Immunoaffinity Purification of Plasma Membrane with Secondary

Nov 17, 2006 - Mascot software was used to analyze the data against IPI-mouse protein database. Nonredundant proteins (248) were identified, of which ...
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Immunoaffinity Purification of Plasma Membrane with Secondary Antibody Superparamagnetic Beads for Proteomic Analysis Lijun Zhang, Xi’e Wang, Xia Peng, Yingjuan Wei, Rui Cao, Zhen Liu, Jixian Xiong, Xingfeng Ying, Ping Chen, and Songping Liang* Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Science, Hunan Normal University, Changsha 410081, P.R. China Received February 27, 2006

Plasma membrane (PM) has very important roles in cell-cell interaction and signal transduction, and it has been extensively targeted for drug design. A major prerequisite for the analysis of PM proteome is the preparation of PM with high purity. Density gradient centrifugation has been commonly employed to isolate PM, but it often occurred with contamination of internal membrane. Here we describe a method for plasma membrane purification using second antibody superparamagnetic beads that combines subcellular fractionation and immunoisolation strategies. Four methods of immunoaffinity were compared, and the variation of crude plasma membrane (CPM), superparamagnetic beads, and antibodies was studied. The optimized method and the number of CPM, beads, and antibodies suitable for proteome analysis were obtained. The PM of mouse liver was enriched 3-fold in comparison with the density gradient centrifugation method, and contamination from mitochondria was reduced 2-fold. The PM protein bands were extracted and trypsin-digested, and the resulting peptides were resolved and characterized by MALDI-TOF-TOF and ESI-Q-TOF, respectively. Mascot software was used to analyze the data against IPI-mouse protein database. Nonredundant proteins (248) were identified, of which 67% are PM or PM-related proteins. No endoplasmic reticulum (ER) or nuclear proteins were identified according to the GO annotation in the optimized method. Our protocol represents a simple, economic, and reproducible tool for the proteomic characterization of liver plasma membrane. Keywords: mouse liver • plasma membrane • proteomics • immunoaffinity purification • MALDI-TOF-TOF • ESIQ-TOF

Introduction Plasma membrane (PM) proteins play pivotal roles in various physiological processes of the liver, such as signal transduction, molecular transport, and cell-cell interactions. PM proteins include neurotransmitter receptors, G-proteins, carriers, and voltage-gated ion channels. Many of them display region- and time-specific expression patterns, therewith determining network specificity and information processing. Therefore, a characterization of the protein composition of PM with proteomics is important in studying the functions of PM,1-4 the molecular mechanism of PM biogenesis,5,6 and the treatment of disease.7 Profiling PM proteins has proven to be particularly challenging due to the difficulty of purifying PM, the low abundance of PM proteins, and the difficulties in resolving and identifying them. Plasma membrane was usually enriched by (i) ultracentrifugation in sucrose medium based on density differences between plasma membrane and other subcellular organelles,6,8,9 (ii) silica bead enrichment,7,10 (iii) affinity enrichment,4,11,12 and * To whom correspondence should be addressed: College of Life Sciences, Hunan Normal University, Changsha, 410081, P.R. China; Tel: 86-731-8872556; Fax: 86-731-886-1304; E-mail: [email protected].

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Journal of Proteome Research 2007, 6, 34-43

Published on Web 11/17/2006

(iv) aqueous two-phase purification.1,13,14 The purity of the membrane fraction prepared by ultracentrifugation, lectin column, and silica beads is not satisfactory because of the contamination by other cellular organelles such as mitochondria and ER. Affinity enrichment with biotinylation is very good for cell samples,12 but is not suited for mouse liver tissue. Aqueous two-phase systems separate PM according to membrane surface properties such as charge and hydrophobicity, but generally there is contamination from other membranes, especially ER. Usually further separation by subsequent use of the affinity ligand wheat germ agglutinin (WGA) coupled to dextran15,16 is needed. Here, we describe a method integrating sucrose density gradient centrifugation and immunoisolation using anti-flotillin and anti-Na+/K+-ATPase (PM special antibodies). This immunoisolation is facilitated by antibodies directed against special membrane proteins, then antibodies bound to immunoaffinity beads to select membrane fractions. It has been successfully applied to the purification of peroxisomes,17,18 tubulovesicles,19 microsomal membrane,20 Golgi,21 and PM.22,23 We have optimized the protocol for PM purification using Dynabeads, comparing four methods. We have determined the optimum number of membrane, antibodies, and beads for proteome 10.1021/pr060069r CCC: $37.00

 2007 American Chemical Society

research articles

Immunoaffinity Purification of Plasma Membrane Table 1. Signal Intensity of Marker Proteins Indicated by Western Blot Analyzed by Quantity One 4.1.0. Comparison of four methods (repeat for three times)

anti-Na+/K+-ATPase anti-flotillin anti-NADH ubiquinol oxidoreductase 39

Change of membrane amount

Change of amount of beads

CPMa

PPM4

PPM3

PPM2

PPM1

5 mgb

10 mg

20 mg

0.5 × 107c

1.0 × 107

2.0 × 107

1 1 1

3.13 ( 0.26 3.02 ( 0.45 0.49 ( 0.15

3.26 ( 0.41 2.84 ( 0.35 0.41 ( 0.10

3.03 ( 0.50 2.34 ( 0.36 0.55 ( 0.17

2.30 ( 0.30 2.12 ( 0.32 0.62 ( 0.18

1 1 1

2.09 2.69 2.26

2.41 4.38 4.50

1 1 not detected

2.20 1.50 not detected

6.05 2.31 signal detected

a Volume in PPM 1,2,3, and 4 was divided by the volume of CPM. b Volume from the fractions of 10 mg and 20 mg CPM was divided by the volume of that of 5 mg CPM. c Volume from the fractions of 1.0 × 107 and 2.0 × 107 beads was divided by the volume of that of 0.5 × 107.

analysis through western blotting and mass spectrometric analysis. The optimized method resulted in a 3-fold enrichment of plasma membrane and a 2-fold decrease of contamination from mitochondria compared with the density gradient centrifugation method. Almost no endoplasmic reticulum (ER) or nuclear proteins were found in the PM preparation. The purified PM proteins were extracted and subjected to SDSPAGE separation and MS/MS analysis.

Materials and Methods Materials. Cellection pan mouse IgG kit and magnetic plate were obtained from Dynal Biotech ASA (Oslo, Norway). Proteomics sequencing grade trypsin, DTT, IAA, TFA, HEPES, and sucrose were obtained from Sigma-Aldrich (St. Louis, MO). Immobilon transfer membranes (PVDF) were from Millipore (Bedford, MA). Bio-Rad DC protein assay kit and Sypro Ruby protein blot gel stain were bought from Bio-Rad Laboratories (Hercules, CA). Anti-Flotillin monoclonal antibody, antioxphosphoplex monoclonal antibody and Horseradish peroxidase-conjugated anti-mouse IgG were obtained from BD Bioscience (San Jose, CA USA). Anti-Na+/K+-ATPase monoclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Immobilized pH gradient (IPG) DryStrips (3-10 linear), IPG buffer, and Silver nitrate kit were purchased from Amersham Pharmacia-Biotech (Uppsaia, Sweden). Acrylamide, bis-acrylamide, urea, glycine, Tris, CHAPS, and SDS were from Amresco (Solon, OH). HPLC-grade acetonitrile, acetone (chromatogram grade), and other chemicals (analytical grade) were from Chinese National Medicine Group Shanghai Chemical Reagent Company (shanghai, China). Water was obtained from a Milli-Q Plus purification system (Millipore, Bedford, MA). C57 mice (9 weeks) were purchased from Hunan Medical University (Changsha, China). Preparation of Mouse Liver Plasma Membranes. Crude plasma membrane (CPM) was purified according to the procedure described by Fieischer S.24 In three ultra-centrifugations, the fractions at the sucrose mediums of 0.25/1.60 M, 0.25/1.45 M, and 0.25/1.35 M were obtained in turn. The PM at 0.25/1.35 M sucrose interface was collected and washed. A specimen of the sample was analyzed by electron microscopy; the rest was stored at -80 °C in storage buffer (50 mM HEPES, 1 mM PMSF). Dynabeads Coating for Direct Selection of PM. Cellection Dynabeads (2 × 107) were washed three times with phosphatebuffered saline (PBS) according to the manufacturer’s protocol. PM-specific antibodies (anti-flotillin (0.5 µg) and anti-Na+/K+ATPase (0.5 µg)) diluted in phosphate-buffered saline (PBS) with or without 0.1% BSA were bound to Dynabeads (2 × 107). The PM-enriched fractions (CPM) (30 mg) from the sucrose density gradient were incubated with the magnetic beads suspended in PBS with or without 0.1% BSA at 4 °C for 4 h. After

incubation, the bead complex was collected by placing the reaction tube in a magnet stand. The complex was washed three times with PBS with or without 0.1% BSA. All purified PM fractions (PPMs) were analyzed by western blotting. The PPMs from the procedure with or without BSA treatment were named PPM1 and PPM2, and used for electron microscopy and proteome analysis. Dynabeads Coating for Indirect Selection of PM. PM special antibodies, anti-flotillin (0.5 µg) and Na+/K+-ATPase (0.5 µg), diluted in PBS with or without 0.1% BSA were added to the CPM suspensions (30 mg) and incubated for 4 h at 4 °C. After incubation, the complex was washed three times with PBS with or without 0.1% BSA to remove unbound antibody and then resuspended in PBS with or without 0.1% BSA and added to Cellection Dynabeads (2 × 107). After incubation for 1 h at 4 °C, the bead complex was collected by placing the reaction tube in a magnet stand and washed three times with PBS with or without 0.1% BSA. The PPMs from the procedure with or without BSA treatment were named PPM3 and PPM4 and used for electron microscopy and proteome analysis. Optimization of the Number of CPM and Dynabeads. The bead quantity was varied from 2 × 107, 1 × 107, to 0.5 × 107 with a constant number of antibodies (0.5 µg of every kind) and CPM (20 mg). On the other hand, the quantity of CPM was decreased from 20 to 10 to 5 mg with a constant number of beads (0.5 × 107) and antibodies (1.0 µg of every kind) constant. All conditions are listed in Table 1. After extraction, half was used for western blotting and the other half for SDSPAGE. Analysis by Quantity One 4.1.0. The signal intensity of marker proteins in the western blot was analyzed by Quantity One 4.1.0 (Bio-Rad) (a software for measuring the quantities of 1-D electrophoresis gel bands). The volume of each band equals the area multiplied by the intensity with the blank volume subtracted. Electron Microscope Analysis of the PPMs. PPM2 and 4 were fixed with 2.5% glutaraldehyde overnight at 4 °C, washed with phosphate-buffered saline and dehydrated with alcohol (25, 50, 75, 95, and 100% in turn). Samples were then washed with isopentyl acetate, dried at freezing, stained with gold, and examined with the scanning electron microscope -JSM-6360LV (JEOL, Tokyo, Japan). SDS-PAGE Separation and Western Blot Analysis. Proteins were extracted by incubating in 30-50 µL of SDS sample buffer (0.1 M Tris-HCl, pH 6.8, 20% glycerol, 4% SDS and 10% mercaptoethanol) for 30 min at 65 °C. After quantification with Bio-Rad RC/DC kit, the proteins were separated by SDS-PAGE using 11% gels. For immunoblotting analysis, the proteins were transferred to PVDF membrane. The membrane was blocked with 5% dry milk in TBST (25 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature or overnight at 4 Journal of Proteome Research • Vol. 6, No. 1, 2007 35

research articles °C and then incubated with appropriate primary antibody in TBST with 5% dry milk for 1 h at room temperature. After washing with TBST three times (5 min every time), the membrane was incubated with HRP-conjugated secondary antibody at 1:500 dilution in TBST with 5% dry milk at room temperature for 1 h, washed with TBST for three times (5 min every time), and detected by LumiGLO Chemiluminescent Substrate Kit from KPL (Gaithersburg, USA). Primary antibodies were diluted using following conditions: anti-Na+/K+-ATPase, 1:5000, anti-flotillin 1:250, anti-Grp 94, 1:1000, and anti-NADH ubiquinol oxidoreductase 39, 1:1000. For proteome analysis, the gels were stained with Colloidal Coommassie blue. Trypsin Digestion and Analysis by NanoHPLC Tandem Mass Spectrometry. The protein bands were excised by hand, and in-gel digested as described in our published papers.25 The digested peptides were analyzed by ESI-Q-TOF (Waters/ Micromass, Manchester, UK) according to our previous described.26 Search parameters for protein identification were set as follows: enzyme, trypsin; allowance of up to one missed cleavage peptide; mass tolerance, 1.0 Da and MS/MS mass tolerance, 0.5 Da; fixed modification parameter, carbamoylmethylation (C); variable modification parameters, oxidation (at Met); auto hits allowed (only significant hits were report); results format as peptide summary report. Proteins were identified on the basis of peptides whose ions scores exceeded the threshold, p < 0.05, which indicates identification at the 95% confidence level for these matched peptides.27 In this study scores above 37 were validated without any additional manual inspection. Those proteins, for which one peptide had a score higher than 37, and a second peptide had a score between 20 and 37, were systematically checked and/or interpreted manually to confirm or negate the MASCOT suggestion. If proteins were identified by a single peptide, and had more than one spectrum, then only the spectrum with the highest score was manually inspected. For a protein to be confirmed (1) the assignment had to be based on four or more y-or b-series ions (e.g., y4, y5, y6, y7) and (2) the protein molecular mass had to be consistent with gel migration data. MALDI-TOF-TOF. The tryptic mixed peptides from SDSPAGE gels were loaded onto AnchorChip target plate according to the previous described.28 Molecular weight information of peptides was obtained by using a MALDI-TOF-TOF mass spectrometer (UltroFlex I, Bruker Daltonics) equipped with nitrogen laser (337 nm) and operated in reflector/delay extraction mode for MALDI-TOF peptide mass fingerprint (PMF) or LIFT mode for MALDI-TOF/TOF with a fully automated mode using the flexControl software. An accelerating voltage of 25kV was used for PMF. Calibration of the instrument was performed externally with [M + H]+ ions of angiotensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (clip 1-17 and clip 18-39). Each spectrum was produced by accumulating data from 100 consecutive laser shots, and the spectra were interpreted with the aid of the Mascot Software (Matrix Science Ltd, London, UK). The peaks with S/N g 5, resolution g 2500 were selected and used for LIFT from the same target. A maximum of 6 precursor ions per sample were chosen for MS/MS analysis. In the TOF1 stage, all ions were accelerated to 8 kV under conditions promoting metastable fragmentation. After selection of a jointly migrating parent and fragment ions in a timed ion gate, the ions were lifted by 19 kV to a high potential energy in the LIFT cell. After further acceleration of the fragment ions in the second ion source, their masses could simultaneously be analyzed in the reflector with 36

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high sensitivity. LIFT spectra were interpreted with the Mascot software. PMF and LIFT datasets were combined via BioTools 2.2 software (Bruker) and used for protein identification. The parameters are following: mass tolerance in PMF of 50 ppm, MS/MS tolerance of 1.0 Da and 1 missing cleavage sites and cysteines modified by carbamidomethylation. The protein identifications were considered to be confident when the protein score of the hit exceeded the threshold significance score of 58 (p < 0.05). Bioinformatics. All identified proteins were analyzed by a perl script, which was written in-house to parse Mascot output files (html files) into XML files suitable for subsequent data analysis. All identified proteins have a IPI database accession number and many of these proteins have assigned Gene Ontology (GO) numbers.29 Use this GO number to retrieve the protein information in the database. Predictions for putative transmembrane domains (TMDs) in all identified proteins were carried out using the transmembrane hidden Markov model (TMHMM) algorithm,30 available at http://www.cbs.dtu.dk/ services/TMHMM. The average hydropathy31 for identified proteins and peptides was calculated using the ProtParam software available at http://cn.expasy.org. Proteins with positive GRAVY values are considered to be hydrophobic and those with negative values, hydrophilic.

Results Purity of Antibody-Enriched Plasma Membrane. Membrane prepared by ultracentrifugation methods typically contains a significant number of materials from other cellular organelles including mitochondria and ER, so one of our present goals was to obtain highly purified PM. To this end, we enriched PM based on immunoisolation. Four methods were carried out, including direct and indirect immunity selection of PM with or without BSA (shown in Figure 1A-C). Four fractions (PPM1, PPM2, PPM3, and PPM4 were obtained, PPM1 and PPM2 from direct selection (Figure 1B) and PPM 3 and PPM4 from indirect selection (Figure 1C). Dynabeads coated without PM and with PPM2 or PPM4 were observed with the scanning electron microscope to examine the immunoisolation process (Figure 1D-F). Plasma membrane was found to be enriched through antibody hooks and no free membrane was observed in the outer space of Dynabeads (Figure 1E,F). To examine the enrichment of PM and the removal of contamination by other cellular organelles, organelle-specific protein markers were detected by probing CPM and PPM 1, 2, 3, and 4 with organelle-specific antibodies by western blot analysis. When equal amounts of protein lysates of those samples were applied to an SDS-PAGE gel, the signals that corresponded with PM-specific proteins (Na+/K+-ATPase and flotillin) were found to be similar in PPM1, 2, 3, and 4 and stronger than that in CPM control. The quantity of each band was analyzed by Quantity One 4.1.0 (Bio-Rad Laboratories, Hercules, Calif.), which is a powerful software for measuring the intensities of 1-D electrophoresis gel bands. The results showed that the PM was enriched ∼3-fold in these 4 fractions (marked by Na+/K+-ATPase and flotillin). Mitochondrial contamination in all the enriched fractions was reduced 2-fold as shown by a mitochondrion-specific antibody, NADH ubiquinol oxidoreductase 39. The PM was more enriched in PPM3 and PPM4 (as shown in Figure 2A and Table 1). Almost no signal of GRP 94 (ER-specific protein) was detected in PPM 1, 2, 3, and 4, which is much less than that in CPM. The SDS-PAGE gels (Figure 2A) show a stronger BSA band (parallel with marker

Immunoaffinity Purification of Plasma Membrane

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Figure 1. Strategy for antibody supermagnetic beads enrichment of plasma membrane proteins. (A) Dynabeads coated with second antibody. (B) Direct selection of PM. Primary antibodies were incubated with beads, followed by incubation with CPM. (C) Indirect selection of PM. Primary antibody was incubated with CPM, followed by incubation with beads. (D) Scanning electron micrographs of Dynabeads coated with second antibody, ×5000. (E) Scanning electron micrographs of Dynabeads coated with PPM2, ×5000. (F) Scanning electron micrographs of Dynabeads coated with PPM4, ×5000.

protein band (66.2KDa)) in PPM1 and PPM3. This would affect proteome analysis, so BSA should be removed from the protocol for proteome analysis. Comparing the electrophoresis profiles of PPM 1, 2, 3, and 4 with CPM, a few differences were noticed (Figure 2A). Several bands with lower molecular weight (MW) disappeared during the purification, whereas some bands with higher MW were clearly concentrated. Taken together, the result indicated that indirect selection using the protocol without BSA (PPM4) is the best of all, giving the biggest enrichment, least contamination, and the weakest BSA band. Optimization of the Number of CPM and Dynabeads. The scanning electron micrographs (Figure 1 E,F) showed that the beads are not fully coated with CPM. Furthermore, strong signals of PM-specific proteins were found in the sample unbound by beads through western blot analysis (data not shown). Therefore, the amount of CPM and Dynabeads should be optimized. The protocol of indirect selection of PPM without BSA was used for the optimization experiment. The number of beads was varied from 2 × 107, to 1 × 107, to 0.5 × 107 with PM (20 mg) constant (as shown in Table 1). Protein signals from

the western blot were reduced proportionately with the reduction of beads. Protein bands in each lane of SDS-PAGE are explicit as shown in Figure 2B. The beads could be reduced 4-fold and 0.5 × 107 beads are enough for PM proteome analysis (including SDS-PAGE and western blot analysis). This is much less than that (1 × 108) described by Kikuchi M.18 On the other hand, for the optimization of CPM, the CPM was varied from 20 to 10 to 5 mg with a constant number of beads (0.5 × 107) and antibodies (1.0 µg every kind) (as shown in Table 1). The intensity of proteins in the SDS-PAGE and western blot analysis were reduced gradually with the reduction of CPM. The protein bands from SDS-PAGE were explicit (Figure 2C). Protein Identification. To confirm that the CPM could be reduced to 5 mg for proteome analysis, three middle intensity protein bands were cut, in-gel digested, and submitted to MALDI-TOF-TOF analysis (shown in Figure 2C, marked by numbers and arrows). Proteins from each band were accurately identified by PMF or PMF combined LIFT as marked with italics in Table S1, Supporting Information. These proteins included sodium/potassium-transporting ATPase alpha-1-chain precurJournal of Proteome Research • Vol. 6, No. 1, 2007 37

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Figure 3. Physicochemical characteristics of the identified 248 proteins.

Figure 2. SDS-gels and western blot of CPM purified by four different methods and the variation of the amounts of CPM and Dynabeads. (A) Equal amounts of proteins (5 µg) from CPM control and PPMs were separated by SDS-PAGE, stained by silver nitrate (upper large panels), or transferred to PVDF membrane and analyzed by western blot (lower strips). The blots were probed with antibodies against organelle-specific proteins: antiNa+/K+-ATPase (MW: 112 KDa) and anti-flotillin (MW: 48 KDa) for plasma membrane; anti-NADH-ubiquinol oxidoreductase 39 (MW: 39 KDa) for mitochondria; anti-GRP 94 (MW: 94 KDa) for endoplasmic reticulum. (B) Optimization of Dynabeads. The numbers of beads were reduced from 2 × 107 to 1 × 107 to 0.5 × 107 with the amount of antibodies (0.5 µg of every kind) and CPM (20 mg) constant. Half of the extracted proteins was used for western blotting and the other half for SDS-PAGE. (C) Optimization of the amount of CPM. The amount of CPM was decreased from 20 to 10 to 5 mg with the number of beads (0.5 × 107) and antibodies (1.0 µg of every kind) constant. Half of the extracted protein was used for western blotting and the other half for SDS-PAGE. Numbers and arrows in the gel from 5 mg CPM indicated the protein bands cut for MALDI-TOF-TOF analysis.

sor and adapter-related protein complex 2 alpha 2 subunit identified from band 1; B230312I18Rik protein, Keratin, type I cytoskeletal 13 and Keratin, type I cytoskeletal 14 (band 2); and microsomal glutathione S-transferase-1 (band 3). To determine the extent of contamination of the plasma membrane fraction by other cellular organizations and cytosolic proteins and know what proteins are located on PM, the proteins were extracted from PPM2 and PPM4 (30 µg) and used for proteome analysis. Proteins were separated by SDS-PAGE and stained by Colloidal Coomassie Blue. All protein bands were cut and subjected to in-gel digestion. The resulting peptide mixtures were analyzed by MALDI-TOF-TOF and ESIQ-TOF as described under the Experimental Procedures section. Raw data were analyzed by Mascot MS/MS ion search against the mouse IPI v3.07, nonredundant database, using the stringent criteria mentioned above. In total, 248 nonredundant proteins were identified, of which 174 were from PPM2 and 133 from PPM4 (Supplementary Table S1, Supporting Information). 38

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Figure 4. Classification of proteins identified in mouse liver plasma membrane. (A) and (C) Percentage of the identified proteins classified according to GO annotation terms in PPM2 and PPM 4, respectively; (B) and (D) Expected primary subcellular localization of the characterized proteins in PPM 2 and PPM4, respectively.

Physicochemical Characteristics of the Identified Proteins. The 248 identified proteins were classified according to different physicochemical characteristics such as molecular weight (MW), hydrophobicity (GRAVY value), and TM domain predicted by TMHMM. The protein distribution patterns are shown in Figure 3. Two proteins with molecular weight below 10 KDa and 27 (10.9%) proteins with molecular weight above 100 KDa were identified. The largest molecular weight obtained is 536.3 KDa, beyond the general 2D-PAGE separation limits. Some of these proteins with higher molecular weight have no function and location annotation. Many proteins with high molecular weight are apparently enriched in PPM, and some proteins with larger molecular mass may be low-abundant components that are enriched after immunoaffinity purification. The hydrophobic property of protein expressed as the GRAVY index was usually analyzed in PM proteome analysis. Of the identified 248 proteins, 26 (10.5%) hydrophobic proteins (with positive GRAVY values up to 0.503) were identified (Figure 3). The ratio is similar to that obtained by 2D-LC in the work of Jiang et al.32 For the theoretical TM domains predicted by

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Immunoaffinity Purification of Plasma Membrane Table 2. The Identified Plasma Membrane or Membrane Proteins in Mouse Liver Plasma Membrane IPI number

query

description

MW

Gravya

TMb

scoresc

fractionsd

IPI00347096 IPI00109275 IPI00115454 IPI00117042 IPI00121209 IPI00122548 IPI00124499 IPI00124771 IPI00130460

1 1 1 1 1 1 1 1 1

57228 35104 35911 49944 30569 31207 57802 40063 51486

-0.42 0.112 0.012 -0.802 -0.772 -0.307 -0.538 0.048 -0.253

0 1 4 0 0 0 0 2 0

40 67 49 38 47 46 41 62 60

PPM4 PPM2 PPM2 PPM4 PPM4 PPM2 PPM2 PPM2 PPM2

IPI00131366 IPI00139301 IPI00221797

1 1 1

59832 61971 59932

-0.488 -0.462 -0.509

0 0 0

39 39 39

PPM2 PPM2 PPM2

IPI00222228

1

58629

-0.471

0

38

PPM4

IPI00265371

1

35551

-0.013

2

46

PPM2,4

IPI00322209 IPI00331459 IPI00347110 IPI00406377 IPI00462250 IPI00473320 IPI00110885

1 1 1 1 1 1 2

54531 57377 59502 50658 33083 15427 33782

-0.602 -0.354 -0.466 -0.588 0.033 -0.218 -0.293

0 0 0 0 3 0 0

71 38 37 38 46 56 95

PPM2,4 PPM4 PPM2,4 PPM4 PPM2,4 PPM2 PPM2

IPI00114593 IPI00114778 IPI00114780 IPI00115117 IPI00118678 IPI00120212

2 2 2 2 2 2

42334 56276 56532 38475 60930 42596

-0.228 -0.109 -0.132 -0.193 -0.027 -0.087

0 0 0 0 0 0

84 53 42 75 51 87

PPM2 PPM4 PPM4 PPM2 PPM2 PPM2

IPI00121079 IPI00121550 IPI00128489 IPI00131584 IPI00131695 IPI00133240

2 2 2 2 2 2

34203 35571 56542 33347 70700 29634

-0.203 -0.55 -0.15 0.109 -0.381 -0.068

0 1 0 3 0 0

95 59 46 39 95 92

PPM2 PPM4 PPM4 PPM2 PPM4 PPM2

IPI00308213 IPI00311493 IPI00311809 IPI00322322 IPI00331322 IPI00380781 IPI00407502 IPI00409800 IPI00468220 IPI00475246 IPI00556858 IPI00462140 IPI00117312 IPI00122547 IPI00135651 IPI00230754 IPI00276926 IPI00308162 IPI00348328 IPI00551240 IPI00112947 IPI00136056 IPI00227140 IPI00230365 IPI00112963 IPI00123181 IPI00123276 IPI00134503 IPI00136929 IPI00310131 IPI00317074 IPI00458351

2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4

type-II keratin Kb35 mitochondrial glutamate carrier 1 sideroflexin 1 glial fibrillary acidic protein, astrocyte apolipoprotein A-I precursor voltage-dependent anion channel 3 CDNA sequence BC031593 phosphate carrier protein, mitochondrial precursor NADH-ubiquinone oxidoreductase 51 kDa subunit, mitochondrial precursor 60 kDa protein type II keratin 5 mus musculus 10 days neonate skin cDNA, RIKEN full-length enriched library, clone:4732 mus musculus 10 days neonate skin cDNA, RIKEN full-length enriched library, clone:4732 solute carrier family 25 (Mitochondrial carrier; adenine nucleotide translocator), mem cytokeratin endo A keratin, type II cuticular HB5 type II keratin Kb36 51 kDa protein PREDICTED: similar to adenine nucleotide translocase cytoskeletal beta-actin mus musculus 13 days embryo liver cDNA, RIKEN full-length enriched library, clone:2500 actin, alpha cardiac cytochrome P450 2C37 56 kDa protein stomatin-like protein 2 T-complex protein 1, alpha subunit A NADH-ubiquinone oxidoreductase 39 kDa subunit, mitochondrial precursor NADH-cytochrome b5 reductase sodium/potassium-transporting ATPase beta-1 chain cytochrome p450 mitochondrial carnitine/acylcarnitine carrier protein serum albumin precursor ubiquinol-cytochrome c reductase iron-sulfur subunit, mitochondrial precursor 50 kDa protein keratin, type I cytoskeletal 18 solute carrier family 2, facilitated glucose transporter,member 2 93 kDa protein Microsomal glutathione S-transferase 1 53 kDa protein complement C1r subcomponent precursor cytochrome P450 Ig gamma-1 chain C region secreted form Igh-4 protein complement component 1, r subcomponent keratin, type II cytoskeletal 1b aspartate aminotransferase, mitochondrial precursor voltage-dependent anion-selective channel protein 2 calcium-binding mitochondrial carrier protein aralar2 mitochondrial 2-oxoglutarate/malate carrier protein solute carrier family 25, member 1 calcium-binding mitochondrial carrier protein aralar1 keratin Kb40 beta-actin keratin, type I cytoskeletal 19 keratin, type I cytoskeletal 13 keratin, type I cytoskeletal 14 keratin, type I cytoskeletal 17 alpha-1 catenin nonmuscle heavy chain myosin II-A RIKEN cDNA 2810484M10 cytochrome P450 2C29 gamma actin-like protein adapter-related protein complex 2 alpha 2 subunit mitochondrial dicarboxylate carrier PREDICTED: similar to adenine nucleotide translocase

50551 47344 57469 93625 17466 54200 81504 56563 36252 51659 81527 61379 47780 32340 74819 34173 34252 74922 86156 8759 44515 48066 53176 48286 100728 227414 38797 56378 44029 104947 32150 33278

-0.28 -0.625 0.503 -0.571 0.14 -0.356 -0.495 -0.123 -0.362 -0.279 -0.494 -0.59 -0.231 -0.223 0.022 0.072 -0.108 -0.027 -0.603 -0.264 -0.49 -0.55 -0.54 -0.58 -0.366 -0.84 -0.23 -0.116 -0.207 -0.086 0.044 -0.06

1 0 11 0 3 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 2

36 56 48 44 48 51 49 53 36 36 49 50 61 35 86 78 72 97 125 67 63 63 71 62 83 50 37 82 66 45 36 87

PPM2 PPM4 PPM4 PPM4 PPM2,4; band 3 PPM4 PPM4 PPM4 PPM2 PPM2 PPM4 PPM4 PPM2 PPM2 PPM2,4 PPM2 PPM2,4 PPM2,4 PPM2,4 PPM2,4 PPM4 PPM4, band 2 PPM4′ band 2 PPM4 PPM4 PPM4 PPM2 PPM4 PPM2,4 PPM4, band 1 PPM4 PPM2

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Table 2 (Continued) IPI number

query

IPI00115564

5

IPI00311682 IPI00110827 IPI00114593 IPI00117043 IPI00119685 IPI00122549

5 6 6 6 6 6

IPI00127841 IPI00230540

6 6

IPI00266875 IPI00404804 IPI00453996 IPI00480406

6 6 6 6

IPI00120678 IPI00125813 IPI00130280 IPI00280664 IPI00406187

7 7 7 7 7

IPI00411145

7

IPI00411146

7

IPI00473229

7

IPI00228150 IPI00338536

8 8

IPI00381412 IPI00381413 IPI00554845 IPI00110850 IPI00110851 IPI00121309

8 8 8 9 9 9

IPI00225329

9

IPI00111885

10

IPI00117978

10

IPI00131368 IPI00221528

10 10

IPI00131424 IPI00380436 IPI00316329

11 13 15

IPI00313475 IPI00125853 IPI00119138

17 21 64

description

MW

Gravya

TMb

scoresc

fractionsd

PREDICTED: solute carrier family 25 PREDICTED: solute carrier family 25 sodium/potassium-transporting ATPase alpha-1 chain precursor actin, alpha skeletal muscle actin, alpha cardiac actin, aortic smooth muscle cytochrome P450 27, mitochondrial precursor splice isoform Pl-VDAC1 of voltage-dependent anion-selective channel protein 1 ADP,ATP carrier protein 2 splice isoform Mt-VDAC1 of voltage-dependent anion-selective channel protein 1 43 kDa protein actin, gamma-enteric smooth muscle nonmuscle myosin II-C heavy chain mus musculus 14, 17 days embryo head cDNA, RIKEN full-length enriched library, clone:3 putative Sp100-related protein dipeptidyl peptidase 4 ATP synthase alpha chain, mitochondrial precursor WD repeat membrane protein mus musculus 3 days neonate thymus cDNA, RIKEN full-length enriched library, clone:A63 splice isoform 1 of low-density lipoprotein receptor-related protein 4 precursor splice isoform 2 of low-density lipoprotein receptor-related protein 4 precursor mus musculus 13 days embryo heart cDNA, RIKEN full-length enriched library, clone:D330 splice isoform 1 of mitochondrial inner membrane protein succinate dehydrogenase [ubiquinone] iron-sulfur protein, mitochondrial precursor splice isoform 2 of mitochondrial inner membrane protein splice isoform 3 of mitochondrial inner membrane protein splice isoform 5 of mitochondrial inner membrane protein actin, cytoplasmic 1 actin, cytoplasmic 2 NADH-ubiquinone oxidoreductase 30 kDa subunit, mitochondrial precursor mus musculus 2 days neonate thymus thymic cells cDNA, RIKEN full- length enriched libr ubiquinol-cytochrome-c reductase complex core protein I, mitochondrial precursor cytochrome c oxidase subunit IV isoform 1, mitochondrial precursor keratin, type II cytoskeletal 6A mus musculus 10 days neonate skin cDNA, RIKEN full-length enriched library, clone:4732 carnitine O-palmitoyltransferase II, mitochondrial precursor alpha-actinin 1 mus musculus 0 day neonate head cDNA, RIKEN full-length enriched library, clone:483343 ATP synthase gamma chain, mitochondrial precursor mitochondrial ornithine transporter 1 ubiquinol-cytochrome-c reductase complex core protein 2, mitochondrial precursor

39740

-0.102

3

126

PPM2,4

114221 42366 42334 42381 60910 32505

0.002 -0.232 -0.228 -0.233 -0.247 -0.334

10 0 0 0 0 0

100 104 104 104 115 143

PPM2,4; band 1 PPM2,4 PPM4 PPM2,4 PPM2,4 PPM2,4

33007 30853

0.013 -0.423

2 0

165 143

PPM2,4 PPM2,4

43513 42249 229359 42262

-0.236 -0.219 -0.786 -0.219

0 0 0 0

104 104 36 104

PPM2,4 PPM2,4 PPM4 PPM2,4

24501 88065 59830 146638 39436

-0.15 -0.323 -0.1 -0.226

1 1 0 0 0

30 145 408 40 30

PPM4 PPM4 PPM2,4 PPM2 PPM4

217243

-0.486

1

37

PPM4

210608

-0.479

1

37

PPM4

116385

-0.198

0

40

PPM2

84247 32605

-0.473 -0.381

0 0

127 58

PPM2 PPM2,4

83277 79078 88038 42052 42108 30360

-0.493 -0.466 -0.462 -0.2 -0.199 -0.362

0 0 0 0 0 0

127 127 127 148 148 367

PPM2 PPM2 PPM2 PPM2,4 PPM2,4 PPM2,4

30302

-0.35

0

367

PPM2,4

53420

-0.189

0

139

PPM2,4

19575

-0.412

1

179

PPM2

59510 42319

-0.502 -0.166

0 0

134 55

PPM2,4 PPM2,4

74508 103631 66099

-0.27 -0.6 -0.588

0 0 0

329 63 179

PPM2 PPM4 PPM2,4

32979 33324 48262

-0.163 0.211 -0.057

0 0 0

180 217 534

PPM2,4 PPM2,4 PPM2,4

a Grand average of hydrophobicity. b Predicted number of transmembrane helices returned by TMHMM (http://www.cbs.dtu.dk/services/TMHMM). c Mascot score. d In the column of fractions, PPM2,4 represent the proteins were simultaneously identified in fraction PPM2 and PPM4; Bands 1, 2, and 3 represent the proteins identified by MALD-TOF-TOF from the fraction purified from 5 mg CPM. These lines were shown in italics.

TMHMM, 16.1% (40 out of 248) are integral membrane proteins with at least one predicted TMD; 4 of these have 10 or more TMDs (Table S1, Supporting Information). The number of proteins with GRAVY > 0 and TM domains was not very high. This might be due to limitation in the criteria for the presence of putative alpha-helices and the software for the prediction of GRAVY, which has been expatiated in several papers.9,32 It is questionable whether the efficiency of prediction software still holds when applied to proteome data.33 40

Journal of Proteome Research • Vol. 6, No. 1, 2007

Subcellular Location Annotation. To assess the efficacy of the developed protocol for the enrichment of plasma membrane proteins and to estimate contamination by other cellular organelles, including mitochondria and endoplasmic reticulum (ER) and so on, we classified the 174 identified proteins from PPM2 and 133 proteins from PPM4 according to the gene ontology (GO) annotation. Among the proteins with their location annotated, 58 and 67% are PM or PM-related proteins in PPM2 and PPM4 respectively. The contamination from PPM4

Immunoaffinity Purification of Plasma Membrane

Figure 5. Functional categories of the identified mouse liver plasma membrane proteins.

is less than that from PPM2 (Figure 4A-D). This may be because a primary antibody can usually react with several secondary antibodies, and the binding affinity of the primary antibody with the secondary antibodies is stronger than the primary antibody with antigen. No nuclear protein was found in PPM4. Compared with our proteome analysis of CPM, the ratio of PM or PM-related proteins was greatly increased in PPM4 and PPM2 (data not shown). All PM or PM-related proteins are shown in Table 2. Plasma membrane special markers such as sodium/potassium-transporting ATPase alpha, beta-1 chain, solute carrier family 25, and so on were identified. As shown in Table 2, the 105 PM proteins include integral or lipid-anchored membrane proteins, cytoskeletal proteins and proteins externally associated with plasma membrane (data not shown). These proteins (81%) were identified by two or more peptides. For example, sodium/potassium-transporting ATPase alpha-1 chain precursor, which is a protein with 10 transmembrane domains, was identified by 5 peptides. Among the 105 PM proteins, 36 were identified as being from both PPM2 and PPM4; however, 34 were only identified from PPM2 and 35 from only PPM4 (Table 2). The results indicated that the direct and indirect methods can complement each other in identifying PM proteins. Functional Annotation. The identified proteins were functionally categorized based on universal GO annotation terms. Two hundred eight (83.9%) of the identified proteins were mapped to at least one annotation term with the GO molecular function category, including binding proteins (36%), catalytic activity proteins (32%), structural molecules (12%), transporter activity proteins (6.0%), and so on (Figure 5). Similar functional classification was found in PPM2 and PPM4.

Discussion For subcellular proteome research, there are usually two challenges: one is the purification of organelles, and the other is the identification of hydrophobic proteins. In the case of plasma membrane proteome research, these problems are more intractable because of overlap of PM density with other organelles and contamination with less hydrophobic proteins from cytoplasm. Previous PM purification methods, such as centrifugation, silica bead enrichment, and aqueous two-phase systems

research articles purification, have inherent limits, particularly serious contamination from other organelles. Affinity enrichment with biotinylation is a good method for purification of PM, but it has two limitations,12 including its relatively low yield and its restriction to membrane proteins from cultured cells. Morciano et al.22 and Lawson et al.23 used immunoaffinity to purify PM and obtained highly pure PM. However, less than 100 proteins were identified in each fraction. No optimization of experimental method, such as variation of CPM and beads was carried out. We present here an optimized immunoaffinity protocol for PM proteins, based on second antibody superparamagnetic bead affinity-enrichment. As shown in Figure 1, monoclonal primary antibodies (anti-Na+/K+-ATPase and anti-flotillin, two kinds of PM-specific integral membrane protein antibodies) were incubated with crude PM or magnetic beads containing second antibody. The primary antibody complex was then incubated with beads or crude PM. The bead-captured PM can be selected by the magnet. The purity of the affinity-enriched membrane fractions was determined by analysis of organellespecific proteins. Immunoblotting analysis demonstrated that the affinity-enriched membrane fractions from PPM 2 and 4 resulted in a 3-fold enrichment of plasma membrane proteins and a 2-fold decrease of mitochondria-specific proteins compared with PM from sucrose density gradient centrifugation. The fraction of proteins from the PM (67%) is higher than with previously reported methods such as sucrose density centrifugation,9 affinity two-phase partitioning,34 and so on. No proteins from the ER, previously found as the main contamination of liver and hepatoma PM,35 were found in this work. Because the experimental procedure relies on the isolation of plasma membrane sheets and subsequent proteome analysis, it is necessary to optimize the experiment procedures to suit proteome analysis. After comparing four kinds of methods, the indirect selection of PM without BSA was thought to be the optimized method. To optimize the economy of the work, that is to use the minimum sample and reagents, a systemic analysis was performed, determining a suitable number of beads (0.5 × 107), CPM (5 mg), and antibodies (1.0 µg) for mouse liver proteome analysis to give obvious and identifiable electrophoresis bands. CPM (5 mg) can be easily obtained using less than 500 mg of liver. It is cheap for proteome analysis using this number of beads and antibodies. Even with the stringent quality criteria of 3-fold enrichment of PM markers and 2-fold depletion of specific markers of mitochondria compared with PM from sucrose density gradient centrifugation, there were still many mitochondrial proteins identified. Most of these may have more than one subcellular location.10,36 Mann et al.37 have pointed out that 41% of all organellar proteins are found in more than one location. Historically, it is not unusual for proteins initially categorized as organelle-specific to be later discovered elsewhere in the cell. For example, ATP synthase beta subunit was initially assigned to mitochondria but later was found to be expressed also at the cell surface, prompting the suggestion that the ATP synthase complex might be located in PM rafts as well as in the mitochondria.38 Prohibitin, which is a mitochondrial protein according to GO, could be located to PM through interaction with proteins from PM.39 NipSnap1 protein was annotated as a mitochondria protein. However, as a membrane of the NIPSNAP family with putative roles in vesicular trafficking, it may be located on PM.40 Journal of Proteome Research • Vol. 6, No. 1, 2007 41

research articles In summary, we reported a powerful method for the proteomic analysis of plasma membrane proteins, involving antibody affinity purification of plasma membrane, SDS-PAGE separation, and protein identification by MALDI-TOF-TOF and ESI-Q-TOF. Western blotting analysis showed that plasma membrane specific proteins were greatly enriched, mitochondria was reduced, and ER was almost removed. We obtained a high-quality plasma membrane express profile. Given the importance of the PM proteome for therapeutic drug design, these methods should expedite the discovery of protein drug targets.

Acknowledgment. This work was supported by a grant from National 973 Project of China (2001 CB5102), a grant from CHLPP (2004 BA711A11), National Natural Science Foundation of China (30000028, 30240056), and a project of PCSIRT (IRT0445). Supporting Information Available: Supplementary table S1, all identified proteins from mouse liver plasma membrane in this study. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Schindler, J.; Lewandrowski, U.; Sickmann A.; Friauf, E.; Nothwang, H. G. Proteomic analysis of brain plasma membranes isolated by affinity two-phase partitioning. Mol. Cell. Proteomics 2006, 5, 390-400. (2) Navarre, C.; Degand H.; Bennett, K. L.; Crawford, J. S.; Mortz, E.; Boutry, M. Subproteomics: identification of plasma membrane proteins from the yeast Saccharomyces cerevisiae. Proteomics 2002, 2, 1706-1714. (3) Marmagne, A.; Rouet, M. A.; Ferro, M.; Rolland, N.; Alcon, C.; Joyard, J.; Garin, J.; Barbier-Brygoo, H.; Ephritikhine, G. Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome. Mol. Cell. Proteomics 2004, 3, 675-691. (4) Zhang, W.; Zhou, G.; Zhao, Y.; White, M. A.; Zhao, Y. Affinity enrichment of plasma membrane for proteomics analysis. Electrophoresis 2003, 24, 2855-2863. (5) Watarai, H.; Hinohara, A.; Nagafune, J.; Nakayama, T.; Taniguchi, M.; Yamaguchi, Y. Plasma membrane-focused proteomics: dramatic changes in surface expression during the maturation of human dendritic cells. Proteomics 2005, 5, 4001-4011. (6) Adam, P. J.; Boyd, R.; Tyson, K. L.; Fletcher, G. C.; Stamps, A.; Hudson, L.; Poyser, H. R.; Redpath, N.; Griffiths, M.; Steers, G.; et al. Comprehensive proteomic analysis of breast cancer cell membranes reveals unique proteins with potential roles in clinical cancer. J. Biol. Chem. 2003, 278, 6482-6489. (7) Oh, P.; Li, Y.; Yu, J.; Durr, E.; Krasinska, K. M.; Carver, L. A.; Testa, J. E.; Schnitzer, J. E. Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy. Nature 2004, 429, 629-635. (8) Blonder, J.; Terunuma, A.; Conrads, T. P.; Chan, K. C.; Yee, C.; Lucas, D. A.; Schaefer, C. F.; Yu, L. R.; Issaq, H. J.; Veenstra, T. D.; Vogel, J. C. A proteomic characterization of the plasma membrane of human epidermis by high-throughput mass spectrometry. J. Invest. Dermatol. 2004, 123, 691-699. (9) Zhang, L.; Xie, J.; Wang, X.; Liu, X.; Tang, X.; Cao, R.; Hu, W.; Nie, S.; Fan, C.; Liang, S. Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins. Proteomics 2005, 5, 4510-4524. (10) Durr, E.; Yu, J.; Krasinska, K. M.; Carver, L. A.; Yates, J. R.; Testa, J. E.; Oh, P.; Schnitzer, J. E. Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture. Nat. Biotechnol. 2004, 22, 985-992. (11) Alexandersson, E.; Saalbach, G.; Larsson, C.; Kjellbom, P. Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking. Plant Cell Physiol. 2004, 45, 1543-1556. (12) Zhao, Y.; Zhang, W.; Kho, Y.; Zhao, Y. Proteomic analysis of integral plasma membrane proteins. Anal. Chem. 2004, 76, 18171823. (13) Srivastava, R.; Pisareva, T.; Norling, B. Proteomic studies of the thylakoid membrane of Synechocystis sp. PCC 6803. Proteomics 2005, 5, 4905-4916.

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Zhang et al. (14) Lindberg, S.; Banas, A.; Stymne, S. Effects of different cultivation temperatures on plasma membrane ATPase activity and lipid composition of sugar beet roots. Plant Physiol. Biochem. 2005, 43, 261-268. (15) Persson, A.; Johansson, B.; Olsson, H.; Jergil, B. Purification of rat liver plasma membranes by wheat-germ-agglutinin affinity partitioning. Biochem. J. 1991, 273(Pt 1), 173-177. (16) Abedinpour, P.; Jergil, B. Isolation of a caveolae-enriched fraction from rat lung by affinity partitioning and sucrose gradient centrifugation. Anal. Biochem. 2003, 313, 1-8. (17) Luers, G. H.; Hartig, R.; Mohr, H.; Hausmann, M.; Fahimi, H. D.; Cremer, C.; Volkl, A. Immuno-isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting. Electrophoresis 1998, 19, 1205-1210. (18) Kikuchi, M.; Hatano, N.; Yokota, S.; Shimozawa, N.; Imanaka, T.; Taniguchi, H. Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease. J. Biol. Chem. 2004, 279, 421-428. (19) Calhoun, B. C.; Goldenring, J. R. Two Rab proteins, vesicleassociated membrane protein 2 (VAMP-2) and secretory carrier membrane proteins (SCAMPs), are present on immunoisolated parietal cell tubulovesicles. Biochem. J. 1997, 325 (Pt 2), 559564. (20) Srinivasan, S.; Traini, M.; Herbert, B.; Sexton, D.; Harry, J.; Alexander, H.; Williams, K. L.; Alexander, S. Proteomic analysis of a developmentally regulated secretory vesicle. Proteomics 2001, 1, 1119-1127. (21) Henley, J. R.; McNiven, M. A. Association of a dynamin-like protein with the Golgi apparatus in mammalian cells. J. Cell Biol. 1996, 133, 761-775. (22) Morciano, M.; Burre, J.; Corvey, C.; Karas, M.; Zimmermann, H.; Volknandt, W. Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis. J. Neurochem. 2005, 95, 1732-1745. (23) Lawson, E. L.; Clifton, J. G.; Huang, F.; Li, X.; Hixson, D. C.; Josic, D. Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes. Electrophoresis 2006. (24) Fleischer, S.; Kervina, M. Subcellular fractionation of rat liver. Methods Enzymol. 1974, 31, 6-41. (25) Zhang, L.; Liu, X.; Zhang, J.; Cao, R.; Lin, Y.; Xie, J.; Chen, P.; Sun, Y.; Li, D.; Liang, S. Proteome analysis of combined effects of androgen and estrogen on the mouse mammary gland. Proteomics 2006, 6, 487-497. (26) Cao, R.; Li, X.; Liu, Z.; Peng, X.; Hu, W.; Wang, X.; Chen, P.; Xie, J.; Liang, S. Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins. J. Proteome Res. 2006, 5, 634-642. (27) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20, 3551-3567. (28) Gobom, J.; Schuerenberg, M.; Mueller, M.; Theiss, D.; Lehrach, H.; Nordhoff, E. Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics. Anal. Chem. 2001, 73, 434-438. (29) Ashburner, M.; Ball, C. A.; Blake, J. A.; Botstein, D.; Butler, H.; Cherry, J. M.; Davis, A. P.; Dolinski, K.; Dwight, S. S.; Eppig, J. T. et al: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 2000, 25, 25-29. (30) 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. (31) Kyte, J.; Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 1982, 157, 105132. (32) Jiang, X. S.; Zhou, H.; Zhang, L.; Sheng, Q. H.; Li, S. J.; Li, L.; Hao, P.; Li, Y. X.; Xia, Q. C.; Wu, J. R.; Zeng, R. A high-throughput approach for subcellular proteome: identification of rat liver proteins using subcellular fractionation coupled with twodimensional liquid chromatography tandem mass spectrometry and bioinformatic analysis. Mol. Cell. Proteomics 2004, 3, 441455. (33) Nakai, K. Review: prediction of in vivo fates of proteins in the era of genomics and proteomics. J. Struct. Biol. 2001, 134, 103116. (34) Schindler, J.; Lewandrowski, U.; Sickmann, A.; Friauf, E.; Nothwang, H. G. Proteome analysis of brain plasma membranes isolated by affinity two-phase partitioning. Mol. Cell. Proteomics 2005.

research articles

Immunoaffinity Purification of Plasma Membrane (35) Josic, D.; Brown, M. K.; Huang, F.; Callanan, H.; Rucevic, M.; Nicoletti, A.; Clifton, J.; Hixson, D. C. Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins. Electrophoresis 2005, 26, 2809-2822. (36) Bagshaw, R. D.; Mahuran, D. J.; Callahan, J. W. A proteomic analysis of lysosomal integral membrane proteins reveals the diverse composition of the organelle. Mol. Cell. Proteomics 2005, 4, 133-143. (37) Foster, L. J.; de Hoog, C. L.; Zhang, Y.; Zhang, Y.; Xie, X.; Mootha, V. K.; Mann, M. A mammalian organelle map by protein correlation profiling. Cell 2006, 125, 187-199. (38) Bae, T. J.; Kim, M. S.; Kim, J. W.; Kim, B. W.; Choo, H. J.; Lee, J. W.; Kim, K. B.; Lee, C. S.; Kim, J. H.; Chang, S. Y. et al. Lipid raft

proteome reveals ATP synthase complex in the cell surface. Proteomics 2004, 4, 3536-3548. (39) Rajalingam, K.; Wunder, C.; Brinkmann, V.; Churin, Y.; Hekman, M.; Sievers, C.; Rapp, U. R.; Rudel, T. Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration. Nat. Cell Biol. 2005, 7, 837-843. (40) Buechler, C.; Bodzioch, M.; Bared, S. M.; Sigruener, A.; Boettcher, A.; Lapicka-Bodzioch, K.; Aslanidis, C.; Duong, C. Q.; Grandl, M.; Langmann, T. et al. Expression pattern and raft association of NIPSNAP3 and NIPSNAP4, highly homologous proteins encoded by genes in close proximity to the ATP-binding cassette transporter A1. Genomics 2004, 83, 1116-1124.

PR060069R

Journal of Proteome Research • Vol. 6, No. 1, 2007 43