From the Institute of Biomedical Sciences and Technology,
Sickle Cell Disease Research Center, ¶ Department of Molecular and Cell Biology, and || Center for Biotechnology and Bioinformatics, University of Texas at Dallas, Richardson, TX 75083-0688; and ** Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390
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ABSTRACT |
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Because of the ease in obtaining RBCs and because they lack internal organelles, the plasma membrane of this cell type has been studied extensively. The functions of hemoglobin are also well documented. Based on four decades of study, the identity, function, and topology of many RBC membrane proteins have been determined (13). With the advent of modern mass spectrometry (MS) and associated proteomic techniques, determination of the RBC proteome is now plausible. This kind of approach is a necessary first step in understanding how the RBC proteome becomes altered in various hematologic disorders. With this goal in mind, we utilized ion trap tandem MS to analyze the entire human erythrocyte proteome (plasma membrane and cytoplasmic proteins). We identified 181 unique RBC proteins, half of which reside in the plasma membrane and half in the cytoplasm. Moreover, we were able to not only identify the proteins but to also categorize most of them according to function.
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EXPERIMENTAL PROCEDURES |
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To maximize the number of identified RBC proteins, membrane-associated proteins, as well as cytoplasmic proteins, were separated and digested into several fractions; each fraction was analyzed as a separate sample. The RBC membranes, cytoplasmic proteins, inside out vesicles (IOV), and membrane skeleton proteins ("spectrin extract") were prepared as described (4) with the following modifications. Five milliliters of PBS-washed and packed RBCs were resuspended in 10 ml of PBS and incubated (gently shaken) with 1.5 mg of tosylphenylalanyl chloromethyl ketone-treated trypsin (Worthington, Lakewood, NJ) for 2 h at room temperature to digest exposed domains of membrane proteins. The digested material and the trypsinized RBCs were separated by centrifugation at 2000 x g for 10 min. The digested material was collected (sample 1) and cleared by centrifugation at 32,000 x g for 15 min at 4 °C. The separated trypsin-treated RBCs were washed with 10 volume of PBS three times and resuspended (final volume 32 ml) in ice-cold lysis buffer (5 mM NaPO4, pH 7.6, 1 mM EDTA). The lysed cells and the soluble cytoplasmic proteins were separated by centrifugation at 32,000 x g for 10 min at 2 °C. The cytoplasmic proteins (35 mg protein/ml) were collected and cleared by recentrifugation. Lysed RBCs were further washed with 10 volume of ice-cold lysis buffer (six times) to prepare cell membranes (ghosts,
4 mg protein/ml). Membrane skeleton proteins and IOVs were prepared as follows. The membranes (
5 ml) were washed in 10 volumes of the ice-cold spectrin extraction buffer (0.1 mM EDTA, pH 8.0), then resuspended in 20 ml of the same buffer and incubated at 37 °C for 30 min to dissociate membrane skeleton. The extracted membrane skeleton proteins (spectrin extract) and IOV were separated by centrifugation at 250,000 x g for 30 min at 4 °C. Spectrin extract (
0.2 mg/ml) was collected. The tight IOV pellet was gently resuspended in bicarbonate buffer (100 mM ammonium bicarbonate, pH 8.2) to the original membrane volume of 5 ml (
3 mg protein/ml).
The IOV were further diluted twice (1.5 mg protein/ml) in the bicarbonate buffer and incubated with 0.1 mg/ml trypsin for 15 h at 37 °C to digest exposed domains of IOV proteins. The digested IOV were separated from corresponding supernatant containing tryptic peptides (sample 2) by sedimentation at 100,000 x g for 30 min at 4 °C. In the second set of experiments, the total IOV proteins were solubilized and digested with 0.2 mg/ml trypsin under similar conditions at 16 °C in the presence of 1% precondensed Triton X-114 (5). After digestion, the mixtures were incubated at 30 °C and centrifuged at 1000 x g for 10 min at room temperature to separate aqueous and detergent phases. The aqueous phase containing digested peptides (sample 3) released from IOV were collected and cleared by centrifugation at 100,000 x g for 30 min.
Spectrin extract, containing membrane skeleton proteins (0.2 mg/ml), was digested in bicarbonate buffer with 0.05 mg/ml trypsin at 37 °C for 15 h (sample 4).
The cytoplasmic proteins were diluted with 10 mM Tris-HCl, pH 7.8, to 3.7 mg/ml, and 4.6 ml was applied to a Sephacryl S100 HR (bed volume 92 ml) column (1 x 120 cm). The column was equilibrated and eluted with the Tris buffer at 6.0 ml/h flow rate. Three-milliliter fractions were collected, and protein concentrations were determined. Protein was detected in fractions 1131 (samples 525). The samples (57, 1025) were concentrated by vacuum centrifugation. All cytoplasmic samples were incubated with 8 M urea for 1 h at 37 °C, then diluted four times in the bicarbonate buffer (at this point, the protein concentration in different cytoplasmic samples varied from 0.08 to 1 mg/ml) and digested with 0.06 mg/ml trypsin for 15 h at 37 °C.
Digested sample aliquots were reduced with 2 mM dithiothreitol for 1 h and "alkylated" in the dark with 20 mM iodoacetamide for 1 h at 37 °C in the bicarbonate buffer. The reaction was stopped by addition of 200 mM 2-mercaptoethanol.
Mass Spectrometry
Digested samples were analyzed by microcapillary liquid chromatography in line with tandem MS (µLC/MS/MS) using a Surveyor high-performance liquid chromatography (HPLC) system connected to a LCQ DECA XP ion trap mass spectrometer with an electrospray ionization source (ThermoFinnigan, San Jose, CA). Proteins in the tryptic digest (20 µl) were separated by reverse-phase chromatography on a C18 column (2.1 x 150 mm; Thermo Hypersil-Keystone, Bellefonte, PA) at 200 µl/min flow rate. Water and acetonitrile with 0.1% formic acid each were used as solvents A and B, respectively. The gradient was started and kept for 5 min at 5% B, then ramped to 60% B in 110 or 165 min, and finally ramped to 90% B for another 15 min. The eluted peptides, singly, doubly, or triply ionized (charge state 1+, 2+, or 3+, respectively) at the electrospray source, were analyzed in data-dependent MS experiments ("big three" or "triple play") with dynamic exclusion. In the "big three" experiments, the data acquisition parameters were set such that each analytical event consisted of four consecutive scans: the first, full MS (m/z 3002000) scan was followed by three MS/MS scans on the three most intense peptide ions from the full MS spectrum. In the "triple play" experiments, the first, full MS scan was followed by a zoom (high resolution) scan and an MS/MS scan on the most intense ion from the full scan. A peptide ion, analyzed twice within 30 s, was excluded from re-analysis for 2 min. The spray voltage was set at 4.5 kV; the ion transfer capillary temperature was set at 200 °C; and the normalized collision energy for MS/MS decomposition of peptides was set at 35%.
Database Search
A quality MS/MS spectrum, resulting from a peptide fragmentation, features a unique set of b and y fragment ions characteristic to the peptide. The peptide sequence can be identified through interpretation of its MS/MS spectrum and similarity to the MS/MS spectrum of a known peptide sequence. Each acquired MS/MS spectrum was searched against the nonredundant protein sequence database nr.fasta using the SEQUEST software tool (6, 7). The software creates theoretical peptides for all, or a limited group of, database proteins; calculates corresponding MS/MS spectra; and compares them to an experimental spectrum (submitted for the database search) to find the match. The database search was restricted to 7003500 molecular mass tryptic peptides of human (Homo sapiens) origin. Up to two missed trypsin cleavage sites were allowed, and cysteines, where modified, were considered carbamidomethylated. The acceptable molecular mass difference (mass tolerance) between an experimental and database peptides was set to 1.5 mass unit. Mass tolerance for experimental and calculated MS/MS fragment ions was set to zero. Based on the similarity to the experimental MS/MS spectrum, the software assigns each database peptide the primary score (Sp), then the cross-correlation score (Xcorr) to filter candidate peptides and select a defined number of top hits: database peptides with highest Xcorr scores. Finally, a delta cross-correlation score (dCn, a difference between the top 2 Xcorr values normalized to 1) is calculated. The candidate database peptide with the highest Xcorr score was considered the match if the following identification criteria were met: 1) Xcorr of at least 2.0, 2.2, and 3.5 for singly, doubly, and triply charged peptides, respectively, and 2) dCn of at least 0.1 irregardless of charge state.
All identified peptides were grouped under the proteins of their origin. For each identified protein, the number of identified peptides was counted and the percentage of the covered sequence was calculated, and molecular mass was recorded.
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RESULTS |
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To maximize the number of identified proteins, total RBC protein was divided into two major fractions: membrane-associated proteins, including the membrane skeleton, and cytoplasmic proteins. The two fractions were further divided into subfractions, and the proteins were identified in each subfraction separately as tryptic fragments. For membrane subfractions, trypsin digests were collected from externally exposed proteins, internally exposed proteins, spectrin extract consisting mainly of membrane skeleton proteins, and soluble membrane proteins (missing the spectrin extract). Cytoplasmic proteins were divided into 21 subfractions based on molecular mass using size exclusion chromatography. Thus, the complexity of each sample analyzed was minimized and the abundant hemoglobins were separated from the majority of cytoplasmic proteins.
We were able to identify 181 protein sequences, which are organized in Tables I and II. Proteins found in the membrane fractions are presented in Table I with spectrin extract proteins indicated by an asterisk. Proteins found in the cytoplasmic fractions are listed in Table II. Data for each identified protein include protein description assigned by SEQUEST, protein identification number (Gi), percent of the covered amino acid sequence, number of identified peptides, and molecular mass. Ninety one unique sequences are listed in Table I for the plasma membrane fractions and 91 unique sequences are itemized in Table II for the cytoplasmic fractions. As expected, some proteins were found in more than one fraction. For example, spectrin subunits, protein 4.1, and tropomyosin 3 were found in the spectrin extract as well as in other membrane subfractions. The sequence coverage (number of identified peptides) for protein 4.1 or tropomyosin 3 was of comparable level in the spectrin extract and IOV fractions. As for spectrin subunits, the sequence coverage was much higher in the spectrin extract than in the other fractions. Hemoglobin subunits were found in both cytoplasmic and membrane fractions. However, the sequence coverage was much higher in the cytoplasmic fractions than in the membrane fractions. For this reason, hemoglobin subunits are listed only in Table II. Glyceraldehyde-3-phosphate dehydrogenase, on the other hand, is listed in both tables because coverage of its sequence was of comparable level in both the membrane and cytoplasmic fractions.
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We grouped the 181 identified proteins into different categories as summarized in Fig. 1. Number and percent of proteins included in each category as well as protein positions in Tables I and II are presented in Table III. Proteins that are described as similar to X (example similar to tropomyosin 4) are included under unknown proteins in Table III and Fig. 1.
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All 14 identified proteins of the ubiquitin-proteasome system, including six proteasome subunits, were found in the cytoplasmic fractions (Table II). Ubiquitin-activating enzyme was detected with highest sequence coverage (10 identified peptides), followed by polyubiquitin and ubiquitin isopeptidase T with three identified peptides each. All but two of eight identified proteasome subunits were detected with a single peptide.
The category of unknown proteins includes hypothetical proteins whose existence was predicted from the genome sequence; proteins whose existence was shown only at the transcriptome level of different human cell types where the sequences were deduced from cDNAs; and unknown proteins showing similarity to the sequences of known proteins. The majority of the unknown proteins listed in both tables were determined by a single peptide. The primary reasons why some proteins are difficult to identify are low level of expression or extensive post-translational modifications. Nevertheless, we were able to specify 43 unknown proteins, which represents a significant portion (24%) of all the erythrocyte proteins described in Tables I and II.
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DISCUSSION |
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Using gel filtration, we separated the abundant hemoglobins from the majority of the cytoplasmic proteins to facilitate the detection of lower-abundance proteins. Hemoglobin peak fractions, however, would in addition contain a significant number of other cytoplasmic proteins with molecular mass close to that of hemoglobin. In these fractions, where relative abundance of hemoglobin would remain high, many proteins were most likely not detected. An approach that specifically subtracts hemoglobin (for example immunoaffinity) could further expand the analysis of the RBC proteome.
A recent study combining one- and two-dimensional electrophoresis with matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS identified 84 unique RBC membrane proteins (3). They made no attempt to study RBC cytoplasmic proteins. Interestingly, two major RBC glycoproteins, glycophorin A (600,000 copies/RBC) and glycophorin C (
50,000 copies/RBC) (1, 2) were not identified in the recent study (3). Glycosylated transmembrane proteins are known to be underrepresented on one- and two-dimensional gels (16), which makes the µLC/MS/MS technique of great value when trying to obtain a complete proteome analysis.
We believe that our study provides a strong basis for analysis and interpretation of the physiological competence of the RBC and sets the stage for further protein expression and function-based activity profiling not only of normal healthy erythrocytes but also for RBC pathology as well. Indeed, a more thorough proteomic examination involving µLC/MS/MS combined with isoelectrofocusing-SDS-PAGE/MALDI-TOF approaches should afford a means to elucidate the human erythrocyte proteome in its entirety. In turn, a more general understanding and appreciation of the metabolic capability of the RBC and other cells will be realized.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, February 12, 2004, DOI 10.1074/mcp.M300132-MCP200
1 The abbreviations used are: RBC, red blood cell; HPLC, high-performance liquid chromatography; IOV, inside out vesicles; MS, mass spectrometry; LC/MS/MS, liquid chromatography in line with tandem mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; PBS, phosphate-buffered saline.
* This work was supported by a National Institutes of Health Sickle Cell Center Grant (HL070588) Project 1, which was awarded to S. R. G. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Department of Molecular and Cell Biology, Room 3.610, University of Texas at Dallas, 2601 N. Floyd Road, Richardson, TX 75083-0688. Tel.: 972-883-4872; Fax: 972-883-4871; E-mail: sgoodmn{at}utdallas.edu
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REFERENCES |
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