Analysis of Membrane Proteins from Human Chronic Myelogenous

Analysis of Membrane Proteins from Human Chronic Myelogenous Leukemia Cells: Comparison of Extraction Methods for Multidimensional LC-MS/MS Received September 19, 2005

Mariah C. Ruth,†,⊥ William M. Old,‡ Michelle A. Emrick,‡,⊥ Karen Meyer-Arendt,‡ Lauren D. Aveline-Wolf,‡ Kevin G. Pierce,‡ Alex M. Mendoza,‡ Joel R. Sevinsky,‡,⊥ Micah Hamady,| Robin D. Knight,‡ Katheryn A. Resing,‡ and Natalie G. Ahn*,‡,§ Yale University School of Medicine, New Haven, Connecticut 06510, Department of Chemistry and Biochemistry, Department of Computer Science, and Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, 80309

Abstract: An important strategy for “shotgun proteomics” profiling involves solution proteolysis of proteins, followed by peptide separation using multidimensional liquid chromatography and automated sequencing by mass spectrometry (LC-MS/MS). Several protocols for extracting and handling membrane proteins for shotgun proteomics experiments have been reported, but few direct comparisons of different protocols have been reported. We compare four methods for preparing membrane proteins from human cells, using acid labile surfactants (ALS), urea, and mixed organic-aqueous solvents. These methods were compared with respect to their efficiency of protein solubilization and proteolysis, peptide and protein recovery, membrane protein enrichment, and peptide coverage of transmembrane proteins. Overall, ∼50-60% of proteins recovered were membrane-associated, identified from Gene Ontology annotations and transmembrane prediction software. Samples extracted with ALS, extracted with urea followed by dilution, or extracted with urea followed by desalting yielded comparable peptide recoveries and sequence coverage of transmembrane proteins. In contrast, suboptimal proteolysis was observed with organic solvent. Urea extraction followed by desalting may be a particularly useful approach, as it is less costly than ALS and yields satisfactory protein denaturation and proteolysis under conditions that minimize reactivity with urea-derived cyanate. Spectral counting was used to compare datasets of proteins from membrane samples with those of soluble * To whom correspondence should be addressed. Phone: (303) 492-4799. Fax: (303) 492-2439. E-mail: [email protected]. † Yale University School of Medicine. ‡ Department of Chemistry and Biochemistry. | Department of Computer Science. § Howard Hughes Medical Institute, University of Colorado. ⊥ Present addresses: Mariah Ruth, Department of Dermatology, School of Medicine, University of Colorado Health Sciences Center, Denver, CO 80220. Michelle Emrick, Department of Pharmacology, University of Washington, Seattle, WA 98195. Joel Sevinsky, Research Triangle Institute, Research Triangle Park, NC 27709. 10.1021/pr050313z CCC: $33.50

 2006 American Chemical Society

proteins from K562 cells, and to estimate fold differences in protein abundances. Proteins most highly abundant in the membrane samples showed enrichment of integral membrane protein identifications, consistent with their isolation by differential centrifugation. Keywords: mass spectrometry • leukemia • membrane proteins • urea • acid labile surfactant

Introduction Chronic myelogenous leukemia (CML) is diagnosed in 6000 people every year. The primary defect in this disease is a constitutively active tyrosine kinase, generated by fusion of BCR and the ABL kinase in blood cell progenitors.1 The K562 erythroleukemia cell line, derived from a CML patient, has been widely used as model for development of therapeutic drugs for CML, such as imatinib mesylate (STI-571, Gleevec), which inhibits myeloid and blast crisis by targeting BCR-ABL.2 K562 cells are also intriguing as a model for differential cell responses to variable signaling activation. Constitutive activation of the MAP kinase (ERK1/2) pathway in K562 cells is necessary and sufficient for cell cycle arrest and differentiation into a megakaryocyte-like cell fate,3-5 whereas inhibition of ERK1/2 or BCRABL induces erythroid differentiation, and simultaneous inhibition of ERK1/2 and BCR-ABL leads to cell death.6,7 To define the proteome of K562 cells, we used a shotgun proteomics strategy involving solution proteolysis followed by multidimensional liquid chromatography and automated peptide sequencing by data dependent MS/MS data acquisition,8-10 which has emerged as a leading approach to analyze the composition of complex protein mixtures. Our previous analysis of soluble proteins from K562 cells led to the development of new computational algorithms for data analysis, which improve the sensitivity of data capture and accuracy in peptide sequence assignments to MS/MS spectra, while minimizing redundant protein identifications due to isoforms.11 These include an algorithm named MSPlus which minimizes false negative and false positive assignments by evaluating results from multiple search programs and applying filters based on peptide chemistry, and Isoform Resolver, which uses a peptidecentric search strategy to match peptides to proteins and Journal of Proteome Research 2006, 5, 709-719

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Published on Web 02/03/2006

Membrane Protein Preparations for LC-MS/MS

minimize redundancies caused by protein and peptide isoforms. High confidence identification was achieved for >5000 proteins within the soluble proteome of K562 cells. In an effort to extend the depth of proteome sampling, we wished to examine the composition of membrane proteins in the K562 cell system by shotgun proteomics. Sample handling for multidimensional LC-MS/MS is more difficult with membrane proteins than soluble proteins, due to the requirement for efficient protein solubilization and the MS incompatibility of ionic and nonionic detergents.12 Various methods for proteolysis of membrane proteins have been reported, as recently reviewed.13,14 These include protein solubilization with formic acid followed by CNBr, solubilization with organic solvents (e.g., methanol) followed by trypsin, or denaturation of membrane associated proteins on liposomes in suspension (e.g., urea, guanidinium hydrochloride) and subsequent digestion in situ.15-21 In addition, acid labile surfactant (ALS) compounds have detergent properties that enable efficient membrane protein solubilization and enhance tryptic digestion.22,23 After protein digestion, the ALS molecule can be hydrolyzed under acidic conditions into products that are charge neutral and negatively charged, providing mass spectrometric compatibility. Each method contributes variably to proteolysis, peptide recovery, and determination of peptide sequences from membrane proteins. For example, an earlier study of bacteriorhodopsin showed that proteolysis in ALS was significantly more efficient than proteolysis in 2M urea.24,25 In contrast, a shotgun proteomics analysis of various organelles showed similar protein and peptide recovery following proteolysis in urea following protein alkylation, compared to ALS without protein alkylation.26 To date, various extraction methods have not been extensively compared with respect to efficacy in recovering membrane proteins in shotgun proteomics experiments. In this study, we examined the protein composition of K562 membranes, comparing four protocols for their relative efficiency in solubilization and proteolysis as well as recovery and representation of membrane-associated proteins. Three methods use published protocols for membrane protein extraction in ALS, urea, and methanol. The fourth adds a rapid desalting step following urea extraction, in place of dilution. We found that proteolysis by trypsin is most complete in membrane preparations solubilized with ALS or urea, compared to organic solvent. We also found that extraction with 8 M urea followed by rapid desalting yielded efficient proteolysis and recovery of membrane proteins compared to extraction with ALS or extraction with urea followed by dilution to trypsincompatible concentrations. Analysis by multidimensional LCMS/MS identified 1309 proteins, 55% of which were known or predicted to be integrally membrane-associated or expected to copurify with membrane subfractions. These datasets showed significant enrichment in membrane protein identifications, compared to a previous dataset from the same cell line, of proteins extracted to enrich soluble components.

Materials and Methods Cell Culture and Sample Preparation. K562 erythroleukemia cells were grown in suspension in spinner cultures at a density of 5 × 105 cells/mL in RPMI media supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin and 100 µg/ mL of streptomycin as previously described.3,27 Cells were washed by centrifugation at 4630 × g for 15 min, resuspended in ice cold PBSM (phosphate buffered saline, 4.9 mM MgCl2), and recentrifuged at 2850 × g. Cells were disrupted by hypo710

Journal of Proteome Research • Vol. 5, No. 3, 2006

technical notes tonic lysis, by resuspension in Buffer A (10 mM Hepes, pH 8.0, 2 mM phenylmethylsulfonyl fluoride, 1.5 mM MgCl2, 1 mM KCl, 1 mM dithiothreitol (DTT), 1 mM benzamidine, 20 µg/mL pepstatin A, 10 µg/mL leupeptin, 10 µg/mL aprotinin) with 2 s of vortexing, and incubation on ice for 20 min. Lysis was confirmed microscopically, and lysates were centrifuged at 1450 × g for 10 min at 4 °C to remove intact nuclei. The postnuclear supernatant was removed, added to 0.11% volume of 300 mM Hepes, pH 7.6, 1.4 M KCl, 30 mM MgCl2, and stored at -80 °C. 4.5 × 109 cells yielded ∼40 mL of postnuclear supernatant with protein concentration ∼8 mg/mL. The postnuclear extract was further processed to enrich for membrane proteins. The sample was centrifuged at 200 000 × g for 30 min at 4 °C. The soluble fraction, containing cytosolic proteins, was discarded and the pellet containing the membrane fraction (P1) was washed in a high salt solution to remove remaining soluble proteins. The pellet was suspended in 10 mL of Buffer B (150 mM NaF, 3.3 mM Na3VO4, 250 mM NaCl, 140 mM potassium phosphate, pH 7.6, 4 mM DTT) by passage through a 1 mL syringe with a 23 G needle, then centrifuged at 200 000 × g for 30 min at 4 °C. The second pellet (P2) was resuspended in 6 mL of Buffer B and centrifuged again. The third pellet (P3) was then suspended in 4 mL of ice cold 200 mM sodium carbonate, pH 11, incubated at 4 °C with rocking to strip off peripheral membrane associated proteins before centrifugation at 150 000 × g for 1 h at 4 °C. The fourth pellet (P4) was resuspended in 8 mL 200 mM sodium carbonate and sonicated for 3 × 10 s at 4 °C (Branson, microtip) to break up vesicles containing soluble or peripheral membrane proteins. The lysate was then incubated for 30 min at 4 °C with rocking before centrifugation at 150 000 × g for 30 min, 4 °C, to yield the fifth (P5) membrane pellet. The P5 pellet was then resuspended in 2 mL 1.2 M NH4HCO3, pH 8.3, to bring the pH to neutral. Centrifugation at 200 000 × g for 30 min at 4 °C yielded the final (P6) membrane pellet. 4.5 × 109 cells yielded 16 mg of protein in the final membrane pellet. Membrane Protein Extraction and Digestion: (1) 1% f 0.4% Acid Labile Surfactant (ALS): The P6 pellet (∼4 mg protein) was resuspended in 1 mL 1% (w/v) RapiGest (sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1propane sulfonate, Waters) dissolved in 50 mM NH4HCO3. The solution was heated at 100 °C for 10 min to enhance protein solubilization per the manufacturer’s instructions, then proteins were reduced by adding DTT to 4 mM and incubating for 10 min at 50 °C. The solution was cooled and proteins were alkylated by adding iodoacetamide to 14 mM, incubating for 30 min in the dark at 4 °C, and quenching the alkylation reaction by further addition of 3 mM DTT. The solution was then diluted to 0.4% ALS by adding 1.5 mL of 50 mM NH4HCO3. Samples were immediately proteolyzed at 37 °C in the presence of 1 mM CaCl2 with 3% (w/w) unmodified porcine trypsin (Wako, #207-09891), added in 1% aliquots at t ) 0, 6, and 12 h. After digestion, the ALS was hydrolyzed by adding HCl to 40 mM, followed by incubation at 37 °C for 1 h. The solution was spun at 2500 × g for 10 min to remove particulates. The peptide solution was brought to 1% formic acid and desalted using a reversed phase peptide trap (Oasis HLB, Waters). Peptides were eluted in 70% acetonitrile (ACN)/ 0.1% formic acid and lyophilized once to remove organics. (2) 8 M f 0 M Urea: The P6 pellet (∼4 mg protein) was resuspended in 1 mL 8 M urea (Fisher, electrophoresis grade) added within 1 h after dissolving in water. Proteins were reduced and alkylated as above, then rapidly desalted on a size

technical notes exclusion column (PD10, Amersham) equilibrated with 100 mM NH4HCO3. Immediately after desalting, trypsin was added to initiate proteolysis as described above. After digestion, particulates were removed by low speed centrifugation and peptides were desalted on the peptide trap. (3) 8 M f 1.6 M Urea: The P6 pellet (∼4 mg protein) was resuspended in 1 mL 8 M urea, and proteins were reduced and alkylated as above. Immediately after alkylation, the sample was diluted to 1.6 M urea by the addition of 100 mM NH4HCO3. Proteins were trypsinized as above, followed by removal of particulates and desalting on the peptide trap. (4) 60% Methanol: The P6 pellet (∼4 mg protein) was resuspended in 1 mL 50 mM NH4HCO3 and incubated for 20 min at 90 °C. 1.5 mL of methanol was added to yield 60% (v/v) organic solvent. The sample was then reduced and alkylated, followed immediately by trypsinization, as above. Aliquots were removed at different steps during extraction and following proteolysis, and analyzed by SDS-PAGE, normalizing amounts loaded by the total amount of sample to enable direct comparison of lanes. Protein mixtures were separated on 12.5% acrylamide tris-glycine gels and silver stained by the method of Blum et al.28 Peptide digests were separated on 16.5% tris-tricine gels (Bio-Rad), and silver stained after glutaraldehyde fixation. Briefly, gels were prefixed for 20 min in 50% (v/v) methanol/10% (v/v) acetic acid/0.01875% (w/v) formaldehyde, rinsed once with H2O, and fixed for 30 min in 10% (v/v) glutaraldehyde. After fixation, the gels were washed 3 × 10 min in H2O, reduced for 15 min in 32 mM DTT, and stained in 0.1% (w/v) silver nitrate/0.01875% (w/v) formaldehyde for 15 min. Gels were then rinsed with H2O, developed in 3% (w/v) sodium carbonate/0.01875% (w/v) formaldehyde to desired intensity, and quenched with 2.3 M citric acid. Peptide Chromatography and Data Collection. Lyophilized peptides were dissolved in Buffer C (5 mM K2HPO4, 5% acetonitrile, pH 4) and fractionated by SCX-HPLC (PolySulfoethyl A, 2.1 mm I. D. × 200 mm, Poly LC), equilibrated in Buffer C. Peptides were collected in 20 × 1 mL fractions following elution with a linear gradient of 0-0.5 M KCl in Buffer C. For each SCX fraction, 5% of the total volume was analyzed in a single run by RP LC-MS/MS with m/z scan range 350-1800 Da (“full mass window”), and 15% of the volume was analyzed in 6 runs with overlapping m/z ranges (300-678, 670-798, 790-918, 910-1038, 1030-1278, 1270-1700 Da) (“gas-phase fractionation”).29,30 Reversed-phase separations were carried out on 250 µm I. D. columns fabricated in-house.11 The column was equilibrated with 0.1% formic acid (HPLC Buffer A) at 10 µL/min and the peptides were eluted at 4 µL/min with the following gradient into 70:30 acetonitrile/water + 0.1% formic acid (HPLC Buffer B): 0-18% B in 20 min, 18-27% B in 45 min, 27-50% B in 25 min, and 50-100% B in 22 min. Data collection was carried out using an LCQ Deca XP ion trap mass spectrometer (ThermoElectron). The target value for the ion trap was 8 × 108 ions in MS scan mode and 3 × 108 ions in MS/MS mode. One full-scan mass spectrum was acquired and MS/MS spectra were acquired for the three most intense peaks in the MS spectrum, using a normalized collision energy of 35 units, with dynamic exclusion for 90 s (dynamic exclusion is triggered when the mass spectrometer sequences the same peptide two times in 90 s). After 3 min, the m/z value was removed from the exclusion list. Data Analysis. DTA files were generated from MS/MS spectra using TurboSequest, in centroid mode with intensity threshold ) 10 000, peptide mass tolerance ) 2.5 Da (average mass),

Ruth et al.

allowed grouping of 1-5 scans, and minimum ion count ) 35. DTA files were then searched using Sequest and Mascot as described by Resing et al.,11 allowing 2.5 Da (avg) peptide mass tolerance and 1.0 Da (avg) fragment ion mass tolerance. Searches were carried out by specifying static modification of cysteine, cleavage by trypsin, and allowing up to two incomplete cleavages. As described previously,11 variable covalent modifications or nonspecific cleavages were not enabled, to maximize accuracy in searching the IPI human protein database (v. 3.0, November 2004). To evaluate the frequency of false positive assignments, datasets were also searched against a false protein database created by inverting each protein sequence contained in the normal database.31 Output from search programs were parsed into an Oracle 9i database, and peptide assignments were validated using MSPlus software.11 Briefly, MSPlus compares results from Sequest and Mascot searches and applies heuristic rules for acceptance or rejection of peptide assignments, by evaluating consensus between search programs, XCorr and Mowse scores, ion charges, incomplete cleavage patterns, consistency of peptide sequence with SCX elution, Sequest ion score, and Sequest RSP value. In-house Isoform Resolver software uses peptides validated by MSPlus to construct a protein profile, matching unique peptide sequences with protein accession numbers for all protein entries containing that peptide sequence, grouping proteins according to the variants in which they are present, and computing the minimum number of protein groups.11 Peptides containing one or two amino acid replacements that are often not distinguishable by ion trap mass spectrometers are treated as isoforms by this program. The final output generated by MSPlus and Isoform Resolver lists peptides identified with high confidence and minimum protein groups determined from these sequences. Estimates of fold changes in protein abundances between datasets were quantified from spectral counts as previously described.32 Protein Classifications. Proteins were assigned subcellular location and membrane status based on annotations from GOGetter, a web-based application which assigns cellular component values from Gene Ontology (GO), Conserved Domain Database (CDD), and KEGG databases (bmf.colorado.edu/gogetter/). Proteins classified as ‘membrane’ or ‘integral to membrane’ under GO component were classified as membrane proteins. Proteins without membrane GO annotations were searched manually against Swiss-Prot and Pfam databases to determine membrane association, which added proteins with lipid covalent modifications. Proteins that were still not classified as membrane proteins were searched using TMHMM (www.cbs.dtu.dk/services/TMHMM-2.0/)33 a web based prediction program that predicts transmembrane domains based on primary sequence. IPI addresses were used to compile FASTA files which were analyzed by TMHMM in batch mode. Sequence coverage was visualized with TOPO2 transmembrane protein graphics program.34 Microarray Datasets. K562 cells grown in 10% FBS/RPMI were washed with PBS and total RNA purified by extraction with TRIzol reagent (Invitrogen). First strand and second strand cDNA synthesis, in vitro transcription of biotin-labeled cRNA, and fragmentation were carried out following standard protocols from the Affymetrix Expression Analysis Technical Manual (http://www.affymetrix.com). The samples were hybridized onto U133 Plus 2.0 GeneChip (Affymetrix) and processed at the UCHSC Cancer Center Microarray core facility. Data were corrected for background using MicroArray Suite 5.0 and Journal of Proteome Research • Vol. 5, No. 3, 2006 711


technical notes

Figure 1. Summary of protocols for membrane isolation, solubilization and proteolysis. (A) Isolation of the K562 membrane fraction. K562 cells were lysed hypotonically and post-nuclear pellets consisting of plasma membrane and nonnuclear organelle membranes were washed with high salt and carbonate extraction combined with differential centrifugation. The final pellet (P6) was enriched in membrane proteins, recovering approximately ∼5% of the total starting protein. (B) Four protocols for membrane protein extraction. The P6 membrane pellet (panel A) was divided equally into four parts, then solubilized in 1% ALS, 8 M urea followed by desalting into ammonium bicarbonate, 8 M urea followed by dilution to 1.6 M urea, and 60% methanol. Details are outlined in Materials and Methods. (C) Peptide desalting and chromatography. Peptides from preparations (panel B) were subjected to low speed centrifugation to remove particulate matter, and the soluble peptide mixtures are desalted on a peptide trap to remove detergent and salts. The peptide mixtures were then lyophilized to remove ammonium bicarbonate prior to separation by SCX-HPLC.

normalized using robust multi-array average (RMA) quantile normalization (RMAExpress, version 0.1)35 Genes with “present” calls in Affymetrix datasets were compared to proteins present in shotgun proteomics datasets. This comparison was facilitated using in-house software that matched Affymetrix identifiers with IPI protein identifiers through their common LocusLink IDs.

Results Efficiency of Protein Extraction and Proteolysis. We examined different methods for efficiency of proteolysis of membrane protein preparations. Our previous studies showed that removing protease specificity during data searching led to significant increases in the scoring thresholds for high confidence assignments (“distraction”), due to the effective increase in database size. Therefore, we restricted our attention to methods that would enable trypsin or LysC proteolysis, to limit the number of possible protease cleavage sites. Membrane samples were prepared by hypotonic lysis of K562 cells, removal of nuclei by low speed centrifugation, and collection of the high-speed pellet, which would be expected to contain microsomes and membranes from nonnuclear organelles such as mitochondria, endoplasmic reticulum, Golgi, endosomes, and lysosomes. Membranes were extracted with high salt, then sonicated and washed in the presence of sodium carbonate at pH 11 to remove peripheral membrane associated proteins, followed by pH adjustment by resuspension with ammonium bicarbonate at pH 8.3 (Figure 1A). Silver staining after SDS-PAGE (Figure 2) showed distinct banding patterns of proteins that were removed by high salt washes (S2, S3) or sodium carbonate washes (S4, S5), compared to those in the final pellet (P5). Four methods to trypsinize proteins in the washed membrane fractions were compared (Figure 1B), three of which followed protocols reported by others. First, membrane proteins were solubilized with 1% (w/v) acid labile surfactant (ALS, RapiGest), reduced and alkylated, and diluted to 0.4% ALS prior to trypsinization, as described by Yu et al.23 Second, proteins 712


Figure 2. Separation of protein pools with distinct compositions. K562 proteins were processed as in Figure 1A and proteins in different steps in the extraction were examined by 12.5% SDSPAGE followed by silver staining. Loadings in each lane were adjusted to represent 0.025% of each fraction. The changes in banding pattern as the pellets underwent sequential washing reflect the removal of soluble proteins (S1, S2, S3) and peripheral membrane proteins (S4, S5), yielding an enriched membrane pellet (P5) with a distinct pattern compared to other fractions. Molecular weight standards (kDa) are indicated.

were denatured within liposomes with 8 M urea, reduced and alkylated, diluted with ammonium bicarbonate to 1.6 M urea, and trypsinized, as described by Washburn et al.9 Third, proteins were reduced and alkylated, solubilized by addition of methanol to 60% (v/v), and trypsinized, as described by Blonder et al.16,17 Finally, proteins were denatured by addition of urea to 8 M, reduced and alkylated, and rapidly desalted into 0.1 M ammonium bicarbonate prior to trypsinization. The latter protocol was introduced as a method to minimize potential adventitious reactivity of protein side chains with iodoacetamide or urea-derived cyanate.36-38

technical notes

Figure 3. Efficiency of solubilization and proteolysis by different protocols. (A) P6 proteins solubilized and proteolyzed by four membrane preparation protocols were separated by SDS-PAGE and visualized by silver staining. Lanes 1-4: Proteins extracted by solubilization in 1% ALS (A1) and diluted to 0.4% ALS after heating at 37 °C (A2). Proteins were digested in 3% (w/w) trypsin, yielding a peptide mixture (A3) loaded at 1X or 5X the levels analyzed in lanes 2 and 3. The band at 28 kDa represents undigested trypsin. Lanes 5-8: Proteins extracted in 8 M urea (B1) and desalted into 100 mM ammonium bicarbonate (B2). Peptides after trypsin digestion (B3), loading 1× and 5× the amount in lanes 5 and 6. Lanes 9-12: Proteins extracted in 8 M urea (C1), followed by dilution to 1.6 M urea (C2) and tryptic digestion (C3), loading peptides at 1× and 5×. Lane 12 reveals similar digestion efficiency to the 8 M f 0 M urea method (lane 8), but decreased efficiency compared to ALS (lane 4). Lanes 1316: Proteins extracted by methanol. Membrane proteins were solubilized in 50 mM ammonium bicarbonate (D1) and methanol was added to 60% (v/v) (D2), prior to trypsin digestion (D3). Proteolysis was greatly reduced compared to the other three methods. Molecular weight standards (kDa) are indicated. (B) Solubility of peptides after proteolysis. Digests of membrane proteins extracted with 1% f 0.4% ALS or 8 M f 0 M urea were centrifuged at low speed to remove insoluble particulates. The resulting pellets and supernatants were then separated on tristricine gels and visualized by silver staining with glutaraldehyde fixation to assess the recovery of low mass analytes. Lanes 1: low mass standards. Lanes 2-5: the peptide mixture from ALS extraction before (A3) and after centrifugation to separate supernatant (S) and pellet (P). Lane 5 represents 10× loading of samples in lanes 2-4. Lanes 6-9: the peptide mixture from 8 M f 0 M urea extraction before (B3) and after centrifugation to separate supernatant (S) and pellet (P). Lane 9 represents 10× loading of samples in lanes 6-8. Reduced proteolysis and greater insolubility of peptides were observed in the urea-treated samples compared to ALS.

Proteins were resolved by SDS-PAGE and silver stained in order to compare the efficiency of protein recovery and proteolysis under each condition. Solubilization of proteins with ALS yielded efficient extraction and proteolysis as measured by the disappearance of high mass proteins (Figure 3A, lanes 1-4). Extraction of proteins with 8 M urea led to efficient protein recovery upon desalting into 0 M urea (Figure 3A, lanes 5,6), which was comparable to samples treated with ALS. The

Ruth et al.

extent of proteolysis in this experiment (Figure 3A, lanes 7, 8) was slightly lower than that of samples proteolyzed in ALS, and comparable to samples treated with 8 M urea but instead diluted to 1.6 M urea before trypsin addition (Figure 3A, lanes 9-12). After trypsinization, samples treated with 8 M urea followed by desalting into 0 M urea showed noticably higher turbidity than samples diluted to 1.6 M urea or solubilized with ALS. Therefore, low speed centrifugation of the peptide digest was carried out to assess recovery of peptides within supernatants vs pellets (Figure 1C). Separation of fractions by tristricine PAGE showed higher recovery of peptides into supernatants when samples were digested in ALS than 8 M f 0 M urea (Figure 3B, lanes 3 and 7), where in the latter case, pellets retained 20-30% of the total material from the digest. Aliquots of the membrane proteins that were solubilized and then digested in the presence of 60% methanol were lyophilized to remove organic solvent and analyzed by SDS-PAGE (Figure 3A, lanes 13-16). Both the extraction of proteins and extent of proteolysis were significantly less efficient in the presence of organic solvent compared to proteins solubilized with ALS or urea. The results suggested that peptide recovery is lower with the methanol extraction procedure, therefore this method was not investigated further. Multidimensional LC-MS/MS. Soluble peptides derived from membranes solubilized and proteolyzed with (A) 1% f 0.4% ALS, (B) 8 M f 0 M urea, or (C) 8 M f 1.6 M urea were further processed for mass analysis by desalting on a peptide trap cartridge, lyophilization, and resolution by strong cation exchange (SCX) high performance liquid chromatography (HPLC) (Figure 1C). SCX fractions were then analyzed by RPLC/MS/MS using data dependent data acquisition, collecting data with a m/z window of 350-1800 Da (“full mass window”), as well as over six narrow m/z ranges (“gas phase fractionation”,29,30 see Materials and Methods). Table 1 lists the total numbers of MS/MS spectra files (DTAs) collected and the number of total and unique peptides identified using MSPlus software.11 Isoform Resolver software11 was then used to determine the number of unique proteins, as well as the minimum number of protein isoforms, defined by the protein database entries that could be unambiguously distinguished from other entries by identifying peptides within nonoverlapping regions in their amino acid sequences. Proteins that could not be distinguished in this way were classified into a single isoform group. Datasets collected over a full mass window (“FM”) yielded on average 472 protein groups in each experiment. Datasets collected using FM plus gas-phase fractionation (“GPF”) of SCX fractions yielded on average 755 protein groups per experiment. Spectra were also searched against an inverted protein sequence database,31 to evaluate false positive sequence assignments. Using the same filters in MSPlus as were used for the normal database search, the false discovery rate (FDR) for unique peptides and for proteins identified by one peptide was estimated at 4.2%. Observed proteins, sequence coverage, peptide sequences, observed and predicted masses, and scores are summarized in Supporting Information Tables 1 and 2. Peptide recoveries from methods A (ALS), B (8 M f 0 M urea) and C (8 M f 1.6 M urea) for membrane protein solubilization and digestion were compared, examining results from datasets collected over the full mass window for each SCX fraction (Figure 4A, Table 1A). The numbers of unique peptides were similar between each of the three methods of protein preparation, averaging 1037 peptides for each full mass window Journal of Proteome Research • Vol. 5, No. 3, 2006 713

technical notes


Table 1. Shotgun Proteomics Analysis of Proteins Extracted from Human K562 erythroleukemia cell membranes A. Peptide and Protein Statistics sample

no. of MS/MS spectraa

no. of peptides identified

no. of unique peptides

no. of unique proteinsb

14482 79725

1729 3451

1079 1608

516 742

14209 92100

1602 3254

1028 1447

469 767

15619 216135

1627 11915

1005 3060

431 1309

1% f 0.4% ALS: Full mass window (FM) Gas-phase fractionation + FM 8 M urea f 0 M urea: Full mass window Gas-phase fractionation + FM 8 M f 1.6 M urea: Full mass window Total:

B. Characteristics of Observed Peptides peptide length d

15.9 ( 4.7 14.6 ( 4.3 14.9 ( 4.4

1% f 0.4% ALS 8 M f 0 M urea 8 M f 1.6 M urea

17.0 ( 6.1

percent Cys-containing e

no. of hydrophobic residues f

Membrane Samplec 14.1 16.6 17.5 Soluble Proteinsi 9.7

a

no. of acidic residues g

no. of basic residues h

4.4 ( 1.8 4.1 ( 1.7 4.2 ( 1.7

2.5 ( 1.9 2.1 ( 1.5 2.1 ( 1.6

1.3 ( 0.5 1.4 ( 0.5 1.3 ( 0.5

4.2 ( 2.0

2.6 ( 2.0

2.1 ( 0.9

b

Number of MS/MS files with peptide mass between 900 and 4800 Da. Proteins counted after removing redundancies with Isoform Resolver. c Full mass range dataset only. d Average number of amino acids ( standard deviation. e Percentage of amino acids with at least one Cys (carbamidomethylation). f Average number of Leu, Ile, Phe, Trp, or Val residues ( standard deviation. g Average number of Glu or Asp residues ( standard deviation. h Average number of Arg, Lys, or His residues ( standard deviation. i Peptides from soluble protein samples from K562 cells, described previously.11

Table 2. Proteins Enriched in Membrane Preparations over Soluble Samples RSCa

SCSb

SCMc

gene name

gene ontology

8.86 8.17

3 1

146 48

Transmembrane receptor, integral membrane Transmembrane receptor, integral membrane

7.87 7.73 7.58 7.58 7.49 7.42 7.41 7.41 7.41 7.22 7.22 7.22 7.15 7.00 7.00 7.00 6.93 6.88

0 0 0 0 2 1 0 0 0 0 0 0 1 0 0 0 2 1

21 19 17 17 43 28 15 15 15 13 13 13 23 11 11 11 29 19

Transferrin receptor protein 1 Cation-independent mannose-6-phosphate receptor precursor Solute carrier family 25 A5 (fragment) ATP6V0A1 protein Flotillin-2 Pigment epithelium-derived factor precursor Neutral amino acid transporter b Kinesin 23 protein Thrombospondin 1 precursor Solute carrier family 25 A3 (fragment) Vesicle-associated membrane protein 3 CD81 antigen Inter-alpha-trypsin inhibitor heavy chain H2 Inositol polyphosphate 4-phosphatase IIa Flotillin-1 Rac GTPase activating protein (GAP) 1 Complement component C9 precursor Synaptogyrin-2 4F2 cell-surface antigen heavy chain Solute carrier family 2. Facilitated glucose transporter. Member 1

Transporter activity, integral membrane Ion transporter, integral to membrane Lipid rafts, integral to membrane Protease inhibitor, extracellular Amino acid transporter, integral to membrane Motor activity, cytoskeletal Cell adhesion, extracellular Ion transporter, integral to membrane Membrane fusion, integral to membrane Protein binding, integral to membrane Protease inhibitor, extracellular matrix assoc′d Inositol metabolism, cytoplasmic Lipid rafts, integral to membrane Signal transduction, soluble Complement transmembrane channel Vesicle trafficking, integral to membrane Carrier activity, integral to membrane Transporter activity, integral to membrane

a All proteins with RSC greater than 3.0 are shown. RSC ) log2[(n2 + f)/(n1 + f)] + log2[(t1 - n1 + f)/(t2 - n2 + f)], where n1 and n2 are spectral counts for proteins from soluble and membrane samples, respectively, t1 and t2 are summed spectral counts for all proteins in soluble and membrane samples, respectively, and f is a correction factor equal to 1.25 (ref 32). RSC was calculated for all proteins where n1 or n2 was greater than or equal to 5, and ranked in order of highest positive value, which indicates enrichment in the membrane sample. b Total numbers of peptide spectra for proteins in the soluble sample (n1). c Total numbers of peptide spectra for proteins in the membrane sample (n2).

experiment, and totaling 1805 peptides in the three datasets combined. The three methods also showed comparable numbers of peptides per protein, average peptide length, number of basic, acidic and hydrophobic residues, and recovery of Cyscontaining peptides between the ALS, 8 M f 0 M urea, and 8 M f 1.6 M urea extraction conditions (Table 1B). Of the peptides observed over the three FM experiments, 47% were observed in more than one sample and 25% in all three samples (Figure 4A). A total of 729 proteins were identified from these dataset, of which 56% were observed in more than one sample 714


and 38% were observed in all three samples. Of the 729 proteins, 342 (47%) were identified by two or more of the 1805 peptides observed in all experiments. The two urea-treated samples showed similar yields of peptides and proteins, indicating that desalting into ammonium bicarbonate followed immediately by trypsinization is equally as effective as enzymatic digestion in 1.6 M urea. This implies that the rapid addition of trypsin after desalting enables membrane proteins to be proteolyzed before liposomes aggregate sufficiently to come out of solution.

technical notes

Figure 4. Peptide and protein recoveries from different extraction protocols. (A) Results from FM datasets. The Venn diagrams show unique peptides and minimum protein groups recovered from membrane preparations extracted by ALS, 8 M f 0 M urea, or 8 M f 1.6 M urea protocols. Each method for protein extraction yielded comparable numbers of peptides and proteins, with a total of 1,805 peptides and 729 protein groups identified from all experiments together. (B) Results from GPF+FM datasets. At higher sampling depth, differences in protein recoveries comparing ALS vs urea were not evident.

The proteins captured by the three extraction methods were analyzed for potential membrane localization by searching Gene Ontology (GO) annotations. Those proteins not described by GO as being components of cellular membrane fractions were subsequently examined using TMHMM, a transmembrane membrane domain (TMD) predicting program that predicts transmembrane helices within protein primary sequences. Both methods used for extracting proteins with urea yielded similar percentages of proteins identified as membrane associated (46-47%), based on GO annotations and manual analysis (Figure 5A). The two methods also shared similar percentages of proteins not identified as membrane associated, but nevertheless predicted to contain TMDs by TMHMM (10-11%). Thus, on average 57% of proteins recovered from urea extration were predicted as membrane-associated or membrane-bound for the two urea based methods. These results were comparable to proteins extracted with ALS, identified by GO or manual analysis as membrane-associated (49%) and/or TMHMM (12%). Thus, protocols involving solubilization by urea yielded similar recoveries of membrane proteins compared to solubilization by ALS. Greater sensitivity and depth in protein sampling was obtained by an independent FM analysis together with GPF of SCX fractions, carried out for samples extracted with ALS vs urea. Samples extracted by 8 M f 0 M urea, and not 8 M f 1.6 M, were examined, given that each method yielded similar percentages and numbers of membrane proteins. The GPF + FM data yielded a total of 1,077 proteins and 2,324 peptides identified (Figure 4B, Table 1A). Of these, 432 proteins and 731 peptides were observed in common between ALS and urea extraction methods. Percentages of Cys-containing peptides extracted by ALS vs urea were 17.0% and 14.9%. Each method identified similar number of proteins, with 742 and 767 proteins yielded by ALS and urea extraction, respectively. As observed in the FM datasets collected separately as described above, both

Ruth et al.

methods yielded similar numbers of total and unique peptides identified per protein. GPF + FM datasets were then analyzed for membrane proteins by examination of GO annotations and TMD predictions. ALS extraction resulted in 288 (39%) membrane proteins identified by GO annotations and 80 (11%) identified by TMHMM prediction software (Figure 5B), while urea extraction yielded 269 (35%) proteins identified as membrane bound by GO and 66 (9%) identified as membrane bound by TMHMM. Thus, as observed with the full mass range experiments, urea extraction yielded similar numbers and percentages of membrane proteins compared to ALS extraction. The reduced percentages of membrane proteins in the GPF + FM datasets is most likely attributable to the higher depth of sampling which would detect more soluble proteins within the membrane preparations. Estimation of Membrane Protein Enrichment. To assess the degree of membrane protein enrichment in these samples which were prepared by differential centrifugation away from soluble pools, membrane proteins in nonenriched datasets were evaluated from microarray datasets. Total mRNA was extracted from K562 cells grown under identical conditions in triplicate, and used to generate labeled cRNA, which was then probed against Affymetrix U133 Plus 2.0 GeneChip microarrays. Of the 54 674 probesets, 27 116 were scored as “present” in each of three replicate analyses using MicroArray Suite software. We assumed that these probesets represent proteins most likely expressed in K562 cells. By matching these proteins to corresponding IPI annotations using RefSeq and LocusLink identifiers, and minimizing the number of IPI annotations for any given gene to reduce isoform redundancy, 8017 represented proteins were estimated to be present based on microarray signal. These proteins were then examined by their GO annotations and predicted TMDs, as above. The results estimated that 2038 (25%) of expressed proteins in K562 cells are membrane-associated (Figure 5C). Similar values were estimated for membrane proteins that would be expected by random chance (28%), where in the entire set of 47 094 IPI human database entries, 9425 were described as membrane proteins based on GO annotations (20%, Figure 5C) and 3,605 (8%) of the remaining proteins were predicted to have at least one transmembrane domain by TMHMM. We conclude that protocols for membrane protein enrichment by centrifugation followed by ALS or urea extraction yielded greater percentages of membrane proteins (50-60%) than proteins scored present based on mRNA signal (25%) or represented in the IPI protein database (28%). Integral membrane proteins identified with high peptide coverage were further examined for peptide recovery, comparing GPF + FM datasets of ALS vs urea extraction conditions. Peptide coverage refers to the number of unique peptides that specify a given protein identification. Fourteen proteins were examined that were common to both experiments, contained transmembrane domains, and were represented by the highest number of peptides. Overall, ALS extraction yielded similar peptide coverage and recovery of peptides found within or close to transmembrane regions (Figure 6). In the GPF + FM datasets used to compile Figure 6, 73 peptides were shared between ALS and urea, 54 peptides were unique to ALS, and 30 peptides were unique to urea. In a similar analysis of the FM datasets of ALS vs urea (8 M f 0 M), 60 peptides were shared in common, 32 peptides were unique to ALS, and 35 peptides were unique to urea. Both ALS and urea recovered peptides Journal of Proteome Research • Vol. 5, No. 3, 2006 715


technical notes

Figure 5. Analysis of membrane protein composition. (A) Results from FM datasets. Pie diagrams show membrane-associated proteins predicted from GO annotations, proteins in the remaining set with transmembrane domains (TMDs) predicted by TMHMM software, and proteins with no predicted membrane association or TMDs. ALS extraction yielded similar percentages of proteins with predicted membrane proteins (61%) compared to extraction with 8 M f 0 M or 8 M f 1.6 M urea (57%). (B) Results from GPF+FM datasets. At the higher sampling depth, the efficiency of membrane protein recovery was reduced with either ALS or urea extraction (44-50%), which may reflect greater sampling of soluble protein contaminants present at lower abundance. (C) Estimates of protein compositions in nonmembrane enriched datasets. Shown are compositions of membrane-associated proteins in all genes scored present in microarray analyses, IPI database entries used for searching shotgun proteomics datasets, and proteins identified in previous shotgun proteomics datasets of soluble protein samples.

located close to or within membrane spanning domains (Figure 6, panels E-H). Comparison of Membrane Enriched Proteins to Soluble K562 Proteins. Previous studies identified >5000 proteins from K562 cells by shotgun proteomics.11 These were extracted by sonication of lysates in high salt buffer followed by centrifugation at 200 000 × g to remove insoluble pellets, and were thus presumably more enriched in soluble proteins. For comparison to this “soluble sample” dataset, the current datasets of ALSand urea-extracted proteins (GPF + FM) were combined into a single dataset, referred to as “membrane sample”, where proteins are listed nonredundantly and spectral counts are summed. Between the membrane and soluble sample datasets, 635 proteins were observed in common between membrane and soluble preparations, 442 proteins were unique to the membrane sample, and 5485 proteins were unique to the soluble sample. Of the 6120 proteins in the soluble sample, 1024 (17%) were classified as membrane-associated (Figure 5C), much lower than the 44-50% membrane proteins in the total membrane sample dataset, and consistent with the expected enrichment of membrane proteins within the subset unique to the membrane sample. Semiquantitative methods were then used to assess enrichment of membrane proteins in membrane fractions compared to soluble fractions. Because these datasets were taken on different preparations at different times, methods for quantifying differences in protein abundance by stable isotope labeling of peptides were precluded. Instead, we compared the number of times peptide spectra were recovered for proteins between datasets (“spectral counting”), which has been shown to represent a promising alternative method for enabling labelfree protein quantitation.32,39,40 716


We first examined ALS vs urea extraction methods, comparing the spectral counts of proteins found in common between these datasets, which ranged between 0 and 83 spectra for any protein. As expected, correlations between spectral counts of proteins in the two membrane enriched datasets were observed (Figure 7A), reflecting the similarity in sample preparation between these experiments. However, comparison of the soluble vs combined membrane fractions showed poor correlations overall (Figure 7B). A large number of proteins showed high spectral counts in the membrane dataset, but low spectral counts in the soluble dataset, and vice versa. Such an inverse correlation between many proteins shared between datasets suggested enrichment of certain classes of proteins in each sample. This was examined further by calculating fold changes in protein abundance between membrane vs soluble samples from spectral counts, using an expression which corrects for variable sampling depth as well as discontinuities with spectral count ) 0.32 Proteins were then ranked according to RSC, the log2 ratio of abundance between membrane and soluble samples. The top 20 proteins in this ranking showed 17 integral membrane proteins or extracellular proteins expected to associate with membranes (Table 2). Proteins with RSC g 3 (g8fold greater abundance in membrane samples) and which showed 61% integral membrane or extracellular proteins, 22% ribosomal proteins, and 17% cytoplasmic or cytoskeletal proteins (summarized in Supporting Information Table 3). The analysis confirmed that ALS and urea extraction methods showed biased recovery of membrane associated proteins, with cytosolic or nonmembrane associated proteins in lower amounts. We also observed a partitioning of large vs small ribosomal subunit proteins between each fraction, in which large subunits were more enriched in membrane preparations and small

technical notes

Ruth et al.

Figure 6. Peptide coverage of transmembrane proteins, comparing ALS vs urea extraction methods. Results from GPF + FM datasets were analyzed for the 14 transmembrane proteins with highest peptide coverage that were present in both ALS and 8 M f 0 M urea datasets. Shown in each panel are peptides recovered only under ALS-extraction conditions (red), only under urea extraction conditions (green), and under both extraction conditions (blue). Inspection of each example shows that ALS and urea extractions yield similar peptide coverage for transmembrane proteins, with no significant bias in either method toward membrane spanning regions.

ribosomal subunit proteins were higher in soluble preparations. This could be due to preferential binding with endoplasmic reticulum membranes, or more likely to differential sedimentation conditions between preparations.

Discussion In this study, shotgun proteomics was used to analyze membrane fractions from K562 cells, a cell line often used as Journal of Proteome Research • Vol. 5, No. 3, 2006 717

technical notes


Figure 7. Comparison of spectral counts between membraneenriched vs soluble protein datasets from K562 cells. (A) Comparison of spectral counts between ALS and 8 M f 0 M urea extraction conditions. Spectral counts equal the total number of spectra detected for any peptide from a given protein, and serve as a semiquantitative measure of protein abundance, because peptides from more abundant proteins are detected with higher frequency. Comparison of spectral counts for proteins shared between ALS and urea datasets (GPF + FM) reveals a linear correlation, reflecting similar protein compositions between the two preparations. The slope of the solid line equals 1. (B) Comparison of spectral counts between membrane-enriched and soluble protein datasets. Spectral counts from ALS + urea datasets (GPF + FM) were combined and plotted against datasets of soluble K562 proteins, reported previously.11 Reduced correlations were observed compared to membrane protein comparisons in panel A. This reflects the difference in protein composition between the two datasets, where soluble proteins are present in membrane pools with lower abundance.

a model for CML. The results of this study provide information about the membrane proteome for this cell model, as well as a comparison of efficiencies for membrane protein extraction using various strategies. Of the methods tested, the use of ALS and urea for protein extraction and digestion proved comparable in their effectiveness. Although ALS showed greater solubilization of peptides after proteolysis (Figure 3B), peptide identifications and numbers of integral membrane proteins were generally comparable in shotgun datasets. Thus, urea provides a viable and less expensive alternative to ALS for recovery of membrane proteins, while ALS has the advantage of enabling protein solubilization comparable to SDS and nonionic detergents.24,41 In addition, proteins extracted in 8 M urea and then either desalted to 0 M or diluted to 1.6 M yielded effective proteolysis, with similar yields of membrane proteins. Desalting may be advantageous over dilution, to minimize carbamoylation and adventitious reactivity with iodoacetamide during digestion, or to enable removal of ammonium bicarbonate by lyophilization instead of peptide trap adsorption (the latter which may reduce peptide 718


recovery). Compared to ALS and urea, methanol extraction yielded a significantly higher level of unproteolyzed fragments, most likely due to reduced protease activity in organic solvent. Characteristics of peptides recovered from membrane preparations were similar to those observed in soluble protein digests with respect to peptide length, hydrophobicity, charged residues, and cysteine composition. Significant differences were not observed between different methods. Thus, recovery of peptides appears to occur preferentially from cytosolic or extracellular regions of intrinsic membrane-bound proteins, as suggested by inspection of the 14 transmembrane proteins with highest sequence coverage (Figure 6), and may also reflect selective recovery of peptides from SCX and RP peptide chromatography. Preparation methods involved high salt and high pH washing of membrane fractions, to enrich membrane-associated proteins in final membrane pellets (P6). In multidimensional LCMS/MS datasets collected with full mass range settings, 5761% of proteins were evaluated as membrane-associated, based on GO annotations and transmembrane domain prediction software. This was higher compared to percentages of total expressed genes estimated from microarray datasets. At higher depth of sampling by gas-phase fractionation, lower percentages of proteins were classified as membrane-associated, suggesting greater capture of nonmembrane proteins. Semiquantitative analysis of protein abundance by spectral counting revealed enrichment of integral membrane proteins in membrane samples compared to soluble protein preparations. Abbreviations: RP LC-MS/MS, reversed-phase liquid chromatography and mass spectrometric sequencing; ALS, acidlabile surfactant; HPLC, high-pressure liquid chromatography; TMD, transmembrane domain; FM, full mass range; GPF, gasphase fractionation; GO, Gene Ontology; FDR, false discovery rate; CML, chronic myelogenous leukemia.

Acknowledgment. This work was supported by NIH Grants GM48521 (N.G.A.) and CA87648 (K.A.R.), and by NIH Medical Student Research Fellowship R25 CA47883 (M.C.R.). We are indebted to Michael Browning and Christine Wu (UCHSC, Denver, CO) for insightful conversations and sharing protocols for purifying and handling membrane proteins. We also thank Karen Jonscher and Brian Eichelberger (University Colorado, Boulder) for assistance with data collection, and John Palawek (Yale University) for sponsoring a research internship for M.C.R. Supporting Information Available: Observed proteins, sequence coverage, peptide sequences, observed and predicted masses, and scores are summarized in Supporting Information Tables 1 and 2. Proteins with RSC g 3 (g8-fold greater abundance in membrane samples) and which showed 61% integral membrane or extracellular proteins, 22% ribosomal proteins, and 17% cytoplasmic or cytoskeletal proteins are summarized in Supporting Information Table 3. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Wong, S.; Witte, O. N. The BCR-ABL story: bench to bedside and back. Annu. Rev. Immunol. 2004, 22, 247-306. (2) O’Dwyer, M. E.; Mauro, M. J.; Druker, B. J. STI571 as a targeted therapy for CML. Cancer Invest. 2003, 21, 429-438. (3) Whalen, A. M.; Galasinski, S. C.; Shapiro, P. S.; Nahreini, T. S.; Ahn, N. G. Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase. Mol. Cell. Biol. 1997, 17, 1947-1958.

technical notes (4) Racke, F. K.; Lewandowska, K.; Goueli, S.; Goldfarb, A. N. Sustained activation of the extracellular signal-regulated kinase/ mitogen-activated protein kinase pathway is required for megakaryocytic differentiation of K562 cells. J. Biol. Chem. 1997, 272, 23366-23370. (5) Herrera, R.; Hubbell, S.; Decker, S.; Petruzzelli, L. A role for the MEK/MAPK pathway in PMA-induced cell cycle arrest: modulation of megakaryocytic differentiation of K562 cells. Exp. Cell Res. 1998, 238, 407-414. (6) Jacquel, A.; Herrant, M.; Legros, L.; Belhacene, N.; Luciano, F.; Pages, G.; Hofman, P.; Auberger, P. Imatinib induces mitochondria-dependent apoptosis of the Bcr-Abl-positive K562 cell line and its differentiation toward the erythroid lineage. FASEB J. 2003, 17, 2160-2162. (7) Yu, C.; Krystal, G.; Varticovksi, L.; McKinstry, R.; Rahmani, M.; Dent, P.; Grant, S. Pharmacologic mitogen-activated protein/ extracellular signal-regulated kinase kinase/mitogen-activated protein kinase inhibitors interact synergistically with STI571 to induce apoptosis in Bcr/Abl-expressing human leukemia cells. Cancer Res. 2002, 62, 188-199. (8) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., 3rd. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 1999, 17, 676-682. (9) Washburn, M. P.; Wolters, D.; Yates, J. R., 3rd. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 2001, 19, 242-247. (10) Lipton M.S.; Pasa-Tolic, L. Anderson, G. A.; Anderson, D. J.; Auberry, D. L.; Battista, J. R.; Daly, M. J.; Fredrickson, J.; Hixson, K. K.; Kostandarithes, H.; Masselon, C.; Markillie, L. M.; Moore, R. J.; Romine, M. F.; Shen, Y.; Stritmatter, E.; Tolic, N.; Udseth, H. R.; Venkateswaran, A.; Wong, K. K.; Zhao, R.; Smith, R. D. Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 11049-11054. (11) Resing, K. A.; Meyer-Arendt, K.; Mendoza, A. M.; Aveline-Wolf, L. D.; Jonscher, K. R.; Pierce, K. G.; Old, W. M.; Cheung, H. T.; Russell, S.; Wattawa, J. L.; Goehle, G. R.; Knight, R. D.; Ahn, N. G. Improving reproducibility and sensitivity in identifying human proteins by shotgun proteomics. Anal. Chem. 2004, 76, 35563568. (12) Swiderek, K.; Alpert, A. J.; Heckendorf, A.; Nugent, K.; Patterson, S. D. Structural analysis of proteins and peptides in the presence of detergents: Tricks of the trade. J. Biomol. Techniques 1997, 8, 17-25. (13) Wu, C. C.; Yates J. R. The application of mass spectrometry to membrane proteomics. Nat. Biotechnol. 2003, 21, 262-267. (14) Whitelegge, J. P.; Gomez, S. M.; Faull, K. F. Proteomics of membrane proteins. Adv. Prot. Chem. 2003, 65, 271-307. (15) Wu, C. C.; MacCoss, M. J.; Howell, K. E.; Yates, J. R. A method for the comprehensive proteomic analysis of membrane proteins. Nat. Biotechnol. 2003, 21, 532-538. (16) Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D. Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry. J. Proteome Res. 2002, 1, 351-360. (17) Blonder, J.; Conrads, T. P.; Yu, L. R.; Terunuma, A.; Janini, G. M.; Issaq, H. W.; Vogel, J. C.; Veenstra, T. D. A detergent-and cyanogen bromide-free method for integral membrane proteomics: application to Halobacterium purple membranes and the human epidermal membrane proteome. Proteomics 2004, 4, 3145. (18) Brugiere, S.; Kowalski, S.; Ferro, M.; Seigneurin-Berny, D.; Miras, S.; Salvi, D.; Ravanel, S.; d’Herin, P.; Garin, J.; Bourguignon, J.; Joyard, J.; Rolland, N. The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions. Phytochemistry 2004, 65, 1693-1707. (19) Fan, X.; She, Y. M.; Bagshaw, R. D.; Callahan, J. W.; Schackter, H.; Mahuran, D. J. A method for proteomic identification of membrane bound proteins containing Asn linked oligosaccharides. Anal. Biochem. 2004, 332, 178-186. (20) Han, D. K.; Eng, J.; Zhou, H.; Aebersold, R. Quantitative profiling of differentiation-induced microsomal proteins using isotopecoded affinity tags and mass spectrometry. Nat. Biotechnol. 2001, 19, 946-951. (21) Nielsen, P. A.; Olsen, J. V.; Podtelejnikov, A. V.; Andersen, J. R.; Mann, M.; Wisniewski, J. R. Proteomic mapping of brain plasma membrane proteins. Mol. Cell. Proteomics 2005, 4, 402-408.

Ruth et al. (22) Hixson, K. K.; Rodriguez, N.; Camp, D. G.; Strittmatter, E. F.; Lipton, M. S.; Smith, R. D. Evaluation of enzymatic digestion and liquid chromatography-mass spectrometry peptide mapping of the integral membrane protein bacteriorhodopsin. Electrophoresis 2002, 23, 3224-3232. (23) Ross, A. R.; Lee, P. J.; Smith, D. L.; Langridge, J. I.; Whetton, A. D.; Gaskell, S. J. Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant. Proteomics 2002, 2, 928-936. (24) Yu, Y. Q.; Gilar, M.; Lee, P. J.; Bouvier, E. S.; Gebler, J. C. Enzymefriendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins. Anal. Chem. 2003, 75, 60236028. (25) Yu, Y. Q.; Gilar, M.; Gebler, J. C. A complete peptide mapping of membrane proteins: a novel surfactant aiding the enzymatic digestion of bacteriorhodopsin. Rapid Commun. Mass Spectrom. 2004, 18, 711-715. (26) Arnold, R. J.; Hrncirova, P.; Annaiah, K.; Novotny, M. V. Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver. J. Proteome Res. 2004, 3, 653657. (27) Sevinsky, J. R.; Whalen, A. M.; Ahn, N. G. Extracellular signalregulated kinase induces the megakaryocyte GPIIb/CD41 gene through MafB/Kreisler. Mol. Cell. Biol. 2003, 24, 4534-4545. (28) Blum, H.; Beier, H.; Gross, H. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 1987, 8, 93-99. (29) Spahr, C. S.; Davis, M. T.; McGinley, M. D.; Robinson, J. H.; Bures, E. J.; Beierle, J.; Mort, J.; Courchesne, P. L.; Chen, K.; Wahl, R. C.; Yu, W.; Luethy, R.; Patterson, S. D. Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest. Proteomics 2001, 1, 93-107. (30) Yi, E. C.; Marelli, M.; Lee, H.; Purvine, S. O.; Aebersold, R.; Aitchison, J. D.; Goodlett, D. R. Approaching complete peroxisome characterization by gas-phase fractionation. Electrophoresis 2001, 23, 3205-3216. (31) Moore, R. E.; Young, M. K.; Lee, T. D. Qscore: an algorithm for evaluating SEQUEST database search results. J. Am. Soc. Mass Spectrom. 2002, 13, 378-386. (32) Old, W. M.; Meyer-Arendt, K.; Aveline-Wolf, L.; Pierce, K. G.; Mendoza, A.; Sevinsky, J. R.; Resing, K. A.; Ahn, N. G. Comparison of label free methods for quantifying human proteins by shotgun proteomics. Mol. Cell. Proteomics 2005, 4, 1487-1502. (33) Krogh, A.; Larsson, B.; von Heijne, G.; Sonnhammer, E. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 2001, 305, 567-580. (34) Johns, S. J. TOPO2, Transmembrane protein display software (www.sacs.ucsf.edu/TOPO2/). (35) Irizarry, R. A.; Hobbs, B.; Collin, F.; Beazer-Barclay, Y. D.; Antonellis, K. J.; Scherf, U.; Speed, T. P. Exploration, normalization, and summaries of high-density oligonucleotide array probe level data. Biostatistics 2003, 4, 249-264. (36) Stark, G. R.; Stein, W. H.; Moore, S. Reactions of the cyanate present in aqueous urea with amino acids and proteins J. Biol. Chem. 1960, 235, 3177-3181 (37) Cerami, A.; Manning J. M. Potassium cyanate as an inhibitor of the sickling of erythrocytes in vitro. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 1180-1183. (38) Fruchter, R. G.; Crestfield, A. M. The specific alkylation by iodoacetamide of histidine-12 in the active site of ribonuclease. J. Biol. Chem. 1967, 242, 5807-5812. (39) Pang, J. X.; Ginanni, N.; Dongre, A. R.; Hefta, S. A.; Opiteck, G. J. Biomarker Discovery in Urine by Proteomics. J. Proteome Res. 2002, 1, 161-169. (40) Liu, H.; Sadygov, R. G.; Yates, J. R., 3rd. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem. 2004, 76, 4193-4201. (41) Konig, S.; Schmidt, Ol.; Rose, K.; Thanos, S.; Besselmann, M.; Zeller, M. Sodium dodecyl sulfate versus acid-labile surfactant gel electrophoresis: Comparative proteomic studies on rat retina and mouse brain. Electrophoresis 2003, 24, 751-756.

PR050313Z Journal of Proteome Research • Vol. 5, No. 3, 2006 719

Analysis of Membrane Proteins from Human Chronic Myelogenous

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