Highly Efficient Depletion Strategy for the Two Most Abundant Erythrocyte Soluble Proteins Improves Proteome Coverage Dramatically Jeffrey H. Ringrose,† Wouter W. van Solinge,‡ Shabaz Mohammed,† Martina C. O’Flaherty,† Richard van Wijk,‡ Albert J. R. Heck,† and Monique Slijper*,† Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands, and Department of Clinical Chemistry and Hematology, Laboratory for Red Blood Cell Research, University Medical Center Utrecht, Utrecht, The Netherlands Received February 7, 2008
Abstract: In-depth human erythrocyte proteome studies are severely hampered by the presence of hemoglobin and carbonic anhydrase-1, which account for more than 98% of the total erythrocyte soluble protein content. We developed a specific depletion approach that resulted in a drastic increase in the number of identified proteins. This depletion technique is valuable for proteome studies of human erythrocyte disorders with unknown etiology and of tissue samples that contain blood. Keywords: human erythrocyte proteome • hemoglobin depletion • carbonic anhydrase-1 depletion • erythrocyte disorder proteomics • IEX-chromatography • Ni(II) chromatography • multi-dimensional liquid chromatography
Introduction The most important function of the erythrocyte is oxygen transport. To travel through narrow capillaries, the erythrocyte is extremely deformable, which is enabled by the loss of cytoplasmic organelles and nucleus. Consequently, any defect in protein function which compromises erythrocyte function or viability cannot be compensated for by new protein synthesis. Several genetic and acquired defects in erythrocyte protein function exist that lead to serious hematological disorders; however, the etiology of many of these disorders is still unknown.1–3 Therefore, it is important to gain more insight into the erythrocyte protein content, both in health and disease. In recent years, a number of studies have been performed using state of the art proteomics methods for in-depth analyses of the erythrocyte proteome,4–8 which has recently been reviewed.9 However, as is the case with plasma and serum, also the erythrocyte proteome is heavily dominated by a very small number of proteins responsible for the bulk protein mass.10 This hampers identification of biologically interesting low expression level proteins. In the case of plasma, sample preparation can be supplemented by several commercially * To whom correspondence should be addressed. Mrs. M. Slijper, Ph.D., Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. Fax, (+31) 30 2518219; e-mail,
[email protected]. † Utrecht University. ‡ University Medical Center Utrecht.
3060 Journal of Proteome Research 2008, 7, 3060–3063 Published on Web 05/22/2008
available methods to deplete for several high-abundance proteins.11,12 No such available approach exists for the specific depletion of hemoglobin (Hb) together with carbonic anhydrase-1 (CA-1), which accounts for approximately 97% and 1% of the erythrocyte proteome, respectively. We exploited the affinity of hemoglobin for Ni(II) to develop the first step of a very efficient depletion method.13 We combined this method with ion exchange (IEX) chromatography to deplete for carbonic anhydrase-1. Using this strategy, we were able to drastically improve the dynamic range of protein detection and the number of identifications in erythrocyte soluble protein samples.
Materials and Methods Preparation of Erythrocyte Soluble Protein Fraction. Packed cells were prepared by passing 20 mL of freshly isolated EDTA-blood from a normal healthy donor over 4 columns containing 5 mL of R-cellulose according to the method of Beutler et al.14 With this method, the sample contained typically 0.01% white blood cells, 1.3% thrombocytes and 1.7% reticulocytes as analyzed by Cell-Dyn 1800 and Cell Dyn Sapphire (Abbott Diagnostics) systems. After washing four times with 100 mM isotonic (310 mOsm) phosphate-buffer, pH 7.4, cells were lysed with 7.6 mM hypotonic (20 mOsm) phosphatebuffer, pH 7.4, according to Dodge et al.15 containing complete protease inhibitors (Roche, Basel, Switzerland) for 1 h at 4 °C. Lysate was centrifuged three times at 20 000g for 10 min and the supernatant represented the erythrocyte soluble protein fraction. Hb Depletion. Typically, 200 mg of erythrocyte soluble protein fraction in 2 mL of buffer containing 50 mM phosphate, pH 8.0, 300 mM NaCl, and 5 mM imidazole was passed over an 8 mL Ni-NTA Super flow (Qiagen, Venlo, Netherlands) column at 38 cm · h-1 at 4 °C. The flow-through containing usually 3 mg of protein represents the Hb-depleted fraction. CA-1 Depletion. IEX chromatography was used for CA-1 depletion by passing 1 mg of hemoglobin-depleted protein sample over in-series connected Mini-Q and Mini-S columns (GE-Healthcare, Diegem, Belgium) in 20 mM Tris, pH 8.0, at 300 cm · h-1. The CA-1-depleted fraction was collected in 150 µL by step-elution with 500 mM NaCl. 10.1021/pr8001029 CCC: $40.75
2008 American Chemical Society
Erythrocyte Soluble Proteins’ Strategy Improves Proteome Coverage In-Gel Digestion. Coomassie-stained SDS-PAGE gels were cut into 20 equal pieces, which were subjected to trypsin digestion as described by Romijn et al.16 LC-MS. Tryptic digests were analyzed using an Agilent 1100Series LC system coupled with an LTQ or hybrid LTQ-FT-ICR (Thermo Electron, Germany) mass spectrometer as described by Krijgsveld et al.17 The LC system was equipped with a 20 mm Aqua C18 (Phenomenex, Torrance, CA) trapping column (packed inhouse, i.d., 50 µm; resin, 5 µm) and a 254 mm ReproSil-Pur C18-AQ (Dr. Maisch GmbH, Ammerbuch, Germany) analytical column (packed in-house, i.d., 50 µm; resin, 3 µm). Trapping was performed at 5 µL min-1 for 10 min, and elution was achieved with a gradient of 0-45% B in 45 min, 45-100% B in 1 min, 100% B for 4 min. The flow rate was passively split from 0.4 mL min-1 to 100 nL min-1. Nanospray was achieved using a coated fused silica emitter (New Objective, Cambridge, MA) (o.d., 360 µm; i.d., 20 µm, tip i.d. 10 µm) biased to 1.7 kV. In the case of the LTQ-FT-ICR, the mass spectrometer was operated in data-dependent mode to switch between MS and MS/MS. Full scan MS spectra were acquired from m/z 350 to m/z 1500 in the FT-ICR with a resolution of R ) 100 000 at m/z 400 after accumulation to a target value of 2 × 106 in the linear ion trap. The two most intense ions were fragmented in the linear ion trap using collisionally induced dissociation at a target value of 10 000. In the case of the LTQ, the mass spectrometer was operated in the data-dependent mode to switch between MS and MS/MS. Survey full scan MS spectra were acquired from m/z 350 to m/z 1500 and the two most intense ions were sequentially isolated for accurate mass measurements by a SIM scan over a 10 Da mass range and accumulation to a target value of 10 000. Database Search and Validation. Spectra were processed with Bioworks 3.4.0 (Thermo, Bremen, Germany) and the MS/ MS data were analyzed with Mascot search engine version 2.2.0 (Matrix Science, London, U.K.) and X! Tandem (www.thegpm.org; version 2006.04.01.2) using the IPI-Human-3.28 and 3.25 databases, respectively, setting carbamidomethyl-cysteine as fixed and oxidized-methionine, deamidation of asparagine and acetylated protein N-termini as variable modifications, allowing two missed cleavages. Peptide tolerance was set to 0.5 Da and MS/MS tolerance to 0.9 Da for the LTQ, and 15 ppm and 0.9 Da for the LTQ-FT-ICR. Scaffold (version 01.06.17, ProteomeSoftware, Portland, OR) was used to probabilistically validate peptide and protein identifications. Peptide identifications were accepted when reaching 95% probability as specified by Peptide Prophet.18 Protein probabilities were assigned by Protein Prophet19 and identifications accepted when reaching 99.9% probability. At identification confidences of 99.9%, the false discovery rates (FDR) for each experiment were determined by Mascot using the automatic decoy function which generates and tests peptides with random sequence of the same length against the IPI-Human-3.28 database. The FDR for the experiments with the nondepleted sample, Hb/CA-1 doubly depleted fraction, the Ni(II) bound fraction, and the IEX flow through fraction were 0.3, 0.6, 0.7, and 0.6%, respectively. Homologous proteins containing similar peptides that could not be differentiated were grouped to satisfy principles of parsimony.
Results and Discussion The main constituent of the erythrocyte soluble protein fraction is hemoglobin (Hb) as is illustrated by a very abundant band representing Hb monomer at approximately 16 kDa in Figure 1 (lane I). Only few other weak bands are visible, which
technical notes
Figure 1. Coomassie-stained SDS-PAGE analysis of the depletion of Hb and CA-1 from the erythrocyte soluble protein fraction. Lane I, total erythrocyte soluble protein fraction (nondepleted start sample); lane II, Ni(II)-column bound fraction, eluted with 100 mM imidazole buffer; lane III, Hb-depleted fraction, collected as flow through of the Ni(II)-column step; lane IV, CA-1 containing fraction, collected as flow through of the IEX chromatography step; lane V, Hb and CA-1 doubly depleted erythrocyte soluble protein fraction, collected in one step via high-salt elution in the course of IEX chromatography. For proper assessment, 30 µg of protein was applied to all lanes.
were shown to represent mainly catalase, carbonic anhydrase-1 and carbonic anhydrase-2 (CA-1 and CA-2), and peroxiredoxin as analyzed by mass spectrometry (Supplementary Table 1). Hemoglobin is a known contaminant in His-tagged protein purification from Hb-containing samples using immobilized nickel (Ni(II)), and moreover a Ni(II) binding site on the Hb beta chain has been identified.13 To take advantage of this knowledge, and to explore if immobilized Ni(II) is suited to efficiently deplete for Hb, Ni(II) affinity chromatography was applied to the erythrocyte soluble protein fraction. It was immediately apparent from the red color of the column that the immobilized Ni(II) retained Hb very efficiently, while the flow through fraction was transparent. Since it is known that other proteins besides Hb exhibit affinity for Ni(II), we eluted all Ni(II) bound proteins with 100 mM imidazole. SDS-PAGE analysis, as illustrated in Figure 1, shows both an efficient depletion of Hb (lane II) and a significant increase in the number of detected bands after Hb-depletion (lane III). Besides the Hb-monomers bands, five other protein bands were visible (Figure 1, lane II). With the use of LTQ-MS/MS, 20 proteins could be identified in this Hb fraction (Supplementary Table 1). However, although Hb-depletion already showed a considerable improvement, another very abundant protein now appeared around 30 kDa, which was identified as CA-1 (Figure 1, lane III). Depletion of this CA-1 from Hb-depleted samples was accomplished using IEX chromatography, where, under appropriate conditions, the CA-1 was detected in the flow through fraction (Figure 1, lane IV). The IEX flow through fraction shown in Figure 1, lane IV, showed 16 protein bands. We could identify 39 proteins in this fraction with LTQ MS/MS analyses (Supplementary Table 1). The IEX-bound fraction was eluted from the columns by a high salt-step and represents the soluble erythrocyte fraction depleted for both Hb and CA-1. As can be seen in Figure 1 (compare lanes I and V), our combined depletion strategy resulted in a considerably increased number of visible protein bands for equal sample quantities, while also well-resolved Journal of Proteome Research • Vol. 7, No. 7, 2008 3061
technical notes
Ringrose et al.
Figure 3. Coomassie-stained SDS-PAGE image showing efficiency of Hb depletion from a mouse lung cell lysate. Lane I, 25 µg of protein of a nondepleted sample; lane II, 25 µg of protein of a Hb-depleted sample. A total of 250 µg of protein was depleted using a 1.5 mL Ni(II) Sepharose column.
Figure 2. Increase in number of detected proteins after depletion of Hb and CA-1. The histogram shows for the nondepleted compared to the doubly depleted sample the total number of identified spectra (black boxes; I), of Hb spectra (gray; II), and number of identified proteins (III). This shows that the total number of identified spectra and proteins significantly increases, while the number of identified Hb-spectra considerably decreases due to efficient depletion of hemoglobin.
protein bands emerge that were obscured by Hb and CA-1 in the start sample. This combined depletion strategy for both Hb and CA-1 was also highly reproducible. To compare both the nondepleted and the doubly depleted samples, equal amounts of erythrocyte soluble protein samples were analyzed by LTQ-FT-ICR MS/MS after SDS-PAGE separation and in-gel tryptic digestion. As is shown in Figure 2, both the number of identified spectra and the number of identified proteins increased dramatically by using our depletion method. Moreover, the number of Hb-spectra decreased to a large extend, indicating that the Ni(II) Sepharose depletion step was very efficient in removing Hb, which accounts for approximately 97% of the erythrocyte soluble protein fraction. The number of identified proteins from our soluble erythrocyte protein sample increased from 167 proteins prior to depletion to 677 proteins after depletion (Supplementary Table 1). In all subfractions together, that is, Ni(II)-bound, IEX-flow through, and Hb/CA-1 depleted fraction, a total of 700 unique proteins were identified (Supplementary Table 1). The here presented double-depletion approach provides thus both an amplification in dynamic range of protein detection and a dramatic increase in the number of identified proteins. To demonstrate the potential of our strategy for other proteome studies that are hampered by substantial quantities of hemoglobin and/or CA-1, we show in Figure 3 the depletion of Hb from a mouse lung cell lysate. As can be concluded from this SDS-PAGE image, for which equal protein quantities were loaded in both lanes, the relative amount of most proteins other than Hb increased significantly after depletion of Hb. In future experiments, the protein molecular background of erythrocyte disorders will be determined. This will be per3062
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formed by assessing differential levels of the erythrocyte proteins, using fluorescent labeling in combination with multidimensional LC methods as described here or by similar protein separation methods. Since our erythrocyte sample contained small amounts of lymphocytes, thrombocytes and reticulocytes (approximately 3% in total, see Material and Methods), some of these identified proteins might be located in these blood cells rather than in erythrocytes. Approaches as described by Pasini et al.7 and Goodman et al.20 may provide cleaner erythrocyte samples. Nevertheless, detected differential proteins related to erythrocyte disorders should always be further validated, using, for example, immunohistochemistry or confocal immuno-fluorescence microscopy. In conclusion, the presented double depletion approach allows a significant higher dynamic range of erythrocyte protein detection, and an over 4 times increase in identified proteins. Our strategy has potential in any study which is hampered by the presence of hemoglobin or carbonic anhydrase-1. Importantly, it opens new perspectives to uncover erythrocyte disorders with to date unknown etiology.
Acknowledgment. This work was supported by The Netherlands Proteomics Centre (www.netherlandsproteomicscentre. nl). Supporting Information Available: Supplementary table of identified soluble proteins from human erythrocytes before and after Hb and CA-1 depletion. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Provan, D.; Weatherall, D. Red cells II: acquired anaemias and polycythaemia. Lancet 2000, 355 (9211), 1260–8. (2) van Wijk, R.; van Solinge, W. W. The energy-less red blood cell is lost: erythrocyte enzyme abnormalities of glycolysis. Blood 2005, 106 (13), 4034–42. (3) Weatherall, D. J.; Provan, A. B. Red cells I: inherited anaemias. Lancet 2000, 355 (9210), 1169–75. (4) Bhattacharya, D.; Mukhopadhyay, D.; Chakrabarti, A. Hemoglobin depletion from red blood cell cytosol reveals new proteins in 2-D gel-based proteomics study. Proteomics: Clin. Appl. 2007, 1 (6), 561–564. (5) Kakhniashvili, D. G.; Bulla, L. A., Jr.; Goodman, S. R. The human erythrocyte proteome: analysis by ion trap mass spectrometry. Mol. Cell. Proteomics 2004, 3 (5), 501–9. (6) Low, T. Y.; Seow, T. K.; Chung, M. C. Separation of human erythrocyte membrane associated proteins with one-dimensional
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