Aqueous Two-Phase Partitioning for Proteomic Monitoring of Cell Surface Biomarkers in Human Peripheral Blood Mononuclear Cells Henrik Everberg,† Ragna Peterson,† Sabina Rak,‡ Folke Tjerneld,† and Cecilia Emanuelsson*,† Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-22100 Lund, Sweden, and Department of Respiratory Medicine and Allergology, Sahlgrenska University Hospital, Gothenburg University, Sweden Received December 20, 2005
For proteomic monitoring of processes such as allergy or inflammation an efficient pre-fractionation strategy is required. We isolated plasma membranes from human peripheral blood mononuclear (PBM) cells by aqueous two-phase partitioning. After 1DE combined with LC-MS/MS, several cell surface marker proteins and in total 60 different plasma membrane proteins (out of 84 identified proteins, i.e., 72%) were detected. Plasma membranes obtained were from only one human donor, the procedure is therefore applicable for individual patient screening. Keywords: allergy • plasma membrane proteins • cell surface biomarkers • aqueous two-phase partitioning • prefractionation • proteomics
Introduction A large number of protein-protein interactions are involved in allergic and inflammatory responses. Soluble proteins such as antigens, antibodies, and cytokines interact with receptor proteins in specialized white blood cells (leukocytes). In a complex chain of events, different cells and receptor proteins become up- and down-regulated. The different leukocyte types are distinguished by surfaceexposed receptors, commonly called cluster of differentiation (CD) antigens. For example, exposed on the surface of all leukocytes is the common leukocyte antigen CD45. On the surface of specialized so-called natural killer T-cells there is a combination of CD45, CD11a, CD11c, and CD86.1 The separation of different subclasses of human leukocytes by flow cytometry, based on the presence or absence of such CDantigens, is fundamental in immunology. To investigate the reactions involved in allergic responses or inflammation, fluorescence-labeled monoclonal antibodies can be utilized to measure changes in a number of specific cell surface proteins, such as for example the IgE receptor FcγRI, CD14, and the co-stimulatory molecules CD80, CD86, and CD28 on T-cells.2 However, by this technique only a limited set of predetermined cell surface proteins can be measured at any one time. Changes in other important proteins can be overlooked, and cause-effect relationships therefore may remain unclear. The detection of a large number of proteins and changes in their expression levels by proteomics3-5 would permit the monitoring of a large number of cell surface proteins simultaneously. The cell-surface proteins can be enriched by isolation of the plasma membranes from the cells, also serving * To whom correspondence should be addressed. Tel: +46-46-2224872. Fax: +46-46-2224534. E-mail:
[email protected]. † Lund University. ‡ Gothenburg University.
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to reduce sample complexity and allow proteomic detection also of less abundant proteins. Crude membrane preparations1,6 or plasma membrane domains (lipid rafts) isolated by sucrose gradient centrifugation7 from cultured natural killer (NK) T-cells have been analyzed by proteomic approaches. Also, sucrose gradient-isolated plasma membrane preparations of other peripheral blood mononuclear (PBM) cell types, such as cultured monocytes,8 T-9 and B-lymphocytes10 or bovine neutrophils11 have been studied by proteomics. With cultured cell-lines or animal disease models relatively large amounts of starting material is available. However, to be useful with patient screening, we recognized a need for a rapid and efficient preparation procedure for plasma membranes from smaller amounts of starting material. Here, we report on the preparation of plasma membranes from PBM cells obtained from samples from single human donors, based on a protocol developed for preparation of leukocyte plasma membranes by an aqueous two-phase system,12,13 i.e., two aqueous but immiscible phases formed due to the presence of a low concentration of polymers, PEG and dextran.14 The membranes partition between the two phases and/or the interphase, depending on their different surface properties.15 This method is well suited for low amounts of starting material, and has been used with several cell types to obtain plasma membranes, which have been characterized by electron micrographs and marker enzyme activity measurements.12,13,16-24 In our purification protocol, a carbonate washing step was included to remove contaminating organelles or proteins trapped inside the plasma membrane vesicles. By this procedure, 90 µg of enriched plasma membranes were obtained from human PBM cells within 1 h, starting from 50 mL whole blood withdrawn from one single human donor. By LC-MS/MS, 60 10.1021/pr050469z CCC: $33.50
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Proteomic Monitoring of Cell Surface Biomarkers
different plasma membrane proteins were detected. Several of these were CD-antigen cell surface marker proteins. This rapid plasma membrane preparation can be used to find novel cell surface biomarkers involved in disease progression and therapy response.
Methods Peripheral Blood Mononuclear Cell Preparation and Homogenization. Patient samples, obtained within a study approved by the Ethical board at Gothenburg University, were withdrawn and PBM cells were isolated by Percoll centrifugation as previously described.2 One blood donation (50 mL) typically yielded 2 × 108 PBM cells. Cells were treated according to a procedure adapted from Svensson et al.13 Cells were first pelleted by centrifugation (1000 × g, 10 min) and washed twice with PBS and resuspended in hypotonic cell swelling buffer consisting of 10 mM Tris-HCl, pH 7.4; 10 mM KCl; 1.5 mM MgCl2; 2 mM mercapto-ethanol, at a concentration of 2 × 107 cells/ml buffer. The cell suspension was incubated on ice for 1.5 h, thereafter disintegrated in a Dounce homogenizer with 22 single strokes. The homogenate was centrifuged at 8500 × g, 4 °C for 15 min. The supernatant (cytosolic protein fraction) was collected, and the pellet which contained membranes, was further purified by two-phase partitioning. Aqueous Two-Phase Partitioning and Alkaline Stripping. An aqueous two-phase system containing 4.8% (w/w) poly(ethylene glycol) (PEG Mr: 8000), 3.8% (w/w) dextran (Mr: 500 000), 90 mM sodium phosphate buffer, pH 6.5 and 0.1 mM NaCl was premixed as described by Brunette and Till12 in a separation funnel, and left to phase separate overnight at 4 °C. All components were weighed in and the total weight of the system was 820 g. The top and bottom phases were isolated separately and the interphase discarded. The crude membrane pellet after homogenization and centrifugation was resuspended in 5 mL of premixed top phase. An equal volume of bottom phase was added; the suspension was thoroughly mixed and centrifuged at 8500 × g at 4 °C for 10 min for phase separation and removal of cell debris. The supernatant (i.e., the whole two-phase system) was transferred to a new tube, whereas the pellet was discarded. The centrifugation was repeated and the plasma membrane vesicles, partitioned at the interphase, were collected by a Pasteur pipet. The collected membrane suspension was diluted five times with 50 mM Tris-HCl, pH 7.3, supplied with protease inhibitors (Complete EDTA-free protease inhibitor cocktail, Roche Diagnostics GmbH, Mannheim, Germany) and centrifuged at 8000 × g, 4 °C for 15 min. The plasma membrane pellet was resuspended in 200 µL 50 mM Tris-HCl, pH 7.3, with Complete EDTA-free protease inhibitor cocktail and stored at -80 °C until use. The protein concentration was assayed by the BCAmethod.25 The yield of PBM plasma membranes from one patient blood donation (50 mL whole blood, ∼2 × 108 PBM cells) was typically 90 µg of protein. Contaminating membranes and soluble proteins trapped inside the membrane vesicles were removed by alkaline stripping by incubation in 0.1 M Na2CO3, pH 11.5 on ice for 15 min followed by centrifugation 165 000 × g for 30 min.26 SDS-PAGE and Excision of Samples for In-Gel Digestion. All samples were prepared for electrophoresis by PlusOne SDSPAGE cleanup kit (Amersham Biosciences, Uppsala, Sweden) according to the manufacturers instructions and 25 µg of protein was applied per lane onto NuPAGE Bis-Tris pre-cast gradient gels 4-12% from Novex (San Diego, CA) with 3-(N-
research articles morpholino) propane sulfonic acid (MOPS) as running buffer. The gels were stained with colloidal Coomassie27 overnight and destained in water. Gel plugs were excised from protein bands for mass spectrometric protein identification. For LC-MS/MS, an entire gel lane was cut into 36 sections of different size depending on the staining intensity. Larger sections were taken from weakly stained regions, and smaller sections were excised from strongly stained ones. In-Gel Digestion. Excised gel pieces were destained in 50 µL 50 mM NH4HCO3, 50% ethanol for 60 min. Volumes stated below refer to gel plugs; for gel slices volumes were increased by a factor of 5. The liquid was removed and 50 µL ethanol was added for 15 min to shrink the gel pieces. The liquid was removed and the gel pieces dried in a SpeedVac concentrator (Savant, Farmingdale, NY). The shrunken gel pieces were rehydrated in 10 µL digestion buffer containing 25 mM NH4HCO3, and 12.5 ng/µL of sequencing-grade trypsin (Promega, Madison, WI) on ice for 45 min, ensuring total immersion of the sample. After incubation at 37 °C overnight the supernatant was collected. To further extract peptides, the gel pieces were incubated in 20 µL 10 mM NH4HCO3, 50% ethanol, 0.5% trifluoroacetic acid (TFA) for 60 min at 37 °C. The gel pieces were spun down and the supernatant was collected and pooled with the supernatant from the overnight incubation. The samples were dried using the SpeedVac concentrator. Sample Preparation for MS and MS/MS Analysis prior to Liquid Chromatography Separation. Dried peptide samples were redissolved in 5 µL 0.1% TFA and 0.5 µL was applied onto a stainless steel MALDI-target. The solvent was allowed to completely evaporate before application of 0.5 µL of matrix solution containing 5 mg/mL R-cyano-4-hydroxy cinnamic acid, 50% acetonitrile, 0.1% TFA and 50 mM citric acid for suppression of matrix signals.28 The samples were allowed to dry prior to MS-analysis. Liquid Chromatography. Dried peptide samples were redissolved in 10 µL 0.1% TFA and 5 µL was applied to reversed phase nanoLC using an 1100 Series Nanoflow LC system (Agilent technologies, Waldbronn, Germany). The mobile phases used for separation were composed of A: 1% (v/v) acetonitrile and 0.05% (v/v) TFA, and B: 90% (v/v) acetonitrile and 0.04% (v/v) TFA. 5 µL of the in-gel digested peptide extract was loaded on the precolumn (Zorbax 300 SB C18, 5 × 0.3 mm) from glass vials at a flow rate of 0.050 mL/min A. After 10 min the separation column (Zorbax 300 SB C18, 150 × 0.075 mm) was connected to the nanopump, flow-controlled by a micro 6-port/2-position module. The separation was carried out at a flow rate of 200 nL/min with the following elution profile: 1011 min, 0-20% B; 11-51 min, 20-80% B; 51-52 min, 80-100% B; 52-55 min, concentration held at 100% B; 55-58 min, 100-0% B. Fractions were collected on MALDI targets between 25 and 49 min with 15 s intervals, using the 1100 Series LC micro collection/spotting system. A matrix solution consisting of 5 mg/mL R-cyano-4-hydroxy cinnamic acid, 50% acetonitrile, 0.1% TFA was added post-column manually onto the dried peptide fractions and was allowed to dry. The matrix solution also included 50 mM citric acid for suppression of matrix signals28 and two standard peptides (angiotensin I, m/z: 1296.685; neurotensin, m/z: 1672.918) for internal calibration. Recording of MALDI-TOF MS and MS/MS Data. MALDITOF MS and MS/MS spectra were recorded automatically using a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, CA) mass spectrometer at positive reflector mode. The obtained Journal of Proteome Research • Vol. 5, No. 5, 2006 1169
research articles spectra from direct spotting experiments were internally calibrated using two trypsin auto-proteolytic peptides with the m/z values 842.51 and 2211.104 Da. For LC-MS/MS experiments precursor peptides were selected by using the job-wide interpretation function in the Peak Explorer software excluding masses derived from tryptic autoproteolysis and commonly occurring keratin peptides. A spotto-spot difference between precursor masses of (50 ppm was set as requirement for precursor selection and a maximum of 7 peptides in each fraction were selected for MS/MS acquisition. The obtained spectra from LC-MS/MS experiments were internally calibrated using two standard peptides (angiotensin I, m/z: 1296.685; neurotensin, m/z: 1672.918) added to the matrix solution. Protein Identification and Data Analysis. Protein identification using MS and MS/MS data acquired from peptide extracts prior to LC separation was performed using the GPS Explorer software, with an in-house installed Mascot search engine (Matrix Science, London, UK)29 searching the Swiss-Prot database.30-32 Parameters specified in the search were: taxa, Human; missed cleavages, 1; peptide mass tolerance in MSmode, (25 ppm; in MS/MS-mode, 0.2 Da; variable modifications, oxidation of methionines. A protein was considered identified when the level of confidence exceeded 95% and was identified several times at the same position on the gel by manual inspection. The acquired MS/MS data from LC-MS/MS experiments were used for database searches using GPS Explorer software and the in-house Mascot search engine in the Swiss-Prot database. Search parameters were set as above and here we considered a protein to be reliably identified when the confidence interval exceeded 99.95% according to Mascot, which means that 0.05% of the identified proteins could be false positives. A FASTA formatted list of the identified proteins was created. The retrieved list was submitted to a topology prediction server using the so-called transmembrane hidden Markov model to predict the number of transmembrane helices in each identified protein (TMHMM 2.0).33 Cellular location of the proteins was assigned according to their annotation in SwissProt and HPRD by manual examination.
Results Plasma Membrane Preparation from Human Peripheral Blood Mononuclear Cells by Aqueous Two-Phase Partitioning. A procedure based on aqueous two-phase partitioning12-15,34 was used for the plasma membrane preparation as schematically outlined in Figure 1. The PBM cell plasma membrane vesicles were enriched at the interphase. To further minimize contamination from soluble proteins and other membranes possibly trapped inside the plasma membrane vesicles, the plasma membranes were then treated with sodium carbonate (pH 11.5)26 prior to size fractionation by SDS-PAGE. The enrichment of plasma membranes was monitored by total protein measurements and the successive removal of contaminating organelles and cytosolic proteins are presented in Table 1. Starting from approximately 2 × 108 PBM cells (∼6 mg total protein) yielded 90 µg protein in the isolated plasma membrane preparation. As expected, different proteins were enriched in the fractions withdrawn during the purification procedure (Figure 2). Several proteins enriched in the purified plasma membrane fraction were identified as plasma membrane proteins, including a number of CD-antigens exposed in various types of blood cells 1170
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Figure 1. Schematic outline of the isolation of human PBM plasma membranes by aqueous two-phase partitioning. PBM cells were homogenized and soluble proteins (cytosolic fraction) removed by centrifugation. The pellet was resuspended in a PEG/ dextran two-phase system where plasma membranes were recovered from the interphase. The isolated plasma membranes were further purified by alkaline stripping (Na2CO3, pH 11.5) to remove soluble proteins attached to the membrane. Proteins in the purified plasma membrane vesicles were separated by SDSPAGE, digested by trypsin, and the extracted peptides were separated by reversed-phase liquid chromatography (RP-LC) and analyzed by tandem mass spectrometry (MALDI-MS/MS) yielding protein/peptide identification as described in Figures 2 and 3 and Tables 2 and 3. Table 1. Removal of Cytosolic and Organellar Protein Content for Enrichment of Plasma Membranesa pre-fractionation step
total protein content (µg)
yield (%)
cell lysate centrifugation (bulk removal) two-phase partitioning (primary washing) alkaline stripping (secondary washing)
6000 1850 510 90
100 31 8 1.5
a The yield of total protein after each step in the proposed prefractionation strategy based on ∼2 × 108 PBM cells was assayed by the BCAmethod.25
and leukocytes. For example, CD11B is an integrin subunit assigned to surface of the NK T-cells. A number of plateletspecific surface proteins such as CD62P, CD41, CD42B, and CD42C were identified suggesting some contamination of platelets in this PBM cell preparation. Rab27B is a protein involved in signal transduction across the plasma membrane of platelets. CD61 is found on the surface of macrophages, CD14 is involved in the binding of lipopolysaccharides (LPS) to monocytes and CD90 is a T-cell specific receptor. Also, a number of human leukocyte antigens (HLA) presenting foreign antigens to the T-cell receptors were identified. This motivated integration of this method into a strategy for further examination in order to identify additional plasma membrane proteins and potential cell surface biomarkers. Identification of Proteins in the Plasma Membrane Fraction by LC-MALDI-MS/MS. To further reduce the sample complexity of the plasma membrane preparation from PBM cells in order to detect and identify an even larger number of proteins, an entire lane of the SDS-PAGE was divided into 36 sections (Figure 3a), which were subjected to tryptic in-gel digestion, and peptide separation by reversed-phase chromatography into 96 fractions for MS/MS analysis. Such off-line LC-MALDI-MS/MS permits detection and identification of
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Figure 2. Primary analysis of protein content in plasma membrane preparation by MALDI-MS/MS. Comparison of protein content in fractions obtained during preparation of plasma membranes from PBM cells, as described in Figure 1, after SDSPAGE (25 µg per lane) and direct spotting MALDI-MS/MS. The fractions containing soluble and cytosolic proteins (left), membrane associated proteins (middle) and purified plasma membranes (right). Several cell surface proteins were identified in the major bands by MS and MS/MS in the plasma membrane preparation as indicated to the right in the figure.
database (HRPD).35,36 The number of plasma membrane proteins with one or more transmembrane regions, as predicted by TMHMM 2.033,37 was 34. The remaining 26 plasma membrane proteins were annotated as peripheral, either as associated to an integral membrane protein or by hydrophobic and/ or electrostatic interaction with membrane lipids. Of the remaining proteins, 11 were mitochondrial and five were endoplasmic reticulum proteins. Three of the detected proteins were annotated as cytosolic. These included actin and myosin, both known to be very abundant and involved in maintaining the structural integrity of the plasma membrane. One protein was annotated as a lysosomal enzyme and three as extracellular proteins. The default setting for considering protein identification reliable in the search engine Mascot is an accumulative score corresponding to 95% significance. By using this criterion, we obtained 249 protein identifications presented in Additional file 1 and are discussed further below. Potential Unique Marker Peptides for PBM Cell SurfaceSpecific CD Antigen Marker Proteins. Nine different CD antigen marker proteins were identified with >99.95% confidence (Table 3). Some of these CD antigens are common to many types of PBM cells, such as the common leukocyte antigen CD45 (a signaling tyrosine kinase), whereas others are restricted to one or several subtypes, such as CD11b (typical for NK T-cells). In total, 66 different peptides were detected originating from these CD antigen marker proteins. For each protein, several peptides were observed, ranging from four peptides per protein for CD14, CD42B, CD42C to 13 peptides per protein for CD41. All sequences were confirmed by MS/ MS.
Discussion
Figure 3. Further analysis of protein content in plasma membrane preparation by LC-MALDI-MS/MS. A. Proteins in plasma membranes prepared as described in Figure 1 were separated by SDS-PAGE (25 µg, stained with colloidal Coomassie), the entire gel lane was divided into 36 gel sections, which were processed and analyzed by LC-MALDI-MS/MS. B. Cellular localization of the 84 identified proteins listed in Table 2 (confidence interval 99.95%).
many more peptides since the effect of ion suppression from abundant peptides is reduced in each of these 96 fractions. A total of 84 proteins were identified with 99.95% confidence (Table 2) and the pie chart diagram in Figure 3b shows the cellular localization of the proteins. Out of the 84 identified proteins, 60 (72%) were annotated as plasma membrane proteins in the Swiss-Prot30-32 and the human protein reference
Aqueous Two-Phase Partitioning for Isolation of Plasma Membranes. We have here described the use of aqueous twophase partitioning for preparation of a plasma membrane fraction, which after protein size fractionation by SDS-PAGE and LC-MS/MS contained 72% plasma membrane proteins, and nine different CD antigen marker proteins at a 99.95% confidence interval. The two-phase system used here was designed to enrich plasma membranes at the interphase (Figure 1), whereas other membranes partitioned to the bottom phase.12,13 When the material at the interphase was collected, some bottom phase may also be withdrawn, providing an explanation as to why some contamination from other membranes was found (Figure 3, mainly endoplasmic reticulum (ER) and mitochondria). If desired, one partitioning step can easily be repeated to reduce bottom-phase contamination. If desired, the phase system can also be constructed such that the plasma membranes do not partition to the interphase but to the upper phase, while intracellular membranes are found at the interface or in the bottom phase.15,34 Plasma membranes generally have a high partitioning ratio to the top phase compared with intracellular membranes. There is always a risk that contaminating membranes could be trapped inside the plasma membrane vesicles. To minimize this problem, we included the sodium carbonate washing step into our protocol, which is expected to open the vesicles thus releasing their contents.26 Sucrose gradient centrifugation often gives contamination from ER and mitochondria,3,8,38 which will migrate to the same position in the gradient due to their density overlap with the plasma membrane vesicles. The partitioning of biological material in aqueous two-phase systems is mainly governed by Journal of Proteome Research • Vol. 5, No. 5, 2006 1171
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Table 2. Proteins Identified in the Membrane Fraction after Two-Phase Partitioning, Alkaline Stripping, SDS-PAGE, and LC-MS/MSa,b protein name
acc. no.
cellular location
predicted TM-regions
78 kDa glucose-regulated protein (GRP 78) ADP,ATP carrier protein, fibroblast isoform Annexin A2 Placental anticoagulant protein IV Annexin A6 (CPB-II) ATP synthase beta chain Calreticulin Cathepsin G CD11b Integrin alpha-M CD14 Monocyte differentiation antigen CD18 Integrin beta-2 CD36 Platelet glycoprotein IV CD41 Integrin alpha-IIb CD42B Platelet glycoprotein Ib alpha chain CD42C Platelet glycoprotein Ib beta chain CD45 Leukocyte common antigen CD61 Integrin beta-3 Endoplasmin (Tumor rejection antigen 1) Erythrocyte band 7 integral membrane protein Ficolin 1 Fibrinogen alpha/alpha-E chain Fibrinogen beta chain Fibrinogen gamma chain Filamin A (Endothelial actin-binding protein) Guanine nucleotide-binding protein G(i) Guanine nucleotide-binding protein G(k) Guanine nucleotide-binding protein G(q) HLA class II, DRB1-9 HLA class I, 12.4 HLA class I, A-11 HLA class I, B-14 HLA class I, B-40 HLA class I, B-42 HLA class I, B-46 HLA class I, B-39 HLA class I, B-52 HLA class I, B-53 HLA class I, B-73 HLA class I, Cw-1 HLA class I, Cw-6 HLA class I, Cw-7 HLA class I, Cw-12 HLA class II, DP(W2) HLA class II, DRB1-4 HLA class II, DR HLA class II, DW2.2/DR2.2 HLA class II, DR-1 Moesin NADH-cytochrome b5 reductase NipSnap3A protein (NipSnap4) Peptidyl-prolyl cis-trans isomerase B Perforin 1 (Lymphocyte pore forming protein) Polycystic kidney and hepatic disease 1 Prohibitin Protein disulfide-isomerase A3 precursor Radixin Ras-related protein Rab-1B Ras-related protein Rab-11B Ras-related protein Rab-27B (C25KG) Ras-related protein Rap-1b Sideroflexin 1 Voltage-dependent anion-selective channel 1 Acyl-CoA dehydrogenase, ADP,ATP carrier protein Cytochrome c1, heme protein Glycerol-3-phosphate dehydrogenase Malate dehydrogenase Sulfide:quinone oxidoreductase Tricarboxylate transport protein Thioredoxin-dependent peroxide reductase Ubiquinol-cytochrome-c reductase protein 2 Voltage-dependent anion-selective channel protein 2
P11021 P05141 P07355 P08133 P06576 P27797 P08311 P11215 P08571 P05107 P16671 P08514 P07359 P13224 P08575 P05106 P14625 P27105 O00602 P02671 P02675 P02679 P21333 P04899 P08754 P50148 Q9TQE0 P01893 P13746 P30462 Q04826 P30480 P30484 P30475 P30490 P30491 Q31612 P30499 Q29963 P10321 P30508 P13763 P13760 P01903 P01911 P01912 P26038 P00387 Q9UFN0 P23284 P14222 Q8TCZ9 P35232 P30101 P35241 Q9H0U4 Q15907 O00194 P61224 Q9H9B4 P21796 P49748 P12236 P08574 P43304 P40926 Q9Y6N5 P53007 P30048 P22695 P45880
pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm pm mito mito mito mito mito mito mito mito mito mito
0 6 0 0 0 0 0 1 0 1 2 1 1 1 2 1 0 1 0 0 0 1 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 2 0 0 0 1 0 1 0 0 0 0 0 0 0 5 channel 0 6 0 1 0 0 0 0 0 channel
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alternative cellular location
er mito mito er secr
er
cyto
mito, er cyto
cyto ves nucl mito mito
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acc. no.
Voltage-dependent anion-selective channel protein 3 Serum albumin precursor Thrombospondin-1 precursor Transforming growth factor, T-cell suppressor Dolichyl-diphosphooligosaccharide-protein Dolichyl-diphosphooligosaccharide-protein Reticulon 4 (Neurite outgrowth inhibitor) Tapasin precursor (TAP-binding protein) Thromboxane-A synthase Actin, cytoplasmic 2 (Gamma-actin) Heat shock cognate 71 kDa protein Myosin heavy chain, nonmuscle type A Myeloperoxidase precursor
Q9Y277 P02768 P07996 P61812 P04843 P39656 Q9NQC3 O15533 P24557 P63261 P11142 P35579 P05164
cellular location
mito secr secr secr er er er er er cyto cyto cyto lyso
predicted TM-regions
channel 0 0 1 1 2 2 1 4 0 0 0 0
alternative cellular location
nucl
a The level of confidence was set to 99.95% (0.05% false positives) using the Mascot search engine and Swiss-Prot database. Transmembrane helices were predicted by the software TMHMM 2.0. The cellular locations were obtained by manual investigation of annotations in Swiss-Prot and the Human Reference Protein Database. b Acc. no: Swiss-Prot accession number; TM: transmembrane; Channel denotes proteins where the transmembrane region is made up of β-strands; pm: plasma membrane; mito: mitochondrion; secr: secreted protein; er: endoplasmic reticulum; cyto: cytosolic; nucl: nucleus; ves: vesicular transport.
Table 3. Cell Surface-Specific Antigens and Number of Potential Biomarker Peptides Identified in the Plasma Membranes Isolated by Aqueous Two-Phase Systemsa CD-antigen
Swiss-Prot accession no.
no. of peptides
CD11B CD14 CD18 CD31 CD41 CD42B CD42C CD45 CD61
P11215 P08571 P05107 P16671 P08514 P07359 P13224 P08575 P05106
10 4 7 7 13 4 4 6 11
a The level of confidence was set to 99.95% (0.05% false positives) using the Mascot search engine and Swiss-Prot database.
surface properties, therefore this method is suitable for the separation of cellular components with similar density. Aqueous two-phase partitioning is also less time-consuming and laborious than sucrose gradient centrifugation (1 and 18 h respectively) and other methods described for preparation of plasma membranes for proteomics.3,8,38 So far, aqueous two-phase partitioning has not been frequently used for proteomics. For isolation of plasma membranes from plant material, aqueous two-phase partitioning has nevertheless become a standard method,24 and recent proteomic analysis of plasma membranes from Arabidopsis thaliana isolated by aqueous two-phase partitioning resulted in the identification of a large number of known and novel plasma membrane proteins.39 In our hands the amount of cytosolic contamination was low (4%, Figure 3) although cytosolic contamination has been considered to be the main disadvantage of the aqueous twophase partitioning preparation method.3 The cytosolic and other soluble proteins were removed in three steps in our protocol (Figure 1, Table 1). First, in the supernatant when total membranes from PBM cells were pelleted (bulk removal); second, in the phase system (primary washing) and finally in the alkaline stripping (secondary washing) to remove membraneassociated soluble proteins residing inside the membrane vesicles. Sucrose gradient centrifugation was used to isolate plasma membranes from chronic lymphocytic leukemia human B-cells,
but only 27% of the detected proteins were plasma membrane proteins.40 A preparation of T-cell plasma membranes gave 57% plasma membrane proteins.41 Recently, an approach using the so-called Jacobsons pellicle method, where plasma membranes are immobilized on colloidal silica particles to stabilize and increase the density of the membrane vesicles for facilitated isolation, gave 43% plasma membrane proteins.42 Biotinylation of the cell surface and streptavidin beads generated a plasma membrane fraction from cultivated mammalian cells with 42% (14) plasma membrane proteins,38 and when combined with salt washing, 85% plasma membrane proteins.43 Our plasma membrane preparation yielding identification of 72% (60) plasma membrane proteins is thus rapid and efficient for initial enrichment in comparison to other preparation methods used for mammalian material. In protein identification, we set the confidence level to 99.95% to avoid the frequent occurrence of false positive protein identifications using the Mascot default setting of 95%.44,45 Therefore, none of the 84 identified proteins should be false positives. Besides the 60 plasma membrane proteins, 14 are annotated as with another cellular localization. This can either be due to contamination, or to a functional location of these proteins to the plasma membranes. Some proteins may actually have dual cellular locations, as exemplified in Table 2. Prohibitin, voltage-dependent anion selective channel 1 and ATP/ADP translocator are all examples of proteins known to be abundant in other organelles but also expressed in or assigned to the plasma membrane.1,46-48 Several cell surface-specific antigens and potential biomarker peptides were identified. Recently it was calculated that 96% of all protein sequences among the 12 000 human protein sequences in the Swiss-Prot database include unique signature peptides, provided that the experimentally measured peptide mass is accurate enough (
