Proteomic Profiling of Surface Proteins on Th1 and Th2 Cells

Oct 21, 2004 - We utilized mass spectrometry to profile cell surface protein differential expression on primary human. T helper (Th1 and Th2) cells wi...
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Proteomic Profiling of Surface Proteins on Th1 and Th2 Cells Kelly M. Loyet,*,† Wenjun Ouyang,‡ Dan L. Eaton,† and John T. Stults†,§ Protein Chemistry Department, Immunology Department, Genentech, South San Francisco, California Received October 21, 2004

We utilized mass spectrometry to profile cell surface protein differential expression on primary human T helper (Th1 and Th2) cells with the stable isotope labeling by amino acids in cell culture (SILAC) approach. Proteomic and microarray analyses were done concurrently and results were compared for 38 different genes. Although microarray studies displayed wide variability between donors for mRNA expression, these two approaches were shown to be corroborative for most gene products with the exception of a small subset of uncorrelated protein and message levels. The greatest differing Th1 to Th2 ratios were observed for BST2 (bone marrow stromal protein 2) and TRIM (T cell receptor interacting molecule). Both showed greater Th1 expression by proteomic methods, even though mRNA levels were approximately equal for both. To validate this method, we compared protein expression levels of a recently cloned molecule, B and T cell lymphocyte attenuator (BTLA), on Th1 and Th2 cell populations and showed greater protein expression on Th1 cells, which agrees with a previous analysis of higher BTLA mRNA expression in Th1 cells.1 Keywords: human • mass spectrometry • membrane proteins • proteomics • SILAC • Th1 • Th2

Introduction Any immune response involves first, recognition of the pathogen or foreign material and second, a reaction to eliminate it. Immune responses fall into two different categoriess innate or adaptive, adaptive immune responses being highly specific for a particular pathogen and improving with each successive encounter with the same pathogen. Lymphocytes are central to all adaptive immune responses and fall into two basic categoriessB cells and T cells. T cells have a wide range of activities. Cytotoxic T lymphocytes (CD8+) recognize and destroy virus-infected cells while T helper cells (CD4+) aid in the development and activation of other cell types during immune responses. Much work has sought to understand the regulation and development of T helper (Th) cells.2,3 Th cells mobilize counteraction against invading pathogens. Th1 and Th2 cells represent two extremely polarized forms of helper cells. Th1 cells, producing IFN-γ and TNF-R, are capable of activating macrophages and cellular immunity, while Th2 cells, secreting IL-4 and IL-5, favor the induction of B cell maturation, antibody production, and humoral immunity.4-6 Inappropriate Th2 responses or uncontrolled Th1 responses can lead to allergic disorders or autoimmune diseases, respectively; Th1 and Th2 cells can also cross-regulate each other, by which cytokines of one type of T helper cells can repress the development the other type of T helper cells.7,8 Due to the central roles of T helper cells in many autoimmune disorders, * To whom correspondence should be addressed. Protein Chemistry Department, Genentech, 1 DNA Way, M. S. #63, South San Francisco, CA 94080-4990. E-mail: [email protected]. Tel: (605) 225-6689. † Protein Chemistry Department, Genentech. ‡ Immunology Department, Genentech. § Currently at Predicant Biosciences, South San Francisco, CA 94080.

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it is important to find molecules that manipulate the T helper cell development and functions. A great of amount of work has been focused on the cytokines and T cell surface molecules that play critical roles in regulation of T helper cell differentiation and functions.3,5 Although Th1 and Th2 cells have been shown to share the expression of a number of surface molecules, constitutive and stable distinguishing surface markers which are independent of given in vitro conditions have proven elusive.9,10 RNA analysis is most often used for expression profiling, although many studies have shown that mRNA expression does not always correlate with protein expression and sub-cellular localization.11-14 Another study, however, shows the complementary nature of mRNA and protein data.15 Attempts to profile Th1and Th2-specific markers include transcript analysis by microarray,16,17 selective Th1 and Th2 cell flow cytometry analysis of chemokine receptors,18 as well as immunization of mice with Th1 cells and characterization of Th1-specific antibodies.19 Recent proteomic studies characterized Th1 and Th2 cells using two-dimensional gel electrophoresis followed by peptide mass fingerprinting.20,21 Very few membrane proteins were represented in these studies, however. In the wide ranging field of proteomics, the general goals of proteomic studies include identification of a repertoire of proteins representative of a specific cellular state and quantification of comparative states.22 Although proteomic studies were originally performed with two-dimensional gels, mass spectrometry has begun to supersede this procedure and has become an essential tool in the field of proteomics.23,24 The inability of two-dimensional gels to routinely resolve the typically hydrophobic proteins that represent plasma membrane proteins preclude this method for the goal of elucidating 10.1021/pr049810q CCC: $30.25

 2005 American Chemical Society

Protein-MS Analysis of Th1 & Th2 Surface Proteins

surface proteins on Th1 and Th2 cells.25 A recent study has examined the plasma membrane proteins of lymphocytes by cell surface labeling with a water-soluble biotinylation reagent followed by cell lysis, membrane purification, biotinylated protein affinity capture, solution phase isoelectric focusing and SDS-PAGE, and identification by HPLC electrospray/tandem MS, thus identifying 42 or 46 plasma membrane proteins from a murine T cell hybridoma or primary splenocytes, respectively.26 Even more ambitious studies include one which also used HPLC MS/MS to identify over 500 proteins, about 50% which were thought to be membrane proteins, from cell lines of breast cancer origin27 and one on a lung cancer cell line using a similar biotinylation strategy to identify 781 plasma membrane proteins, 67% of which were integral membrane proteins.28 Using isotope-coded affinity tagging (ICAT) to determine expression profiles,29 another human cell line, HL-60 cells, yielded identification of 79 membrane proteins, eight of which had more than 2-fold differential expression upon stimulation.30 We present proteomic efforts to profile primary Th1 and Th2 cell surface proteins from plasma membrane preparations by mass spectrometry. By enriching for plasma membrane proteins followed by trypsinization, and data-dependent LC-ESIMS/MS, approximately one hundred cell surface proteins were identified, and about half of these were quantified. Quantification was performed by stable isotope labeling, which, in combination with mass spectrometry, has been shown to be more accurate than comparison of spot intensities on gels and has greater potential in the study of complex samples.31 The current study used stable isotope labeling in cell culture to profile proteins comparatively on nonstimulated (resting) primary human Th1 and Th2 cell subtypes derived from the whole blood of a healthy individual. In addition, we validated the method with a protein known to be overexpressed in Th1 cells as well as compare proteomic to microarray analyses for expression of a set of 38 surface proteins on primary human Th1 versus Th2 cells.

Experimental Section Purification and Culturing of Primary Th Cells. Human CD4+ T cells were directly purified from whole blood or leukopack from healthy donors by using human CD4+ T cells enrichment kit from Stemcell Technologies Inc. In brief, 50 µL of RosetteSep cocktail was added per mL of blood and mixed. Blood was then incubated at room temperature for 20 min. Sample was diluted with an equal volume of PBS. CD4+ T cells were then collected after a Ficoll density centrifugation separation. CD45RO- naı¨ve CD4+ T cells were further isolated with anti-CD45RO MicroBeads labeling kits from Miltenyl Biotec. Naı¨ve CD4+ T cells were negative purified by AutoMACS (Miltenyl Biotec) following the protocol suggested by the manufacturer. Purity of the cells was examined by staining the cells with anti-human-CD4-FITC antibody and anti-humanCD45RA-Cychrom antibodies (BD Biosciences). Samples were analyzed with a FACSCalibur. In all our experiments, the purity of CD4+ and CD45RA+ T cells was from 88% to 95%. For Th1 and Th2 differentiation, plates were coated with goat-anti-mouse IgG (10 µg/mL, Caltag) for 2 h, 37 °C followed by three washes with PBS, then 5 µg/mL anti-CD28 plus 10 µg/mL anti-CD3 for an additional 2 h at 37 °C. Naı¨ve CD4+ T cells were stimulated at 2-3 million cells per mL plus 1 ng/ mL IL-2 (R&D Systems) in the anti-CD3 coated plates. For Th1 differentiation, IL-12 (5 ng/mL) and anti-IL-4 (400 ng/mL) were added. For Th2 differentiation, IL-4 (25 ng/mL), anti-IL-12

research articles (1 µg/mL), and anti-IFNγ (120 ng/mL) were added (R&D Systems). On day 3, cell cultures were expanded 5-fold by adding fresh media. On day 7, cells were harvested and washed for phenotype analysis by intracellular cytokine staining or further differentiation and expansion under the same conditions as described above. Intracellular Cytokine Staining. For intracellular cytokine staining, 2 million cells per ml were stimulated by phorbol 12myristate 13-acetate (PMA) (50 ng/mL) and ionomycin (1 mM) for 4 h. Brefeldin A (10 µg/mL, Epicenter Technology) was added and incubated for another 2 h before fixation with 2% Formalin. Cells were then permeabilized by 0.5% Saponin and stained with anti-IL-4-PE or anti-IFN-γ-PE or PE-coupled isotype control antibody (BD Biosciences). Samples were analyzed with a FACSCalibur. Stable Isotope Labeling of Th1 and Th2 Protein in Cell Culture. Known integral membrane and surface proteins were targeted for Th1 versus Th2 quantitative analysis using a SILAC approach.32 Briefly, for quantification studies, stable isotope amino acids were incorporated into purified naı¨ve T cells during polarization and growth in culture. Cell culture media used in the differentiation and growth of Th1 and Th2 cells contained either Leu-d3 or Leu-d0, respectively. Equal numbers of Th1 and Th2 cells were combined, followed by plasma membrane preparations and mass spectrometric analysis of peptides containing leucine. Plasma Membrane Preparations. Resting cells were harvested at day seven following stimulation of fully differentiated Th1 or Th2 cells. Membrane preparations were generally composed of approximately 200 million cells and one-quarter of the final solution was analyzed. Cells were lysed under hypotonic conditions (10 mM Tris pH 7.5, 0.5 mM MgCl2, containing protease inhibitors added fresh, 10 min on ice) with an ice-cold dounce homogenizer, 30 strokes. Immediately following homogenization, a high salt membrane preparation buffer (280 mM sucrose, 50 mM Mes pH 6, 450 mM NaCl, 10 mM MgCl2) was added at an equal volume. Post-nuclear supernatants were spun over a 35% sucrose cushion to obtain the membrane band. Membrane pellets were washed extensively with PBS, incubated for 30 min on ice with a high pH wash (0.025 M Na2CO3, pH 11) to reduce cytoskeletal proteins,33 then washed two to three times more in PBS. Membrane pellets were solubilized with 0.5% SDS in 100 mM HEPES pH 8 and reduced with 2 mM TCEP at 60 °C for 15 min, then alkylated with 10 mM iodoacetamide for 20 min at room temperature in the dark. In preparation for deglycosylation, membrane proteins were precipitated with chloroform/methanol,34 followed by resuspension in 100 mM HEPES pH 8 with brief pulse probe sonication (5 pulses) then addition of 0.05% zwittergent 3-16. Deglycosylation with 500 units of PNGase F for 1 h at 37 °C was followed by proteolysis with 1 µg of trypsin overnight at 37 °C. Mass Spectrometry. Tryptic peptides were analyzed as previously described35 with capillary RP-HPLC operating at a flow rate of approximately 300 nanoliters per minute using an 11 cm × 100 µm ID C18 PicoFrit column (New Objective) with a 15 µm tip, in-line with ESI ion-trap mass spectrometry using a Thermo Finnigan LCQ Deca. The peptides were analyzed by human protein sequence database searching with the SEQUEST program. The Dayhoff database was used, which is an internal protein database including sequences from Swiss-Prot, the Protein Identification Resource (PIR), translated coding regions from Genbank and the patent databases. Potential identities Journal of Proteome Research • Vol. 4, No. 2, 2005 401

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Figure 1. Differentiation of Th1 and Th2 cells. IFN-γ and IL-4 are cytokine markers for Th1 and Th2 cells, respectively. (A) Flow cytometry analysis of Th1 and Th2 intracellular staining with anti-IFN-γ and anti-IL4. (B) RNA expression analysis of IFN-γ and IL-4 in resting and activated Th1 and Th2 cells.

were based on predefined scoring criteria36 of dCn greater than or equal to 0.1, Xcorr scores for singly charged molecules greater than or equal to 1.9, for doubly charged molecules greater than or equal to 2.2, and triply charged residues greater than or equal to 3.75. Matches for scores near the borderline were visually inspected. LCQ Deca was operated in triple play data dependent scanning mode with parameters as previously described.35 Labeled peptides from Th1 and Th2 cells were co-purified in membrane preparations and compared with either a shotgun approach, using Triple Play data dependent acquisition software (Thermo Finnigan) in which the largest m/z peaks are subject to MS/MS analysis, or a guided single ion monitoring (SIM) approach by trapping specific peptides in the ion trap for MS/MS analysis.35 Microarray Analysis. Affymetrix microarrays were used. Fully differentiated Th1 and Th2 cells with or without anti-CD3 and anti-CD28 stimulation, as described before, were used to purify RNA with Qiagen’s RNeasy Mini Kits. RNA samples were treated with DNase to remove residual contamination of DNA. The preparation methods of cRNA and hybridization/scanning of the arrays were provided by Affymetrix. Briefly, 5 µg of total RNA was converted into double-stranded cDNA using a cDNA synthesis kit (SuperScript Choice, GIBCO/BRL) and a T7-(dT)24 oligomer primer (Operon Technologies, Inc.). Double-stranded cDNA was purified by phenol-chloroform extraction (Phase Lock Gel Light, Eppendorf Scientific, Inc.) and ethanol precipitation. After second-strand synthesis, labeled cRNA was generated from the cDNA sample by using a T7 RNA polymerase and biotin-labeled nucleotide in an in vitro transcription (IVT) reaction (Enzo Diagnostics). The labeled cRNA was purified on an affinity resin (RNeasy Mini Kits, Qiagen). The 402

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labeled cRNA amount was determined with absorbance measurement at 260 nm, using the convention that 1 OD at 260 nm corresponds to 40 µg/mL of RNA. 20 µg of cRNA was fragmented by incubating at 94 °C for 30 min. in 40 mM Trisacetate pH 8.1, 100 mM potassium acetate and 30 mM magnesium acetate. Samples were then hybridized to the arrays at 45 °C for 19 h in a rotisserie oven set at 60 rpm. Arrays were washed, stained and scanned in the Affymetrix Fluidics station and scanner. Data analysis was performed using the Affymetrix GeneChip Analysis software. Pairwise comparisons were made. The genes were ranked according to concordance in the pairwise comparison, and a Mann-Whitney pairwise comparison test was used to calculate significance.

Results and Discussion Differentiation of Human Th1 and Th2 Cells. T helper cells play important regulatory roles in immune responses during pathogen invasion and autoimmune diseases. In previous proteomic studies, T cell lines, such as Jurkat T cells, have been used 37, however, these cell lines differ significantly from primary cells in terms of their dependency on TCR signaling for their proliferation capacity and their inability to further differentiate to T helper subtypes. In these studies, the use of purified human T cells allowed identification of physiologically relevant Th1 and Th2 proteins. To obtain fully developed Th1 and Th2 cells, naı¨ve CD4+ T cells were activated 2-3 rounds by anti-CD3 and anti-CD28 plus differentiation cytokines as described in the methods. The phenotypes of the Th cells were analyzed by intracellular cytokine staining. As shown in Figure 1A, over 48% of the Th1 cell population produced IFN-γ, whereas only 3% produced

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IL-4. On the other hand, about 26% of the Th2 cell population secreted IL-4 and only 14% secreted IFN. Similar data were obtained by gene chip analysis of the same cells with or without activation, as shown in Figure 1B. Other known Th1 and Th2 specific genes also demonstrated skewed expression profiles in the gene chip analysis as expected (data not shown). These data demonstrated that our in vitro differentiated T cells demonstrated true T helper cell phenotypes. Plasma membrane preparations were made from harvested Th1 and Th2 cells as explained in the methods. Using an LCQ ion trap mass spectrometer in data dependent MS/MS mode, peptides were selected for collision-induced dissociation (CID), and these spectra were searched against the Dayhoff (see methods) human protein database using SEQUEST. For initial studies, to validate the preparation method and to identify candidate membrane proteins for quantification, Th1 and Th2 cells were prepared separately from seven different donors. Fourteen different mass spectrometric analyses were performed, seven each of Th1 and Th2 membrane preparations. To cast a wide net in identifying potential integral plasma membrane proteins, all peptides with charge-dependent SEQUEST scores over a certain threshold as previously described36 and stated in methods, were selected for inclusion in a combined catalog list of potential proteins in Th1 and Th2 cells. The potential proteins were combined into one list since the selection of peptides using a shotgun approach (see methods) is a random selection of CID spectra and would not be considered indicative of differences between the two cell subsets. An estimate of the false positive rate was determined by repeating a representative database search against a human database of reversed sequences and showed that 4.5% of resulting nonsense matches met the significance criteria used for the study (data not shown). Integral plasma membrane proteins consistently seen in two or more preparations were targeted for further quantification. Categorization of Detected Proteins. The majority of peptides identified from any single experiment were from proteins known be localized to the plasma membrane (PM)seither integral or plasma membrane-bound, as determined by database annotations or published reports. As shown in Figure 2A and 2B, an average of 71% of peptides were identified as PM or PM-bound in any one experiment. The proteins identified from mass spectrometric peptide determinations yielded a PM or PM-bound protein distribution of 59%. This indicates enrichment of plasma membrane proteins and compares well to a recent study in which 28% of proteins identified from unfractionated brain homogenates were membrane proteins.38 All other cellular compartments had peptide or protein distributions of 12% or less. Although 12% of proteins identified had no function or localization annotations in any database or publication, using in-house localization prediction algorithms we were able to predict cellular localizations for these peptides. Approximately 20% of the uncharacterized proteins were predicted to reside in the plasma membrane with similar percentages attributed to secreted, nuclear, mitochondrial and 7 or 12% to ER/golgi or cytoplasmic respectively (data not shown). As shown in Figure 2C, of the integral plasma membrane proteins identified, the majority of proteins were single-spanning transmembrane domain proteins, yet over 25% contained between two and twelve transmembrane domains. Multi-spanning transmembrane domain proteins are traditionally difficult to analyze due to their hydrophobicity,25 yet a

Figure 2. Distribution of peptides and proteins identified. (A) The average cellular localization distribution of seven independent experiments is shown for peptides identified. (B) The distribution of proteins is shown for these same experiments. For (A) and (B), the sub-cellular localizations of uncharacterized proteins were algorithmically predicted. (C) The distribution of the number of TM domains in the identified PM proteins from a catalog of 14 independent experiments is shown.

relatively large number of multi-spanning membrane proteins were seen in this study. A catalog list of 372 potential proteins in Th1 or Th2 cells (see Supplementary Table, in Supporting Information) reveals an abundance of proteins known to have important immunological roles in T cell signaling. Although potential protein identifications made from only one peptide were put into this list, the list is a compilation of 14 experiments and often other peptides from the same protein were identified in other experiments. In addition, there were many potential hits of Journal of Proteome Research • Vol. 4, No. 2, 2005 403

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Figure 3. Plasma membrane proteins identified by function. (A) For integral membrane proteins identified, the distribution of protein functions is shown. (B) Underlined proteins were subject to further quantification.

proteins that are known to be present in T cells. For example, TCR/CD3, CD4, TRIM (T cell receptor interacting molecule), and LAT (Linker for Activation of T cells) proteins, all known to be present and important as membrane-spanning proteins in T cells, were identified in the study.39 Pathways such as JAKSTAT (JAK-3, STAT1), Ras-Raf (Lck, Raf-1), Ca2+-signaling (IP3 receptor, Annexin VI), TNFR superfamily signaling (TRAF1), and actin-reorganization (Coronin 1a and Ras-related protein RAP1b) were also represented with identified proteins. In addition, 404

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several transcription factors were identified, many with T cell functions as noted by their regulation of and by cytokines. These include Jun-b, NF-ATp Rel (cytokine expression regulation), and STAT1 (regulated by cytokines and growth factors). The proteins identified as cell surface or integral plasma membrane proteins act in a variety of cellular functions. As shown in Figure 3, the largest functional category was signaling proteins (27%) followed by transporter and adhesion (each 20%), and antigen presentation (10%), with smaller numbers

Protein-MS Analysis of Th1 & Th2 Surface Proteins

contributing to hydrolysis, apoptosis, vesicular transport, metabolism, and unknown function. In the largest category of signaling, many peptides were identified from the protein CD45 (almost 50% of all possible CD45 peptides were identified), which occupies 10% of the surface of T and B cells40 and represents millions of sites per cell. On the other hand, cytokine receptors are generally low abundance proteinssmost cells express them on the order of 100 to 1000 receptors per cell41s and were less likely seen. Cytokine receptor CXCR-3 was seen in Th1 cells although not in Th2 cells, as would be expected,42,43 demonstrating the wide dynamic range of this method. Targeting 38 Membrane Proteins for SILAC Quantification and Comparison to RNA Microarray Data. A subset of plasma membrane proteins (see Figure 3B) was quantified with the SILAC technique in which cells were cultured in RPMI (Leu deficient, dialyzed FBS) supplemented with Leu-d3 for Th1 cells or normal Leu for Th2 cells. In this manner, the masses of all leucine-containing peptides were increased by 3 amu per leucine in Th1 cells but not Th2 cells. This enabled concurrent and combined purification of Th1 and Th2 cell membrane preparations after counting equal numbers of harvested Th1 and Th2 cells. The combination of cells from both subtypes into one membrane preparation eliminated the problems of differential losses due to separate plasma membrane preparations. The combined membrane preparation was analyzed by LC-ESI-IT-MS/MS, and the distinct peak heights of individual leucine-containing peptides allowed relative quantification of the identified protein between Th1 and Th2 preparations. All quantitative SILAC data were from a single donor. Using the SILAC method almost complete incorporation of Leu-d3, as shown in Figure 4A, occurred after only 7 days of culturing. This was shown with incorporation into a peptide from CD45 in Th1 cells. Quantifications for these studies were derived from zoom scans, comparing maximal peak heights of peptide pairs at the timepoint of highest peak elution, with the elution time range determined by MS/MS scans. Combining equal amounts of Th1 and Th2 cells (cultured with normal leucine, Leu-d0) prior to the membrane preparation (see Figure 4B), the mass spectrometry analysis of this peptide and many others from CD45 (data not shown) shows that CD45 is expressed in equivalent amounts in Th1 and Th2 cells. On the other hand, as shown in Figure 5, the expression of integrin β 5 is about five times greater in Th2 cells, resulting in a Th1/Th2 ratio of about 0.2. It has been suggested that a particular homing ability of Th2 cells may correlate with the high expression of certain integrins.9 These results for CD45 and integrin beta 5 were obtained in all cell harvests analyzed and were corroborated with RNA data (see below). Using the SILAC approach, 40 integral membrane proteins were quantified in either a shotgun manner or with a single ion monitoring (SIM) targeted approach and 38 were compared to RNA microarray analyses. SIM is different from a shotgun approach in that it specifically monitors one or more peptide m/z ratios instead of randomly analyzing the highest peaks at any given time. The targeted experiment used predetermined elution window SIM with zoom scans and MS/MS collection to target groups of two peptides in three or four different segments with quantification of up to eight peptides per run. The elution timepoint of a given peptide was determined by the MS/MS profile, and the zoom scan readily discerned comparative peptide abundances by peak height. By comparing results for individual peptides between these two methods, they were determined to be equivalent, but generally more peptides

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Figure 4. Efficient stable isotope labeling of amino acids in cell culture. For quantificaton experiments, Th1 cells were cultured with RPMI-1640 medium containing 10% dialyzed fetal bovine serum and Leu-d3, increasing the mass of Leu-containing peptides by 3 amu per leucine in Th1 cells, while Th2 cells were cultured with medium containing normal Leu. Th1 and Th2 cells were grown separately for 7 days and plasma membrane preparations were combined and analyzed by LC-MS/MS. (A) From Th1 cells alone, the singly charged CD45 peptide,ADTTICLK (m/z ) 921.5) shows that nearly all peptide has incorporated the Leu-d3 form, which has an m/z of 924.5, after only 7 days in culture. (B) After combining equal cell numbers of Th1 and Th2 cells, LC-MS/MS analysis of the membrane preparation shows that CD45 is expressed at nearly equal levels in Th1 (m/z ) 924.5) and Th2 (m/z ) 921.5) cells.

could be quantified in a single run with a shotgun approach. Using these formats, Th1 (Leu-d3)/Th2 (Leu-d0) combined PM preparations were analyzed. The Th1/Th2 ratio ranges of at least two and an average of 4.5 determinations per protein (same or different peptides from individual protein) are shown. In addition, the microarray Th1/Th2 ratio analysis for these genes are also shown, with 3 donors per microarray chip and an average of 2.5 microarrays per gene. The comparison of proteomic and microarray data (as shown in Figure 6 for 38 different membrane proteins) indicates that a wide range of protein expression exists between donors, and also that for the proteomic data obtained from one donor, Th1/ Th2 protein quantification generally correlates with the RNA data. The genes for which proteomic and RNA quantification data do not overlap are shown in Figure 6C,D. BST2 and TRIM show slightly greater expression in Th2 cells or slightly greater expression in Th1 cells, resepectively, by RNA analysis but show much higher Th1 expression by proteomic analysis. With the exception of BST2 and TRIM, the other differences are less than 2-fold. It is unknown why the levels of CD3 isotypes (which Journal of Proteome Research • Vol. 4, No. 2, 2005 405

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Figure 5. Integrin, β-5 shows greater Th2 expression. Using the SILAC method, the Th1/Th2 ratio for integrin, β-5 (itb5) is approximately 0.2, as seen in four independent experiments. Shown here is the zoom scan for an SIM experiment with monitoring for the singly charged peptide: LGFGSFVDK, which has an m/z ) 969.5 for Th2 cells and 972.5 for Th1 cells.

Figure 6. Comparison of proteomic versus microarray RNA expression Th1/Th2 ratios for selected genes. See Figure 3B for abbreviations. (A) Proteomic expression analysis, one donor, with bars representing a range consisting of an average Th1/Th2 plus and minus the standard deviation. For all proteins shown, the average is from data for one or more peptides from at least two independent runs, with an average n ) 4.5 determinations per protein. (B) RNA expression analysis with bars representing the range between minimum and maximum average Th1/Th2 plus and minus the standard deviation. Each RNA expression analysis was done with three different donors per genechip and the data shown are from at least one analysis per gene with an average n ) 2.5 genechip analyses. (C) and (D) Summary of proteins for which Th1/Th2 ratios differ between proteomic and RNA expression approach, respectively. 406

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Figure 8. Novel human diagnostic protein shows greater expression in Th2 cells. As seen in two independent membrane preparations of different Th1/Th2 harvests, novel human diagnostic protein #22628, p_abg22637, has greater expression in Th2 cells. Shown is the quantitative analysis of doubly charged peptide: LAAAGADPAGSR, which has an m/z of 528.7 for Th2 cells and 530.2 for Th1 cells, with the quantitative MS chromatogram in the top right corner and the MS/MS for Th2 cell peptide below, which has SEQUEST scores Xcorr ) 2.7 and dCn ) 0.13. The b ions are shown in red and the y ions are shown in blue. The full-length protein has a 95% BLAST homology to human transporter and ion channel 26. Asterisks correspond to b and y ions for a coeluting peptide potentially identified from phosphodiesterase 9A.

Figure 7. BTLA is expressed ∼2.8× greater in primary human Th1 cells than Th2 cells. A readily ionizable BTLA peptide, EINLVDAHLK, was found by LC-MS/MS analysis of plasma membrane preparations from transiently transfected BTLA 293 cells. An SIM experiment, in which the singly and doubly charged ions of this peptide were monitored, was performed on plasma membrane preparations from primary Th1 (Leu-d3) and Th2 (Leud0) cells. (A) Shown is the quantification for seven-day rest Th1 (m/z ) 1157.6) and Th2 (m/z ) 1151.6) BTLA singly charged peptide, four weeks after initial polarization. (B) Th1 and Th2 cells were stimulated for 48 h with plate-bound anti-CD3 and antiCD28 as explained in the methods. These cells were also four weeks past the initial polarization. (C) BTLA expression by flow cytometry. Th1 and Th2 cells were differentiated as previously described. Rested Th1 or Th2 cells three weeks post-differentiation were stained with biotin labeled anti-BTLA antibody or isotype control antibody. The cells were further incubated with streptavidin-PE before the FACS analysis.

contribute to the TCR-CD3 complex) differ by RNA analysis, although by proteomic analysis all show a higher expression in Th2 cells. Whether or not this difference is significant functionally is unknown, yet recent studies have shown that early TCR signaling events are distinct in human Th1 and Th2 cells.44 Engagement of TCR signaling induces internalization

of TCR complexes; whether this internalization process is different between Th1 and Th2 cells is unclear. BST2, a bone marrow stromal cell surface protein which has been shown to be overexpressed in myeloma cells is thought to be involved in pre-B cell growth.45,46 TRIM (T cell receptor interacting molecule) is a TCR-associated transmembrane adaptor protein which is important for intracellular signaling events in T cells.47 SILAC Quantification Validation with BTLA. Recently, a novel gene named BTLA was identified and shown to be highly expressed on B cells and also shows T cell expression.1,48 BTLA is thought to have an inhibitory role in T helper cell signaling. Although our own studies by microarray analysis, and studies by Watanabe et al. using Northern analysis, showed that Th1 cells have increased BTLA RNA expression over Th2 cells, the protein levels were not ascertained.1 Shotgun analysis of either BTLA-transfected or pRK5-mock transfected 293 cell membrane preparations and SEQUEST searching against the BTLA sequence showed a readily ionized BTLA-specific peptide in BTLA-transfected cells which could then be monitored in a single ion monitoring (SIM) format.35 This peptide can be seen in as little as about 300 000 cells, which represents about 135 fmoles of protein, corresponding to approximately 270 000 sites per cell as determined by Scatchard analysis of these transfected cells (data not shown). As seen in Figure 7A and 7B, at the most highly polarized stage (4 weeks post initial differentiation), Th1 cells expressed BTLA protein about 3-fold higher than Th2 cells in both the resting and stimulated states (stimulated with PMA and ionomycin or with anti-CD3 and anti-CD28). At three weeks past differentiation, Th1 cells had about 2-fold higher expression than Th2 cells (Th1/Th2 ) 2) (data not shown). In addition, using a monoclonal antibody against Journal of Proteome Research • Vol. 4, No. 2, 2005 407

research articles BTLA, FACS analysis of Th1 and Th2 cells from a different donor also corroborated the mass spectrometric results. This analysis, done at an early polarization state, only showed slightly higher BTLA expression on Th1 cells in resting cells after 5 days (data not shown), although after two weeks a large difference can be seen (Figure 7C), indicating a trend similar to that shown by mass spectrometry. These data indicate that down-regulation of BTLA expression on Th2 cells depends on a highly polarized state, and confirmed a higher expression of BTLA protein on Th1 cells. Quantification of an Uncharacterized Protein. Although the focus of this study was on proteins for which one could compare protein to RNA quantification data, the method can also be applied to look for novel proteins that have differential expression patterns. As shown in Figure 8, one uncharacterized protein was found to have about a 10-fold greater expression in Th1 vs Th2 cells. The largest number of b and y ions identified a protein that by BLAST has 95% homology to a putative ion channel protein indicating that it is most likely a membrane protein and possibly localized to the plasma membrane. A coeluting peptide, although well below the scoring criteria threshold, was potentially identified as phosphodiesterase 9A. Further experiments to assay the RNA levels for the putative ion channel protein and to determine its functional significance may prove to be informative.

Conclusion Using a proteomic approach and a wide net, many potential proteins were identified in human Th1 and Th2 cells (see Supplementary Table, Supporting Information). On average, over 50% of all proteins identified from Th1 and Th2 plasma membrane preparations were from integral or peripheral plasma membrane proteins. On the basis of the numerical cataloging of different peptides identified from any one analysis of Th1 or Th2 cells, these studies have indicated the differential presence of both expected proteinssmany proteins which have clearly defined roles in the immune systemsas well as a subset of uncharacterized proteins which have unknown functions in Th1 and Th2 plasma membrane preparations (see Figure 8 for one example). Due to their hydrophobicity and large size, membrane proteins are traditionally difficult to analyze.25 Integral membrane proteins, constituting about a third of the proteins encoded by the human genome,49 are an important class of drug targets, particularly for cells of the immune system whose regulation, when gone awry, can lead to various disorders. Of approximately one hundred integral plasma membrane proteins identified, 40 were targeted for Th1 versus Th2 quantitative analysis. Proteomic analyses for 38 proteins were compared to results obtained from microarray expression analysis of RNA from resting Th1 and Th2 cells. The protein ratios largely corroborated RNA expression data, yet these studies have shown a subset of gene products in which the protein and message levels do not agree. Although the results presented here are subject to further confirmation, TRIM, BST2, and the uncharacterized protein homologous to a putative ion channel protein represent candidate targets to examine regulation of Th1 versus Th2 cells. A follow-up study to examine differences in stimulated Th1 versus Th2 cells may yield additional candidate targets. In addition, the techniques presented here may be extended to other primary human cell subsets and/or in comparisons between dynamic and steady-state cell conditions. 408

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