mM arginine (cathode). The following voltage program was used for peptides: 100 V for 10 min, 200 V for 10 min, 500 V for 20 min, and 1000 V for 100 min. After fractionation, solutions were collected from each chamber and the inner wall and membranes were rinsed with 200 µL chamber buffer. The rinses were combined with the sample fractions. LC-MS/MS Analysis. Each of the six IEF peptide fractions was desalted with a PepClean C-18 spin column (Pierce) according to the user guide before LC-MS/MS analysis. Capillary reversed-phase liquid chromatography was performed using an LCQ DecaXP plus ion trap mass spectrometer equipped with an on-line microcapillary HPLC (ThermoElectron, San Jose, CA) that uses two C18 peptide captraps for highthroughput (Michrom BioResources Inc., Auburn CA). The reverse-phase column was a PicoFrit 75 µm I.D. × 10 cm fused capillary with a 15 µm nanoelectrospray tip and packed with 5 µm, 300 Å BioBasic C18 particles (New Objective Inc., Woburn, MA). The samples were dried and resuspended in 10 µL 0.1% formic acid. Samples were injected using a Surveyor autosampler (ThermoElectron). After injecting 10 µL of sample, the peptides were eluted using a linear gradient of water and acetonitrile, both acidified with 0.1% (v/v) formic acid. The gradient increased from 5% acetonitrile, to 60% in 55 min. The column was then washed with 98% acetonitrile for 5 min and water for 26 min at a constant flow rate of 275 nL/min. The ion-trap mass spectrometer was operated in a data-dependent mode in which a full MS scan was followed by MS/MS scans of the three most intensive ions. The ions were automatically selected for collision-induced dissociation (CID). This analysis Journal of Proteome Research • Vol. 4, No. 6, 2005 2127

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Figure 1. LC-MS base-peak chromatograms of sIEF frations.(A) fraction 1 (pH < 3); (B) fraction 2 (pH 3-5); (C) fraction 3 (pH 5-6.5); (D) fraction 4 (pH 6.5-8); (E) fraction 5 (pH 8-11); (F) fraction 6 (pH >11).

was repeated in triplicate for peptides in each pI range separated in the sIEF chambers. Bioinformatics Analyses. The MS/MS spectra obtained from LC-MS/MS were searched against the NCBI human database (www.pubmed.org) using SEQUEST (ThermoElectron). For preliminary protein identification, searches must achieve the criteria: cross-correlation (Xcorr) higher than 1.9 for singly charged ions, 2.2 for doubly charged ions, 2.5 for triply charged ions, and delta correlation (∆Cn) higher than 0.1. To increase filtering rigor, another set of criteria was used: Xcorr higher than 1.9 for singly charged ions, 2.5 for doubly charged ions, 3.75 for triply charged ions, and ∆Cn higher than 0.1. This second set of criteria reduced the number of pepide identifications from 281 to 211.The SEQUEST search results were also processed with PeptideProphet (peptideprophet.sourceforge.net)21 in order to test the validity of identifications based on single peptides.22 One hundred and ninety-two peptides with scores higher than 0.9, in this algorithm were considered to be identified. The same tandem mass spectrometry data was also searched against the Swiss-Prot human database using Mascot for identification.23 Two hundred and twenty-five peptides with scores higher than 30 were considered to be identified with more than 95% confidence. 2128

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Peptide Separation with Conventional Reversed-Phase Liquid Chromatography. To assess the reproducibility of the sIEF separation, a Shimadzu LC system (Columbia, MD) was used to separate peptides from each fraction obtained by solution isoelectric focusing. The column was purchased from Phenomenex (Torrance, CA) and packed with C18 particles. An 80 min elution gradient was used: 95% solvent A (0.1% TFA in water) for 5 min, then solvent B (0.1% TFA in acetonitrile) from 5% to 60% in 55min, 60% to 90% for 10 min, hold for 5 min, and 90% A for 10 min. The effluent was monitored by a UV detector with computer support. HPLC conditions for the LCMS/MS analysis are described in a previous section. Proteolytic H218O Labeling with Immobilized Trypsin. In the comparative proteomic experiment, only one of the peptide samples is labeled with 18O, but the procedure is applied to both samples in order to ensure experimental homology.24 Immobilized trypsin was washed with water and added to peptide solutions in a ratio of 1:5 (v:v). The peptide and immobilized trypsin mixtures were completely dried in a vacuum concentrator. Then one residue was redissolved in 80% H218O and 20% acetonitrile, and the other was redissolved in 80% H216O and 20% acetonitrile. The solutions were gently shaken at room temperature for approximately 5 h on a

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Figure 2. UV Chromatograms of fraction 2 (pH 3-5) from three independent sIEF runs.

benchtop rotator. The labeled and unlabeled peptide pools were stored at -80 °C.

Results and Discussion Peptide Fractionation by Solution Isoelectric Focusing. In this experiment, solution IEF was applied to separate peptides prepared from the insoluble nuclear protein pellet. After isoelectric focusing, each fraction was desalted, and analyzed by LC-MS/MS. Figure 1 shows the base-peak chromatogram of each fraction. Each fraction was eluted through the same column with the same solvent profile. The variation in the chromatograms indicates the varying composition of each fraction and indicates that peptides were successfully fractionated during solution isoelectric focusing. Detergents in solutions can improve protein solubilization, but most of them impede mass spectrometry and stick on RPLC columns. To optimize the subsequent HPLC separation and mass spectrometry analysis, a chamber buffer without any detergent was used. No sample precipitation during sIEF was observed. IPG Buffer (Amersham Biosciences) was added to chamber buffer to increase the conductivity and to help maintain the pH gradient across the device. Because the initial concentration of the IPG Buffer (0.5%, v/v) was very low and dilution occurred during the experiment, the ampholytes in the IPG Buffer were eluted earlier from the reversed-phase HPLC and did not show any interference with peptides. The reproducibility of peptide separation by sIEF was investigated. Three nuclear peptide mixtures were fractionated with solution IEF separately and the same fractions from different runs were characterized by HPLC separation. Figure 2 shows three chromatograms of fraction 2 obtained using a UV detector. Most of the peaks appeared in all of the three runs. The similarity of the elution profiles suggests that the components of the same fractions from the three different solution IEF separations were similar. Peptide Identification Using Tandem Mass Spectrometry. The mass spectrometer was programmed to select the three most intense peptide peaks from the full MS scan for tandem MS analysis. The MS/MS spectra were searched against NCBInr human protein database. The 211 peptides, which passed the higher Sequest criteria defined in the Experimental Section,

Figure 3. Numbers of peptides identified in each sIEF fraction.

Figure 4. Distribution of peptides according to their frequency of appearance in one or multiple fractions.

were tentatively considered to be identified. All peptide pI values were calculated using the pI/MW calculator in www.expasy.org. Figure 3 shows the total number of unique peptides identified in each fraction. Fraction 2, ranging from pH 3 to pH 5, has the highest number of peptides identified followed by fraction 3 pH 5-6.5. The sum of the numbers in the Figure 3 is higher than the total number of peptides identified, which means there are peptides re-identified between fractions. Figure 4 shows the number of chambers in which a peptide was observed. About 80% of the total peptides appeared in only one fraction. The other 20% appeared in adjacent chambers Journal of Proteome Research • Vol. 4, No. 6, 2005 2129

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An et al. Table 1. Comparison of Two-Dimensional IEF-LC-MS/MS with One-Dimensional LC-MS/MS

no. of peptides identifieda no. of proteins identified sample consumption MS spectrum absolute intensity a

Figure 5. Box plot of the pI ranges of peptides in each sIEF fraction.

with close pH ranges. This indicates that most of the peptides were separated according to their pI values. Figure 5 is a box plot of the pI range of peptides in each fraction. The discontinuous lines indicate the pH ranges of the fractions defined by the membranes. The box in each lane contains the middle 50% of the entries. The upper edge of the box indicates the 75th percentile of the data set and the lower edge of the box indicates the 25th percentile. The horizontal line in each box indicates the median value of the data. The upper end of the vertical line through each box indicates the 95th percentile and the lower end indicates the 5th percentile. Individual spots are suspected outliers. Peptides in fractions 2 (pI 3-5) and 5 (pI 8-11) fit exactly in the range; most of the peptides in fraction 3 (pI 5-6.5) are also within the range. The pI values of peptides in fractions 4 and 6 were lower than the defined ranges, while the peptides in fraction 1 were more basic. The pI overlaps between each fraction were minimal except for the last two fractions, supporting the conclusion that the peptides were fractionated according to their pI values. The overlap between fractions 5 and 6 may be caused in part by intrinsic properties of the peptides. The proteins were digested into peptides by trypsin which cut at the C-termini of lysine or arginine with pI values of 9.74 and 10.76, respectively. A peptide with a pI higher than pH 11 should have multiple lysines and arginines. Most of the peptides identified are fully cut with one lysine or arginine at the C-termini. The peptides with extremely basic pIs should be rare. The pH of the cathode buffer was close to 11. Thus, the pH difference between fractions 5 and 6 is small. Eleven peptides were found in both fractions. The IPG Buffer is another factor that affects the pH gradient in the device. The components in the IPG Buffer are ampholytes with pH between 3 and 10, which may affect the pH range of each chamber. Finally, the pI distribution of the peptides themselves should be considered. It is obvious that few peptides appear in the range of pH 7 to 8. Cargile and colleagues also observed this phenomenon, when they studied the pI distribution of E coli. peptides using an IPG strip and confirmed the same paucity in the computer generated distribution of all peptides from the E coli. proteome.7 Comparison of the New Two-Dimensional Solution Separation with a One-Dimensional LC-MS/MS Strategy. The same nuclear peptide mixture was analyzed on the same LC2130

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single LCMS/MS

281 167 4.7 µg 106 - 108 ion counts

54 24 74 µg 104 - 106 ion counts

See Experimental Section for criteria.

MS/MS system, without prefractionation by solution isoelectric focusing. The flow rate, column and other conditions of LCMS/MS and SEQUEST identification were identical to those used for the two-dimensional experiments described above. In summary, 54 peptides corresponding to 24 proteins were identified with the single dimensional LC-MS/MS separation, while in the two-dimensional solution IEF-LC-MS/MS strategy, 281 peptides corresponding to 167 proteins were identified (Table 1). Twenty-eight peptides and 20 proteins were identified in common by 1-D and 2-D methods. Most of the proteins identified in the one-dimensional separation are high abundance proteins, which were identified by multiple peptides. The solution IEF separates the high abundance peptides into several chambers and makes the low abundance peptides more visible for data dependent scanning. In the single dimension strategy, about 74 µg of peptides was consumed; while in the twodimensional method only a total of 4.3 µg was used for the eighteen LC-MS/MS analyses. Although the single LC-MS/ MS requires more sample, the ion intensity was about 100 times lower than that of the two-dimensional strategy (Table 1). This difference in sample consumption resulted from the concentration effect of solution isoelectric focusing. Considering a certain peptide sequence, the molecules are distributed in the whole range of pH 3-10 before isoelectric focusing; after focusing, all of the molecules are concentrated at the same pH point. The local concentration of a peptide is increased by the focusing process and the peptide is more detectable. So solution isoelectric focusing not only simplifies the complex mixture but also concentrates the diluted sample. The twodimensional separation method greatly improves the success of the mass spectrometry based protein probability-based search algorithms.20 Comparative Analysis of Nuclear Proteins of Drug Susceptible and Drug Resistant MCF-7 Cancer Cells. Nuclear proteins extracted from drug susceptible MCF-7 and drug resistant MCF-7/MX cancer cells were studied using O-18 isotope labels.24 Briefly, the proteins were extracted from nuclear pellets with sample Solubilizing Buffer (ProteomeSystems, MA) and digested into peptides. The peptides from drug susceptible cells were labeled with H218O, while the peptides from drug resistant cells were labeled with H216O. The labeled and unlabeled peptides were mixed, fractionated by solution isoelectric focusing and analyzed by LC-MS/MS. The labeled and unlabeled peptide pairs coeluted from the LC column and appeared as isotopic doublets 2 Da apart in the mass spectrum of doubly charged ions. The relative abundances were calculated using the intensities of monoisotopic peaks extracted from mass spectra. A ratio of 16O/18O lower than 0.5 or higher than 2.0 is considered significant. Figure 6 presents several proteins with increased abundances in the drug resistant cell line. Of particular interest are the three heterogeneous ribonucleoproteins found to have higher abundances in the mitoxantrone

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development of this technique is intended to enable the use of O-18 labeling for comparative proteomics. The O-18 labels are introduced into tryptic, Lys-C or Glu-C-protease peptide products24,38 and, ideally, all sample fractionation should take place thereafter. Advantages of the sIEF method include high loading capacity, small sample volumes, the concentration effect of the electrofocusing, and simple sample handling to transfer the samples to LC-MS/MS. One concern, which needs further consideration, is the uncertain correlation between theoretical pH values and pH values observed for peptides at the basic end of the scale. Additional resolution will be achieved with apparati with more than six cells.

Figure 6. Relative changes in protein abundances between the mitoxantrone resistant cell line (MXR) and the drug susceptible cell line (Control).

resistant cell line than in the drug susceptible cell line. The heterogeneous ribonucleoprotiens (hnRNPs) are a large family of nucleic acid binding proteins, which associate with mRNA precursors to form ribonucleoprotein paticles.25 They participate in various processes, including regulation of transcription,26 splicing,27 and telomere-length maintenance.28 HnRNPs have been reported to be overexpressed in several types of cancers.29-31 The reason for the overexpression of hnRNPs in cancers is not clear, but it has been associated with the molecular machinery that regulates telomere formation and stabilization25 and with the control of apoptosis.32,33 It has been reported that hnRNPs are dephosphorylated in the early stage of apoptosis.32 Dephosphorylation may change their activities in stabilizing and splicing mRNA, and result in the upregulation of mRNA and proteins of the caspase family. At higher abundance the hnRNPs in drug resistant cells may not be as sensitive to dephosphorylation and thus be resistant to apoptosis.32 The functions of the proteins identified here to have significantly altered abundances need to be further investigated. These proteins might be biomarkers for diagnosis of the development of drug resistance, or targets for new anticancer drugs. A study has been reported previously from this laboratory of changes in protein abundances in soluble nuclear proteins,20 in which members of the hnRNP family were also detected. However, abundances did not change significantly in the soluble fraction between drug susceptible cells and drug resistance cells. Similarly, nucleophosmin and ATP-synthetase were detected in the soluble nuclear protein fraction from control and resistant MCF-7 cells with minimal or no changes in abundances. Taken together, these observations suggest that the proteins isolated in the insoluble fraction are bound up with other biopolymers, and that these may be different isoforms from those in the soluble fraction. It should be noted that Carrier and co-workers have observed changes in the abundance of pan-nuclear nucleophosmin as a genotoxic-stress response.34 Andersen and co-workers have previously reported ATP-synthase in a nuclear fraction.35 The nuclear mitotic apparatus protein 1 (see Figure 6) is a substrate for activated caspases in apoptosis36 and has been previously associated with drug resistance.37

Conclusions In this study, a free solution isoelectric focusing apparatus was modified and evaluated for peptide separation. The

Acknowledgment. This work was supported by the NIH grant GM21248. We thank Dr. K. H. Cowan (Eppley Cancer Center, University of Nebraska) for MCF-7 cell lines. We also thank Dr. N. Edwards for helpful discussions. Part of the work presented here was carried out in the Proteomics Core Facility of the University of Maryland Marlene and Stuart Greenebaum Cancer Center. References (1) Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9390-9395. (2) Rabilloud, T. Proteomics 2002, 2, 3-10. (3) Santoni, V.; Molloy, M.; Rabilloud, T. Electrophoresis 2000, 21, 1054-1070. (4) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R. 3rd Nat. Biotech. 1999, 17, 676682. (5) Washburn, M. P.; Wolters, D.; Yates, J. R. 3rd Nat. Biotech. 2001, 19, 242-247. (6) Chen, J.; Balgley, B. M.; DeVoe, D. L.; Lee, C. S. Anal. Chem. 2003, 75, 3145-3152. (7) Cargile, B. J.; Talley, D. L.; Stephenson, J. L., Jr. Electrophoresis 2004, 25, 936-945. (8) Cargile, B. J.; Bundy, J. L.; Freeman, T. W.; Stephenson, J. L., Jr. J. Proteome Res. 2004, 3, 112-119. (9) Loseva, O. L.; Gavryushkin, A. V.; Osipov, V. V.; Vanyakin, E. N. Electrophoresis 1998, 19, 1127-1134. (10) Moritiz, R. L.; Schutz, F.; Connolly, L. M.; Kapp, E. A.; Speed, T. P.; Simpson, R. J. Anal. Chem. 2004, 76, 4811-4824. (11) Xie, H.; Bandhakavi, S.; Griffin, T. J. Anal. Chem. 2005, 77, 31983207. (12) Kachman, M. T.; Wang, H.; Schwartz, D. R.; Cho, K. R.; Lubman, D. M. Anal. Chem. 2002, 74, 1779-1791. (13) Herbert, B.; Righetti, P. G. Electrophoresis 2000, 21, 3639-3648. (14) Zuo, X.; Speicher, D. W. Anal. Biochem. 2000, 284, 266-278. (15) Pederson, S. K.; Harry, J. L.; Sebastian, L.; Baker, J.; Traini, M. D.; McCarthy, J. T.; Manoharan, A.; Willkins, M. R.; Gooley, A. A.; Righetti, P. G.; Packer, N. H.; Williams, K. L.; Herber, B. R. J. Proteome Res. 2003, 2, 303-311. (16) Zuo, X.; Speicher, D. W. Proteomics 2002, 2, 58-68. (17) An, Y.; Fu, Z.; Fenselau, C. J. Mass Spectrom. Soc. Jpn. 2005, 53, 1-6. (18) Soule, H. D.; Vazquez, J.; Brennan, M. J. Natl. Cancer. Inst. 1973, 51, 1409-1416. (19) Nakagawa, M.; Schneider, E.; Dixon, K. H.; Horton, J. K.; Kelley, K.; Morrow, C.; Cowan, K. H. Cancer Res. 1992, 52, 6175-6181. (20) Fu, Z.; Fenselau, C. J. Proteome Res. 2005, 4, in press. (21) Veenstra, T. D.; Conrads, T. P.; Issaq, H. J. Electrophoresis 2004, 25, 1278-1279. (22) Keller, A.; Nesvizhskii, A. I.; Koller, E.; Aebersold, R. Anal. Chem. 2002, 74, 5383-5392. (23) Perkins, D. N.; Pappin, D. J. C.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-3567. (24) Yao, X.; Afonso, C.; Fenselau, C. J. Proteome Res. 2003, 2, 147152. (25) Choi, Y. D.; Grabowski, P. J.; Sharp, P. A.; Dreyfuss, G. Science 1986, 231, 1534-1539. (26) Tomonaga, T.; Levens, D. J. Biol. Chem. 1995, 270, 4875-4881. (27) Van der Houven van Oordt, W. J. Cell. Biol. 2000, 149, 307-316. (28) Ford, L. P.; Wright, W. E.; Shay, J. W. Oncogene 2002, 21, 580583.

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