Proteomic Analysis of RBC Membrane Protein Degradation during

Proteomic Analysis of RBC Membrane Protein Degradation during Blood Storage Gian Maria D’Amici, Sara Rinalducci, and Lello Zolla* Department of Environmental Sciences, Tuscia University, Viterbo, Italy Received March 30, 2007

Two-dimensional gel electrophoresis and mass spectrometry were used to identify protein profile changes in red blood cell membranes stored over time under atmospheric oxygen, in the presence or absence of protease inhibitors. New spots with lower molecular masses, ranging between 7 and 15 kDa were observed during the first 7 days storage, while over time, further fragments and highmolecular-mass aggregates appeared, seen as a smearing in the upper part of the gel. Some of the protein changes turned out to be shifts in isoelectric point, as a consequence of chemical oxidations. All these new spots were generated as a result of protein attack by reactive oxygen species (ROS). Protein identification revealed that most of the modified proteins are located in the cytoskeleton. During the first 7 days of storage, oxidative degradation was observed prevalently in band 4.2, to a minor extent in bands 4.1 and 3, and in spectrin. After 14 days, there were new fragments from β-actin, glyceraldehyde-3-phosphate dehydrogenase, band 4.9, and ankyrin, among others. Preliminary proteinprotein cross-linked products, involving R and β spectrin, were also detected. The cross-linked products increased over time. Protein degradation was greatly reduced when oxygen was removed and blood was stored under helium. Interestingly, very few spots were related to enzyme activity, and they were more numerous when oxygen was present, suggesting that some proteases may be oxygen-dependent. Keywords: proteomics • blood storage • Red Blood Cell storage lesion • protein oxidative damage • proteolytic activity • Reactive Oxygen Species

Introduction When whole blood is stored in a preservative medium, there may be morphological, biochemical, and metabolic changes in the red blood cells (RBCs). These changes are collectively known as “storage lesions” and are accompanied by a timedependent decrease in RBC plasticity and concomitant increase in rheological disorders.1 Most of the negative effects of RBC transfusion are associated with these storage lesions, since the RBC cell membrane needs to be flexible to function normally. It is well-known that during storage the morphology of RBCs progressively changes from biconcave disk to spiculated cells (echinocytes); spicules bud off from the echinocyte in the form of microvesicles which contain lipids and haemoglobin (Hb).2,3 The loss in flexibility is only partially reversible and reduces the ability of RBCs to pass through the microcirculatory bed. This leads to a decrease in microvascular flow with local hypoxia and can contribute to hemorrheological disorders in critically ill patients.4,5 “Rejuvenation” solutions have been developed to double the storage life of erythrocytes to up to 12 weeks by doubling the available energy.6 However, the ideal storage protocol has still not been devised, and this is an important priority for manufacturers as well as for blood bank operators. Despite extensive * Corresponding author: Prof. Dr. Lello Zolla, Tuscia University, Largo dell’ Universita` snc, 01100 Viterbo, Italy. Phone, 0039 0761 357 100; fax, 0039 0761 357 630; e-mail, [email protected].

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knowledge of red blood cell metabolism during storage, there are many factors that affect the properties of red blood cells which are still in need of further investigation. Most researchers agree that the etiology of red blood cell membrane lesions appears multifactorial. Two main factors may be involved: active oxygen species and proteolytic enzyme activity. It is wellknown that oxidative mechanisms underlie aging in erythrocytes.7,8 In fact, active oxygen species may be produced from the iron in the heme by the Fenton reaction9 or from neutrophile leukocytes which are involved in free radical production in stored blood.10 During oxidative modification of stored RBCs, glutathione (GSH) and glutathione-peroxidase (GSH-PX) appear to provide the primary antioxidant defense; their decline, concurrent with an increase in oxidative modifications of membrane lipids and proteins, may destabilize the membrane skeleton, thereby compromising RBC survival.11,12 Maintaining cellular reduced glutathione in Adsol-banked RBCs protects membrane proteins from oxidative stress.13 On the other side, proteolytic digestion of membrane proteins may occur either through the action of intracellular enzymes or leukocyte enzymes. It has been reported that the N-terminal cytoplasmic domain of band 3 (CDB3) and spectrin are substrates for the apoptosis executioner caspase 3;14 instead, protein 4.1 and ankyrin are digested by calpain I.15 An increase in haemolysis has been attributed to leukocyte enzymes such as elastase.16 10.1021/pr070179d CCC: $37.00

 2007 American Chemical Society

RBC Protein Fragmentation during Blood Storage

However, it is significant that most studies investigating RBC damage during storage have focused on a small number of proteins, which might be targets for oxidative or proteolytic processes. Wolfe and co-workers demonstrated a progressive oxidative damage of the cytoskeletal protein spectrin,17 while other authors have reported a time-dependent increase in protein clustering and carbonyl modification of band 4.1.11 Damage to human erythrocytes by radiation-generated ‚OH radicals was proven to be induced by molecular changes in the erythrocyte membrane.18 In contrast, few studies have reported use of proteomic techniques to provide a general overview of the effects of storage lesions on RBC membrane proteins. This approach is now possible because in recent years protein separation and structural characterization technologies have improved considerably19 making it possible to reveal any post-oxidative modifications to membrane proteins.20 Recent work in proteomics involving a combination of 2D gel electrophoresis21-23 and 2D nanoHPLC mass spectrometry analysis,24,25 has contributed to the collection of knowledge accumulating on RBC membrane proteins. Only Aminoff et al.26 investigated the variation in pI and Mr values that occurs in aging RBC proteins, but they did not identify the proteins that change. From a clinical point of view, recognizing these changes is important for evaluating factors that may protect RBCs against oxidative damage during storage to improve the quality and shelf life of banked blood. Therefore, the present study was undertaken with the aim of understanding how the RBC proteins change during storage. A proteomic approach was used to detail changes that RBC membrane proteins undergo during 42 days of storage in SAG-M-preserved nonleukodepleted RBC units. Different experiments were performed to differentiate between damage arising from active oxygen species and that due to proteolytic attacks. It was apparent that the time-dependent degradation of membrane proteins was largely a result of oxidative processes.

Materials and Methods Samples. Fresh whole human blood in citrate phosphate dextrose (CPD) anticoagulant was obtained from 10 donors following informed consent. None of the blood donors had a history of diabetes, severe hypertension, or other diseases, which could influence the rheological properties of their blood. Blood from donors was mixed to a final volume of about 800 mL. White blood cell reduction was not performed in order to intentionally include any contribution of leukocytes to proteolytic cleavage and active oxygen species production. After soft spin and manual separation, most of the plasma was removed, and packed RBCs were produced. Subsequently, 25 aliquots of about 30 mL were transferred to 150 mL polyvinyl chloride (PVC) bags (Baxter Healthcare, Round Lake, IL), SAG-M preservative solution (final hematocrit value approximately 0.50) was added to each aliquot, and these were then stored under standard conditions (4 °C).27 Samples of RBCs were removed aseptically for analysis after 0, 7, 14, and 42 days of storage. For studies on proteolytic activity, the Sigma-Aldrich protease inhibitor cocktail for mammalian cells, containing AEBSF, Aprotinin, Bestatin hydrochloride, Pepstatin A, Leupeptin hemisulfate, E-64-[N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide] was used according to manufacturer’s instructions. To differentiate between the effects of oxidative stress and protease activity, packed RBCs were maintained in SAG-M solution for up to 14 days, under helium, at 4 °C, either

research articles in the presence or absence of 1% (v/v) of Sigma-Aldrich protease inhibitor cocktail. Since it is well-known that protein degradation is a random process, it was important to obtain an average figure for the number of spots and calculate its relative variance. Thus, the entire blood sample was first divided into five groups in order to perform five different experiment sets: (i) fresh blood/day 0; (ii) sample stored under helium with protease inhibitors; (iii) sample stored under helium without protease inhibitors; (iv) sample kept in the presence of the protease inhibitor cocktail and without oxygen removal; and (v) sample kept in the absence of the protease inhibitor cocktail and without oxygen removal. Hence, from each group, five separate aliquots and relative electrophoresis runs were provided in an effort to reveal the degree of variation present (Figure 1). For anaerobic storage, oxygen depletion was accomplished by conducting a repetitive gas exchange. Thus, bags containing 30 mL of packed RBCs in SAG-M solution were filled with ultrapure helium and gently agitated horizontally in a 4 °C cold room. The gas in the bag was then expressed out, and the process was repeated five more times. The deoxygenation of haemoglobin was measured spectrophotometrically. RBC Membrane Preparation. Extraction of human erythrocyte membrane proteins was performed based on the conventional method as described by Olivieri et al.28 with some modifications. The erythrocytes were isolated by centrifuging twice at 1000g for 10 min. Packed cells were washed three times in 5 mM phosphate buffer, pH 8.0, containing 0.9% (w/v) NaCl; then, they were centrifuged at 300g for 10 min, at 4 °C. Erythrocytes were lysed with 9 vol of cold 5 mM phosphate buffer, pH 8.0, containing 1 mM EDTA and 1 mM phenylmethanesulfonyl fluoride (PMSF). Membranes were collected by centrifugation at 17 000g, for 20 min at 4 °C, and further washed until free of haemoglobin. Ghosts prepared in this way were used for the following steps. Two-Dimensional Electrophoresis. To remove lipids, proteins were precipitated from a desired volume of each sample with a cold mix of tri-n-butyl phosphate/acetone/methanol (1: 12:1). After incubation at 4 °C for 90 min, the precipitate was pelleted by centrifugation at 2800g, for 20 min at 4 °C. After washing with the same solution, the pellet was air-dried and then solubilized in the focusing solution containing 7 M urea, 2 M thiourea, 2% (w/v) ASB 14, 0.8% (w/v) pH 3-10 carrier ampholyte, 40 mM Tris, 5 mM TBP, 10 mM acrylamide, 0.1 mM EDTA (pH 8.5), 2% (v/v) protease inhibitor cocktail (SigmaAldrich), and 2 mM PMSF. Before focusing, the sample was incubated in this solution for 3 h at room temperature, under strong agitation. To prevent over-alkylation, acrylamide was destroyed by adding an equimolar amount of DTE. A total of 250 µL of the resulting protein solution was then used to rehydrate 13 cm long IPG 3-10 NL (Amersham Biosciences) for 8 h. IEF was carried out on a Multiphor II (Amersham Biosciences) with a maximum current setting of 50 µA/strip at 20 °C. The total product time voltage applied was 40 000 Vh for each strip. For the second dimension, the IPG strips were equilibrated for 30 min in a solution containing 6 M urea, 2% (w/v) SDS, 20% (v/v) glycerol, and 375 mM Tris-HCl (pH 8.8), with gentle agitation. The IPG strips were then laid on a 5-16% T gradient SDS-PAGE gel with 0.5% (w/v) agarose in the cathode buffer (192 mM glycine, 0.1% w/v SDS and Tris to pH 8.3). The anode buffer was 375 mM Tris-HCl, pH 8.8. The electrophoretic run was performed at a constant current (10 Journal of Proteome Research • Vol. 6, No. 8, 2007 3243

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Figure 1. Schematic summary of experimental design. Whole blood from 10 donors was mixed and subsequently divided into 25 aliquots, that is, 5 for each experiment set (fresh blood/day 0: control sample; + He/+ I: sample stored under helium with protease inhibitors; + He/- I: sample stored under helium without protease inhibitors; + I: sample kept in the presence of protease inhibitor cocktail and without oxygen removal; - I: sample kept in the absence of protease inhibitor cocktail and without oxygen removal). 2D electrophoresis gels of RBC membrane proteins extracted from each blood aliquot were run. Total spot number was determined and the arithmetic mean ( standard deviation (SD) was considered.

mA for 60 min, followed by 40 mA until the run was completed). During the whole run, the temperature was set at 13 °C. Proteins were visualized by a double staining procedure: sensitive Coomassie Brilliant Blue G-250 stain29 or Silver staining as described by Chevallet et al.30 Statistical Analysis. Stained gels were digitalized, and image analysis was performed using PDQuest 7.3 software (Bio-Rad, Hercules, CA). Each gel was analyzed for spot detection and background subtraction. Within-group comparison of protein spot numbers was determined by repeated measures analysis; thus, the arithmetic mean of the total spot number ( standard deviation (SD) was considered (see Figure 1). Among-group comparisons were determined by ANOVA (Analysis of Variance) procedure in order to classify sets of proteins that showed a statistically significant difference with a confidence level of 0.05. Moreover, protein spots matching across all the replica maps were selected and analyzed by MS/MS. In-Gel Digestion. Spots from 2D maps were carefully excised from the 2D gel and subjected to in-gel trypsin digestion according to Shevchenko et al.,31 with minor modifications. The gel pieces were swollen in a digestion buffer containing 50 mM NH4HCO3 and 12.5 ng/µL trypsin (modified porcine trypsin, sequencing grade, Promega, Madison, WI) in an ice bath. After 30 min, the supernatant was removed and discarded; then 20 3244

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µL of 50 mM NH4HCO3 was added to the gel pieces, and digestion was allowed to proceed overnight, at 37 °C. The supernatant containing tryptic peptides was dried by vacuum centrifugation. Prior to mass spectrometric analysis, the peptide mixtures were redissolved in 10 µL of 5% FA (formic acid). Peptide Sequencing by NanoRP-HPLC-ESI-MS/MS. Peptide mixtures were separated using a nanoflow-HPLC system (Ultimate; Switchos; Famos; LC Packings, Amsterdam, The Netherlands). A sample volume of 10 µL was loaded by the autosampler onto a homemade 2 cm fused silica precolumn (75 µm i.d.; 375 µm o.d.; Resprosil C18-AQ, 3 µm (AmmerbuchEntringen, DE)) at a flow rate of 2 µL/min. Sequential elution of peptides was accomplished using a flow rate of 200 nL/min and a linear gradient from Solution A (2% acetonitrile and 0.1% formic acid) to 50% of Solution B (98% acetonitrile and 0.1% formic acid) in 40 min over the precolumn in-line with a homemade 10-15 cm resolving column (75 µm i.d.; 375 µm o.d.; Resprosil C18-AQ, 3 µm (Ammerbuch-Entringen, Germany)). Peptides were eluted directly into a high capacity ion trap (model HCTplus Bruker-Daltonik, Germany). Capillary voltage was 1.5-2 kV, and a dry gas flow rate of 10 L/min was used with a temperature of 200 °C. The scan range used was from 300 to 1800 m/z. Protein identification was performed by

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Figure 2. Two-dimensional electrophoresis gels of erythrocyte membrane proteins extracted after 0, 14, and 42 days from blood stored in SAG-M solution under standard conditions (4 °C). No protease inhibitors were added to samples. Maps were stained with silver nitrate. Total protein sample load: 100 µg.

searching in the National Center for Biotechnology Information nonredundant database (NCBInr) using the Mascot program (Matrix Sciences, London, U.K.). The following parameters were adopted for database searches: complete carbamidomethylation of cysteines and partial oxidation of methionines, peptide mass tolerance ( 1.2 Da, fragment mass tolerance ( 0.9 Da, missed cleavages 2. For positive identification, the score of the result of [-10 × Log(P)] had to be over the significance threshold level (P < 0.05). To study post-biosynthetic modifications, peptides were detected by using a quadrupole time-of-flight (Q-TOF) micro hybrid mass spectrometer (model microTOF-Q, Bruker Daltonik, Germany) equipped with a modified ESI-ion source (spray capillary: fused silica capillary, 0.090 mm o.d., 0.020 mm i.d.). The mass spectrometer operated in the positive ion MS mode, and data-dependent analysis was employed for survey scans (m/z 350-1500) to choose up to three most intense precursor ions. For collision-induced dissociation (CID) mass spectrometric (MS/MS) analysis, collision energies were chosen automatically as a function of m/z and charge. The collision gas was argon. The temperature of the heated sample source was 160 °C, and the electrospray voltage was 4000 V. External mass calibration in quadratic regression mode using NaFormiate resulted in mass errors of typically 5-10 ppm in the m/z range 50-2000. Data searching was accomplished with Mascot software (Matrix Sciences, London, U.K.) using the following constraints: only tryptic peptides up to two missed cleavage sites were allowed; 15 ppm tolerance for MS and ( 0.2 Da for MS/MS fragment ions; carbamidomethyl cysteine (C) was specified as a fixed modification, deamidation (NQ) and methionine oxidation (M) were specified as variable modifications. Glutathione Measurements.To evaluate the antioxidant status of the fresh and stored RBCs, GSH concentrations were assayed using procedures described by Raththagala et al.32 This measurement can be performed without any sample preparation beyond isolation of the RBCs from whole blood. RBCs were separated from other formed elements and plasma by centrifugation at 500g at 4 °C, for 10 min. The supernatant and buffy coat were removed by aspiration. Packed RBCs were resuspended and washed three times in 4.7 mM KCl, 2.0 mM CaCl2, 1.2 mM MgSO4, 140.5 mM NaCl, 21.0 mM Tris, and 11.1 mM dextrose with 5% bovine serum albumin, pH 7.4. A calibration curve was calculated by combining 100 µL of a 1.0% hematocrit of control RBCs and varying amounts (0, 4.0, 8.0,

12.0, 16.0, and 20.0 µL) of a 1.0 mM stock solution of standard GSH in separate vials. The reaction mixtures were prepared to a final volume of 1.0 mL with water. Finally, 50 µL of glutathione transferase (GST; 50 units mL-1) and 100 µL of 250 µM monochlorobimane (MCB) were added to the mixtures. Following the incubation period of 10 min, the GSH-MCB fluorescence was measured (ex 370 nm, em 478 nm) for each of the prepared vials. Samples from 14 and 42 storage days were prepared by combining 100 µL of 0.1% hematocrit of RBCs, 900 µL of water, 50 µL of glutathione transferase (GST; 50 units mL-1) and 100 µL of a 250 µM monochlorobimane (MCB).

Results and Discussion It is well-known that the etiology of lesions in red blood cell membranes is multifactorial, involving both active oxygen species and proteolytic enzyme activity. To collect information on the time course of storage lesions and to investigate the relative contributions of protein oxidation and proteolysis to this process, fragmentation of red blood cell membranes was documented by mapping membranes through 2D electrophoresis and protein identification by ESI-MS/MS. The electrophoretic method used33,34 made it possible to detect most of the expected RBC membrane proteins (see later); therefore, reproducible 2D maps were obtained, comparable to those already reported in the literature. Figure 2 shows the silverstained two-dimensional electrophoresis gels of erythrocyte membrane proteins extracted after 0, 14, and 42 days from blood stored in SAG-M solution under standard conditions (4 °C).27 Many protein spots were observed with their number increasing over time. Immediately after drawing the sample, 392 spots were counted, while this value increased to 487 after 14 days of storage and declined to 447 after 42 days. Interestingly, some spots present in the control showed a decrease in intensity of staining over time, whereas other spots showed a decrease in electrophoretic mobility. Both phenomena are usually observed when proteins are exposed to active oxygen species,35,36 suggesting that most lesions could be related to oxidative processes. In fact, the presence of smearing, mobility shift of intrinsic protein bands, aggregate formation, and also protein fragmentation could all be caused by active oxygen species, generated during storage, which modify proteins in the cytoskeleton. Moreover, our data were consistent with previous published works: (i) a gradual increase in the number of spots during the first 14 days of storage was observed, Journal of Proteome Research • Vol. 6, No. 8, 2007 3245

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Figure 3. Two-dimensional electrophoresis gels of maps of the RBC membrane proteins extracted from fresh blood (A), cells stored for 7 (B), and 14 days (C) without removing oxygen and in presence of a specific protease inhibitor cocktail (1% v/v). The numbers shown refer to proteins identified by mass spectrometry and detailed in Tables. The mean number of total spots ( standard deviation is also annotated (upper part, left side). Gels were stained with colloidal Coomassie blue. Total protein sample load: 600 µg.

followed by a decline at 42 days, as already observed by Anniss et al.;37 and (ii) oxidation occurred systematically after 10 days of storage.38 The ultimate aim of our study was to achieve a clear distinction between the damage due to proteolytic enzyme activity and that caused by active oxygen species. To this end, we have conducted two different series of experiments in which: (i) a cocktail of enzyme inhibitors was added to a sample of blood, immediately after it was drawn, and it was stored without removing the oxygen; and (ii) oxygen was first removed from the sample by bubbling helium through it, and this sample was then stored under helium either with or without the addition of enzyme inhibitors. Involvement of Atmospheric Oxygen in Degradation of RBC Membrane Proteins. It was decided to block proteolytic enzyme activity using enzyme inhibitors instead of pre-storage leukocyte depletion, because in this way both intracellular RBC enzymes and those from neutrophyl leukocytes could be inhibited.39 Moreover, to reveal the proteins primarily attacked by active oxygen species, we have focused our investigation on new gel spots observed during the first 14 days of storage, which represent the first steps in the oxidative processes activated during storage, before any chain reactions (arising from an increase in radical species) might have occurred. Figure 3 compares the 2D maps of the RBC membrane proteins extracted from fresh blood (Figure 3A) with those extracted from cells stored for 7 (Figure 3B) or 14 days (Figure 3C). Hence, storage-modulated spots of membrane proteins were cut out of the SDS-PAGE, digested with trypsin, and analyzed by internal peptide sequencing, using mainly RP-HPLC-ESI-MS/ MS. For protein identification by MS, only the differential protein spots constantly present in all replica maps and selected by PDQuest qualitative analysis were considered. The supplemental table reported in Supporting Information gives the protein identification of spots excised from the control map (Figure 3A). We were able to identify most of the proteins we expected to find, indicating that the method of protein extraction used here, as well as the mapping technique, were suitable for red blood cell investigation. Comparison of maps obtained from control or from blood stored for 7 days revealed that some spots showed a decreased staining intensity, while new spots appeared during storage. In particular, in the molecular mass region ranging from 35 up to 100 kDa, most 3246


spots became less abundant, while an initial smearing of some proteins with molecular mass above 130 kDa was detected. Below 60 kDa, new spots, evenly distributed along the pH gradient, were observed at molecular masses ranging between 8000 and 15 000 Da. They may represent fragments from native proteins resulting from active oxygen species attacks. Table 1 lists the MS/MS analyses of spots showing major changes. Four proteins seem to be main targets of ROS attack in the first days of storage: band 4.2, prevalently, band 4.1 and band 3, as well as spectrin, to a minor extent. In detail, our analysis showed the new spot 22 (Figure 3) to be the R-spectrin protein that in the control was found at its molecular mass (spot 01). This indicates that this new spot may be ascribed to a truncated form of spectrin, which has lost some peptides. By analyzing the sequence coverage obtained, it can be seen that all identified peptides are localized at the N-terminal of the protein, suggesting a possible removal of some amino acids at the C-terminal portion. Instead, C-terminal R-spectrin peptides were detected in spot 30. Similarly, spots 20 and 33 contained truncated band 4.1 protein having lower molecular masses than the native one which was present in spot 5 (Figure 3). Interestingly, three different fragments of band 3 were located in: (i) spot 19, containing a band 3 degradation product, lacking 30-35 of the C-terminal amino acids; (ii) spot 31, representing the cytoplasmic domain of the band 3 subunit, and (iii) spot 32, containing a different truncated portion of band 3. Our investigation also detected several new spots having apparent molecular masses close to those of native proteins, but with slightly different isoelectric points. These were, in fact, identified by MS analysis: flotillin 1 protein in spots 07 (control, Figure 3A), 23 and 24 (after 7 days, Figure 3B); aldolase in spots 10 (control, Figure 3A), 27 and 28 (after 7 days, Figure 3B); arginase in spots 11 (control, Figure 3A) and 29 (after 7 days, Figure 3B); β-spectrin in spots 02 (control, Figure 3A) and 18 (after 7 days, Figure 3B). Our observations suggested that a number of amino acids in these proteins had been modified by oxidative processes, resulting prevalently in a more acid isoelectric point of the entire protein without any degradation of the polypeptide chain. In fact, it has been demonstrated that Cys, Met, Trp, His, and Tyr are highly susceptible to oxidation,40 and at least two of these can be charged under physiological conditions (Cys as an anion, and His in a protonated form).

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Table 1. Proteins Identified in 2D Map of RBC after 7 days without Oxygen Removal and in the Presence of Protease Inhibitorsa peptides identified by MS/MS pI predict./exp.

spot

Mr, kDa theor./exp.

18

247.1/227

5.1/5.22

19

102.0/68

5.1/4.94

20

70.2/54

6.3/5.95

21

52.4/54

6.9/7.05

22

281.0/52

4.9/5.90

23

47.5/49

7.1/6.75

m/z

charge state

start-end

sequence

491.86 479.38 458.84 581.91 588.11 651.82 578.93 773.90 767.01 682.38 422.34 752.38 748.94 602.84 702.41 586.38 767.39 599.89 888.53 480.83 588.88 647.43 568.91 715.92 649.39 454.34 805.94 751.11 782.43 681.48 733.94 745.96 938.96 475.28 728.93 527.81 802.85 563.31 546.84 731.89 586.36 617.42 517.33 561.82 492.36 556.36 494.41 856.52 1013.01 718.40 366.41 635.96 571.93 789.96 557.40 569.86 677.53 516.83 658.91 451.34 479.29 679.05 626.34 635.41 600.38 529.37 552.21 485.87 703.49 920.04 562.36 442.88 828.45 734.92 575.84 640.39

(2+) (3+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+)

63-70 232-243 380-387 435-443 463-477 572-583 668-677 765-779 828-840 936-946 1003-1010 1017-1035 1294-1306 1360-1369 1404-1416 1460-1469 1486-1497 1559-1568 1647-1663 1738-1746 1747-1756 1887-1907 1934-1943 1944-1955 2007-2016 2057-2064 57-69 57-74 139-150 161-180 220-233 234-246 247-263 284-292 293-304 296-304 347-360 543-551 412-420 492-505 601-610 77-87 99-107 156-164 165-172 165-173 173-180 213-227 228-247 228-248 277-282 283-295 284-295 296-309 300-309 363-371 375-386 7-16 17-27 28-34 35-41 73-90 80-90 94-104 239-248 259-268 312-321 19-28 59-72 110-124 125-133 127-133 153-166 199-211 212-220 220-230

WVNSHLAR HNLEHAFNVAER VYTPHDGK ETWLSENQR KHEAIETDTAAYEER LMEADIAIQGDK DLTSVLILQR LLSGEDVGQDEGATR ELYQQVVAQADLR WQAFQTLVSER DVAAIQAR ESQQLMDSHPEQKEDIGQR LLTSQDVSYDEAR LWDELQATTK SDDPGKDLTSVNR FLDLLEPLGR DLEDETLWVEER LGHLQSSWDR AQGLLSAGHPEGEQIIR ETGAIGQER VDNVNAFIER TQLVDTADKFR DVSSVELLMKb YHQGINAEIETR MLLEVCQFSRb STASWAER VYVELQELVMDEKb VYVELQELVMDEKNQELRb FIFEDQIRPQDR HSHAGELEALGGVKPAVLTR IPPDSEATLVLVGR ADFLEQPVLGFVR LQEAAELEAVELPVPIR AAATLMSER VFRIDAYMAQSR IDAYMAQSR YQSPOTAKPDSSFYK IFQDHPLQK LDGENIYIR TQTVTISDNADAVKd EQHPDMSVTK VTEEPMGITLKb ILHGGMIHR LPALQMFMRb AQFDYDPK AQFDYDPKK KDNLIPCK ESAGLIPSPELQEWR VASMAQSAPSEAPSCSPFGKb VASMAQSAPSEAPSCSPFGKKb LPAFKR KTLVLIGASGVGR TLVLIGASGVGR SHIKNALLSQNPEK NALLSQNPEK FETVHQIHK IAILDIEPQTLK ETVVESSGPK VLETAEEIQER RQEVLTR YQSFKER VNILTDKSYEDPTNIQGK SYEDPTNIQGK HQSLEAEVQTK QNEVNAAWERc ALSNAANLQR NLAVMSDKVK SPPVMVAGGR HGVPISVTGIAQVK AIMAHMTVEEIYKDRb QKFSEQVFKc FSEQVFK DIHDDQDYLHSLGK VSAQYLSEIEMAK AQRDYELKK KAAYDIEVNTR

Mascot score

NCBI accession number

1353

gi|67782319

spectrin beta

845

gi|4507021

Erythrocyte membrane protein band 3

172

gi|110430928

EPB41 protein

907

gi|4505237

palmitoylated membrane protein

509

gi|115298659

spectrin alpha

gi|5031699

flotillin 1

1622

protein ID [Homo sapiens]

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Table 1 (Continued) peptides identified by MS/MS

spot

Mr, kDa theor./exp.

pI predict./exp.

24

47.5/49

7.1/7.55

25

78.9/47

5.3/5.95

26

77.7/48

8.3/6.08

27

39.7/44

8.3/7.46

28

39.7/44

8.3/7.53

29

34.8/39

7.1/6.94

30

281.0/43

4.9/4.75

31

42.6/42

4.4/4.29

29

3248

m/z

charge state

start-end

sequence

576.35 787.96 709.94 478.89 735.46 542.64 810.39 689.92 546.32 552.83 690.41 608.41 415.36 550.71 485.83 380.98 442.88 734.95 640.89 576.36 709.96 623.40 478.85 735.46 690.00 546.32 698.43 616.90 718.92 550.37 616.98 499.32 380.64 676.35 747.66 544.33 717.90 781.94 470.86 666.96 744.97 549.71 672.44 758.04 382.35 401.37 547.34 566.88 667.24 523.00 703.33 552.40 557.35 621.39 379.83 587.93 643.35 582.31 416.29 811.47 585.67 556.33 623.34 784.78 621.34 563.88 710.40 788.45 734.42 865.97 544.83 693.67 495.30 557.83 805.94 751.11 782.43

(2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (3+) (2+)

221-230 231-244 232-244 254-261 262-274 262-275 303-317 318-330 320-330 331-340 378-392 393-403 404-410 411-425 19-28 125-133 127-133 199-211 220-230 221-230 232-244 247-261 254-261 262-274 318-330 320-330 378-392 78-88 174-187 188-197 289-305 371-378 379-384 288-301 302-320 555-563 2-13 2-14 15-22 29-42 29-43 44-57 88-99 112-134 209-215 305-312 323-331 332-342 29-42 61-69 154-173 7-17 22-32 22-33 33-39 72-83 181-191 197-205 285-291 309-322 2080-2094 2133-2141 2133-2147 2133-2151 2142-2151 2178-2187 2188-2200 2188-2201 2252-2263 2267-2281 2282-2290 2302-2318 2322-2330 2369-2377 57-69 57-74 139-150

AAYDIEVNTR RAQADLAYQLQVAK AQADLAYQLQVAK VQVQVVER AQQVAVQEQEIAR AQQVAVQEQEIARR SQLIMQAEAEAASVRb MRGEAEAFAIGAR GEAEAFAIGAR ARAEAEQMAK ITLVSSGSGTMGAAK VTGEVLDILTR LPESVER LTGVSISQVNHKPLR SPPVMVAGGR QKFSEQVFK FSEQVFK VSAQYLSEIEMAK KAAYDIEVNTR AAYDIEVNTR AQADLAYQLQVAK QQIEEQRVQVQVVER VQVQVVER AQQVAVQEQEIAR MRGEAEAFAIGAR GEAEAFAIGAR ITLVSSGSGTMGAAKb GNNSSNIWALR GVSCSEVTASSLIK VNILGEVVEK NHGVVALGDTVEEAFYK MLDNLGYRb TGYTYR VVTTFASAQGTGGR LLIDEYYNEEGLQNGEGQR NPPENTFLR PYQYPALTPEQK PYQYPALTPEQKK ELSDIAHR GILAADESTGSIAK GILAADESTGSIAKR LQSIGTENTEENRR ADDGRPFPQVIK GVVPLAGTNGETTTQGLDGLSER VLAAVYK ALQASALK AAQEEYVKR ALANSLACQGK GILAADESTGSIAK QLLLTADDR IGEHTPSALAIMENANVLAR TIGIIGAPFSK GGVEEGPTVLR GGVEEGPTVLRK KAGLLEK SVGKASEQLAGK DVDPGEHYILK YFSMTEVDRb TPEEVTR EGNHKPIDYLNPPK QLQKDHEDFLASLARc HLSDIIEER HLSDIIEEREQELQK HLSDIIEEREQELQKEEAR EQELQKEEAR AYFLDGSLLK ETGTLESQLEANK ETGTLESQLEANKR MQHNLEQQIQAK GVSEETLKEFSTIYK HFDENLTGR GLNYYLPMVEEDEHEPK FLDAVDPGR SYITKEDMK VYVELQELVMDEKb VYVELQELVMDEKNQELRb FIFEDQIRPQDR


Mascot score



816

gi|5031699

flotillin 1

273

gi|34785151

ADD2 protein

252

gi|112798

Erythrocyte membrane protein band 4.2

677

gi|28614

aldolase A

161

gi|28614

aldolase A

434

gi|178995

arginase

773

gi|115298659

spectrin alpha

745

gi|14277739

Cytoplasmic Domain of Human Erythrocyte Band-3 Protein

research articles

RBC Protein Fragmentation during Blood Storage Table 1 (Continued) peptides identified by MS/MS

spot

Mr, kDa theor./exp.

pI predict./exp.

32

42.6/31

4.4/4.1

33

70.2/25

6.3/6.00

34

36.2/20

8.2/8.9

35

77.7/21

8.3/6.03

36

77.7/17

8.3/6.37

37

77.7/17

8.3/6.21

38

15.9/14

6.8/6.4

m/z

charge state

start-end

sequence

681.48 733.94 745.95 938.97 475.28 728.94 527.81 751.12 681.66 733.95 745.93 337.24 379.77 700.41 636.96 581.85 403.71 807.64 544.31 477.67 544.34 669.84 715.94 536.81 544.37 440.70 715.92 657.89

(3+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (3+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+)

161-180 220-233 234-246 247-263 284-292 293-304 296-304 57-74 161-180 220-233 234-246 23-28 102-108 146-157 167-183 184-193 6-13 67-80 555-563 652-663 555-563 641-651 652-663 681-691 555-563 629-635 652-663 18-30

HSHAGELEALGGVKPAVLTR IPPDSEATLVLVGR ADFLEQPVLGFVR LQEAAELEAVELPVPIR AAATLMSER VFRIDAYMAQSR IDAYMAQSR VYVELQELVMDEKNQELRb HSHAGELEALGGVKPAVLTR IPPDSEATLVLVGR ADFLEQPVLGFVR GQDLLK QDIVAGR LAPNQTKELEEK SMTPAQADLEFLENAKKb LSMYGVDLHK VGVDGFGRd LVINGNPITIFQER NPPENTFLR FQFTPTHVGLQR NPPENTFLR SVWPENTMCAKb FQFTPTHVGLQR SVTVVAPELSA NPPENTFLR GLIHRER FQFTPTHVGLQR VNVDEVGGEALGR

Mascot score


270

gi|14277739


197

gi|110430928

EPB41 protein

136

gi|31645

96

gi|112798

217

gi|112798

glyceraldehyde-3-phosphate dehydrogenase Erythrocyte membrane protein band 4.2 Erythrocyte membrane protein band 4.2

169

gi|112798

Erythrocyte membrane protein band 4.2

gi|56749856

Hemoglobin subunit beta

81


a Only the spots matching across all the replica maps were selected and analyzed by MS/MS. Spot ID number relates to numbers shown in Figure 3B. The theoretical and experimental values of molecular weight and isoelectric point, the MS/MS data, the Mascot score, the NCBI accession number, and the protein name are also reported. b Peptide with oxidation of methionine (M). c Peptide with Pyro-glu modification from glutamine (Q). d Peptide with deamidation of asparagine.

Oxidation of either or both of these could result in an overall change in charge. It is worth pointing out that our MS/MS analyses revealed a deamidation site for band 4.1 at the level of Asn502 occurring in a fragment product of this protein obtained after 7 days of RBC storage (spot 20, see Table 1). Interestingly, this deamidation site, combined with Asn478, was previously characterized and related to the physiological aging of red cells in circulation.41 In fact, it is well-known that membrane proteins are also the major targets of post-biosynthetic alterations during erythrocyte aging in response to oxidative stress. In particular, deamidation of asparagine residues and isomerization of aspartate residues have been shown to be involved in the molecular damage of cytoskeletal components, such as band 4.1, band 4.2, ankyrin, and of the integral membrane protein band 3.42 Curiously, we also identified a previously unknown deamidation site for glyceraldehyde-3-phosphate dehydrogenase (Asn9, see spot 34, Table 1) that could be related to oxidative stress. Thus, storage of erythrocytes in the presence of oxygen and protease inhibitors induces some protein modifications which were further investigated by mapping cells after 14 days of storage times. As matter of fact, analysis of spots detected after 14 days showed the presence of additional fragments and the formation of aggregates (see Figure 3 and Table 2). Looking at the new degradation products arising from band 4.1, there were other fragments of the same protein (spots 44, 49, and 53), in addition to spots 20 and 33, previously revealed at a shorter storage time. Moreover, at 14 days, previously undamaged proteins appear to be undergoing fragmentation, as we detected the following new degradation

products: β-actin (spots 46 and 52), glyceraldehyde-3-phosphate dehydrogenase (spot 43), ankyrin (spot 41), and band 4.9 (spots 42 and 45). At high molecular mass, our analysis revealed the presence of both R- and β-spectrin in spot 39, as an aggregation product. Results from a previous study are in agreement with this analysis; generation of protein-protein cross-linkages of spectrin, ankyrin, actin, band 3, band 4.1 protein, and glycophorin was observed as a result of protein oxidation.43-45 On this subject, Caprari et al.46 showed that tert-butyl hydroperoxide (t-BHP) induces several kinds of damage, including partial degradation of spectrin and ankyrin with simultaneous HMWA formation, an increase in membrane-bound globin, and the appearance of low molecular weight products.47 Spectrin alterations, its degradation, or irreversible polymerization due to cross-linking cause a redistribution-marginalization of spectrin that was associated with changes in the structure of the junction complex of the membrane skeleton.46 Apparently, aggregation of proteins occurs as a final stage of oxidative degradation of the membrane skeleton of erythrocytes, which is accompanied by global conformational restructuring of protein domains in the membrane skeleton. Thus, it is not surprising that preliminary high molecular weight cross-linked derivates were observed after 14 days of storage and their amounts increased considerably over time (see Figure 2C, 42 days). In agreement, Wolfe et al. showed progressive oxidative damage of the cytoskeletal protein spectrin48 during the 42 days of storage. This spectrin damage affects a correct spectrinactin-protein 4.1 complex assembling and was correlated with a decline in total RBC phospholipid content, with membrane vesiculation, and loss of RBC surface area.49 Journal of Proteome Research • Vol. 6, No. 8, 2007 3249

research articles

D’Amici et al.

Table 2. Proteins Identified in 2D Map of RBC after 14 days without Oxygen Removal and in the Presence of Protease Inhibitorsa peptides identified by MS/MS

spot

Mr, kDa theor./exp.

39

247.1/280

pI predict./exp. 5.15/5.10

40

247.1/150

5.15/4.91

41

207.1/45

5.82/7.42

42

45.6/41

8.9/7.57

3250

Mascot score


VQLIEDR LLTSQDVSYDEAR LWDELQATTK SDDPGKDLTSVNR RVEDQVNVR FLDLLEPLGR DLEDETLWVEER NQTLQNEILGHTPR LGHLQSSWDR AQGLLSAGHPEGEQIIR LQGQVDKHYAGLKDVAEER ETDDLEQWISEK ELVASPOTEMGQDFDHVTLLRb ETGAIGQER ETGAIGQERVDNVNAFIER VDNVNAFIER MQLLAASYDLHR LQTAYAGEK TQLVDTADKFR DVSSVELLMKb YHQGINAEIETR MLLEVCQFSRb STASWAER FAALEKPTTLELK

1278

gi|67782319

spectrin beta

SDDKSSLDSLEALMKb ALKAQLIDER HQTFAHEVDGR DLQGVQNLLK AAVGQEEIQLR LAQFVEHWEK HEALENDFAVHETR VQNVCAQGEDILNK VLQEESQNKEISSK IEALNEKTPSLAK QDTLDASLQSFQQER LPEITDLKDK WEQLLEASAVHR AEDLFVEFAHK DHEDFLASLAR CLLELDQQIK AYFLDGSLLK MQHNLEQQIQAK HFDENLTGR FLDAVDPGR SSDEIENAFQALAEGK SYITKEDMKb VQLIEDR AQEASVLLR LLTSQDVSYDEAR LWDELQATTK SDDPGKDLTSVNR RVEDQVNVR FLDLLEPLGR NQTLQNEILGHTRP LGHLQSSWDR AQGLLSAGHPEGEQIIR LQGQVDKHYAGLKDVAEER VDNVNAFIER TQLVDTADKFR DVSSVELMKb YHQGINAEIETR STASWAER FAALEKPTTLELK EADAATSFLR EIILETTTK KGNTALHIAALAGQDEVVR GNTALHIAALAGQDEVVR LPALHIAAR TAAVLLQNDPNPDVLSK GSSVNFTPQNGITPLHIASR ISEILLDHGAPIQAK DLAAIPKDK STSPPPSPEVWADSR SPGIISQASAPR TSLPHFHHPETSRPDSNIYK

1304

gi|115298659

spectrin alpha

1042

gi|67782319

spectrin beta

378

gi|178646

Ankyrin

674

pi|22654240

Dematin (Erythrocyte membrane protein band 4.9)

m/z

charge state

start-end

sequence

436.84 748.93 602.89 702.37 557.87 586.65 767.43 810.98 599.84 888.53 719.45 746.93 754.13 480.84 706.76 588.86 709.35 490.84 647.42 568.89 715.89 649.85 454.34 730.47

(2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (3+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+)

1249-1255 1294-1306 1360-1369 1404-1416 1423-1431 1460-1469 1486-1497 1520-1533 1559-1568 1647-1663 1664-1682 1698-1709 1710-1729 1738-1746 1738-1756 1747-1756 1790-1801 1862-1870 1897-1907 1934-1943 1944-1955 2007-2016 2057-2064 2065-2077

827.95 570.90 648.83 564.41 607.41 643.91 556.62 794.43 809.95 707.44 883.35 586.45 719.94 653.59 637.38 629.94 563.84 734.37 544.83 495.36 854.95 565.85 436.82 494.37 748.92 602.90 702.43 557.54 586.97 810.54 599.85 888.53 719.42 588.88 647.41 568.89 715.92 454.30 730.49 540.48 524.03 654.93 612.03 480.98 897.67 699.52 535.66 485.87 807.44 592.45 788.41

(2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (3+) (2+)1 (2+) (3+) (3+) (2+) (2+) (2+) (3+)

1420-1424 1484-1493 1541-1551 1744-1753 1783-1793 1794-1803 1861-1874 1875-1888 1889-1902 1903-1915 1970-1984 1985-1994 2017-2028 2041-2051 2084-2094 2101-2110 2178-2187 2252-2263 2282-2290 2322-2330 2353-2368 2369-2377 1249-1255 1263-1271 1294-1306 1360-1369 1404-1416 1423-1431 1460-1469 1520-1533 1559-1568 1647-1663 1664-1682 1747-1756 1897-1907 1934-1943 1944-1955 2057-2064 2065-2077 9-18 68-76 77-95 78-95 74-182 188-204 229-248 287-301 43-51 90-104 105-116 123-142



research articles

RBC Protein Fragmentation during Blood Storage Table 2 (Continued) peptides identified by MS/MS

spot

Mr, kDa theor./exp.

pI predict./exp.

43

36.2/33

8.2/6.84

44

70.2/28

6.3/6.21

45

45.6/31

8.9/7.71

46

42.0/28

5.3/5.79

47

40.6/27

5.0/8.89

48

44.4/26

5.6/6.69

49

70.2/24

6.3/5.15

50

42.6/21

4.4/4.69

51

247.1/20

5.15/7.75

m/z

charge state

Mascot score

start-end

sequence

495.32 739.36 436.85 583.86 670.41 720.91 469.37 491.89 531.38 621.41 749.11 917.46 871.00 706.50 406.28 765.8 724.46 882.47 617.32 665.95 700.45 379.26 637.35 589.87 469.22 491.81 531.30 566.84 581.36 750.39 675.11 607.36 496.03 807.44 454.35 769.35 636.76 861.95 669.35 584.03 741.37 569.46 509.73 457.73 515.88 565.43 436.97 795.46 731.40 604.43 570.35 809.00 697.94 885.01 583.41 659.93 700.44 692.89 378.83 890.97 637.06 645.89 581.89 806.02 751.16 782.47 681.46 475.29 490.86 573.70 647.43 681.82 560.94 715.89 549.22 649.52 454.36

(2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (3+) (3+) (2+) (2+) (3+) (2+) (2+) (3+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (3+) (2+) (2+) (2+) (3+) (2+) (3+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+)

151-160 173-186 237-243 251-259 269-286 274-286 287-295 356-364 370-378 379-389 119-139 146-162 198-215 201-215 228-234 235-248 249-260 310-323 324-334 324-335 146-157 158-163 167-183 184-193 287-295 356-364 370-378 197-206 316-326 360-372 190-206 197-206 215-227 215-228 228-235 236-249 236-252 272-286 315-325 326-340 328-340 344-354 47-60 48-60 51-60 100-109 102-109 110-124 111-124 155-164 165-173 202-216 217-228 5-19 20-29 54-64 146-157 153-163 158-163 167-182 167-183 183-193 184-193 57-69 57-74 139-150 161-180 284-292 1862-1870 1896-1905 1897-1907 1914-1924 1934-1943 1944-1955 1973-1981 2007-2016 2057-2064

ESVGGSPQTK FPAAQPPDPNQPAK QREELSKc MILKEEMEKb SLPDRTPFHTSLHQGTSK TPFHTSLHQGTSK SSSLPAYGR TKLPPGVDR HLSAEDFSR VFAMSPEEFGK VIISAPSADAPMFVMGVNHEKb IISNASCTTNCLAPLAK DGRGALQNIIPASTGAAK GALQNIIPASTGAAK LTGMAFRb VPTANVSVVDLTCR LEKPAKYDDIKK LISWYDNEFGYSNR VVDLMAHMASKb VVDLMAHMASKE LAPNQTKELEEK VMELHK SMTPAQADLEFLENAKKb LSMYGVDLHKb SSSLPAYGR TKLPPGVDR HLSAEDFSR GYSFTTTAER EITALAPSTMK QEYDESGPSIVHRc IKNNDPKLEEVNLNNIR LEEVNLNNIR AYAEALKENSYVK AYAEALKENSYVKK KFSIVGTR SNDPVAYALAEMLKb SNDPVAYALAEMLKENKb LVEALPYNTSLVEMKb FGYHFTQQGPR LRASNAMMNNNDLVRb ASNAMMNNNDLVRb LADLTGPIIPK RLVKGPDPSPOTAFR LVKGPDPSPOTAFR GPDPSPOTAFR TRGTLLALER GTLLALER KDHSGQVFSVVSNGK DHSGQVFSVVSNGK SITLFVQEDR AQLYIDCEK GGVNDNFQGVLQNVR FVFGTTPEDILR VSLLDDTVYECVVEK HAKGQDLLKR TWLDSAKEIKK LAPNQTKELEEK ELEEKVMELHK VMELHK SMTPAQADLEFLENAK SMTPAQADLEFLENAKK KLSMYGVDLHK LSMYGVDLHK VYVELQELVMDEKb VYVELQELVMDEKNQELRb FIFEDQIRPQDR HSHAGELEALGGVKPAVLTR AAATLMSER LQTAYAGEK RTQLVDTADK TQLVDTADKFR DLLSWMESIIR DVSSVELLMK YHQGINAEIETR QHQASEEIR MLLEVCQFSRb STASWAER



567

gi|31645

glyceraldehyde-3-phosphate dehydrogenase

141

gi|110430928

EPB41 protein

123

gi|22654240

dematin (Erythrocyte membrane protein band 4.9)

225

gi|4501885

beta actin

668

gi|4507553

tropomodulin 1

700

gi|538354

thrombospondin

605

gi|110430928

EPB41 protein

334

gi|14277739


455

gi|67782319

spectrin beta


research articles

D’Amici et al.

Table 2 (Continued) peptides identified by MS/MS

spot

Mr, kDa theor./exp.

pI predict./exp.

52

42.0/10

5.3/9.5

53

70.2/24

6.3/6.3

m/z

charge state

start-end

sequence

527.47 581.37 398.35 750.39 659.69 379.74 700.40 589.89

(2+) (2+) (2+) (2+) (2+) (2+) (2+) (2+)

313-326 316-326 329-335 360-372 54-64 102-108 146-157 184-193

MQKEITALAPSTMKb EITALAPSTMK IIAPPER QEYDESGPSIVHRc TWLDSAKEIKK QDIVAGR LAPNQTKELEEK LSMYGVDLHKb

Mascot score



177

gi|4501885

beta actin

172

gi|110430928

EPB41 protein

a Only the spots matching across all the replica maps were selected and analyzed by MS/MS. Spot ID number relates to numbers shown in Figure 3C. The theoretical and experimental values of molecular weight and isoelectric point, the MS/MS data, the Mascot score, the NCBI accession number, and the protein name are also reported. b Peptide with oxidation of methionine (M). c Peptide with Pyro-glu modification from glutamine (Q).

Figure 4. Two-dimensional electrophoresis gels of erythrocyte membrane proteins extracted from blood sample after oxygen removal, and storage under saturating conditions of helium, either with (A) or without (B) addition of protease inhibitor cocktail. The mean number of total spots ( standard deviation is shown (upper part, left side).+ He indicates that samples are stored under helium; + I indicates that a cocktail of protease inhibitors is added to samples; - I means that no enzyme inhibitors are present. Refer to Figure 3 legend for staining and protein loading conditions.

Damage Extent of RBC Membrane Proteins under Anaerobic Conditions. To ascertain whether oxygen plays a major role in protein degradation and just how much of this fragmentation is exclusively due to proteolytic enzymes, we decided to remove oxygen from freshly drawn blood by bubbling helium through and preserving the red blood cells under saturating helium either in the presence or the absence of enzyme inhibitors. We preferred to remove oxygen instead of adding chemical scavengers, since it is well-known that the latter only work in the vicinity of radical production and are quite specific. Thus, 2D gels of proteins extracted from blood samples containing protease inhibitors kept for 7 days under saturating conditions of helium (Figure 4A) were compared with those where oxygen was not removed (Figure 3B). Analysis revealed a spot mean number of 129.4 ( 3.44 (µ ( SD) in the sample under helium; this value was lower than that recorded in the presence of oxygen (161.00 ( 4.85) and very close to that observed for fresh blood (126.60 ( 2.07). This allows one to hypothesize that by eliminating oxygen and blocking most proteases, fragmentation is highly reduced, at least during the first week of storage. To reveal those spots due to enzyme attacks, a blood sample was stored under helium in the absence of enzyme inhibitors. 3252


Thus, proteins extracted from membranes after 7 days of storage under helium were compared to highlight pattern changes associated to the presence of inhibitors (see Figure 4). An extra 7 protein spots were observed, suggesting that a small number of changes were related to protein cleavages. Among these spots, the most abundant were selected and analyzed by MS/MS revealing that they contained fragments derived from band 3 (spots 54 and 55, Figure 3C), glyceraldehyde-3-phosphate dehydrogenase (spot 56), and band 4.1 (spot 57), in agreement with previous works.14-16 Evaluation of the Relative Importance of Factors Contributing to RBC Storage Lesions. Figure 5 displays the total spots counted in 2D gel electrophoresis performed with or without oxygen removal when enzyme inhibitors are present or absent in blood stored for 7 and 14 days. It can be seen that, although significant variability was recorded within each experiment due to the random nature of oxidative processes, in the presence of oxygen, the total spot number is higher, allowing one to surmise that protein oxidization is the main phenomenon in storage lesions, while proteolytic cleavage plays a minor role. Interestingly, when we compared protein spot profiles resulting from enzyme activity in the presence or absence of oxygen,

research articles


recorded in our 2D maps. However, attempts to maintain cellularly reduced glutathione in banked RBC is of limited use, as it only partially protects membrane proteins from oxidative stress.11,13 Similarly, any effort to increase the anti-oxidative capacity of RBC during storage by acting on the anti-oxidant protein activity with which RBCs are well-endowed (such as peroxiredoxin, catalase, and glutathione peroxidase) becomes superfluous when oxygen can be removed and any active oxygen species production is consequently avoided.

Concluding Remarks

Figure 5. Total spots counted in 2D gel electrophoresis performed with or without oxygen removal when enzyme inhibitors are present or absent in blood stored for 7 and 14 days. Results shown are mean ( SD (values refer to ones listed in Figure 1). Symbol meanings as in Figure 4.

there were more spots when oxygen was present than when oxygen is absent, indicating that some proteases seem to be oxygen-dependent. This agrees with recent experimental observations that proteases, such as caspase 2 and 3, are activated in oxidatively stressed erythrocytes.50,51 In this scenario, ROSinduced modifications may produce protein and phospholipid surface remodeling as well as intracytoplasmic network changes by increasing their susceptibility to proteases. Thus, it may be hypothesized that oxidative fragmentation initiates protein degradation while proteases complete the process. The phenomenon is initially reduced by the presence of glutathione inside the erythrocyte, but as its concentration decreases, protein fragmentation increases significantly.11 By using a nondestructive method, which does not require separation of glutathione from the complex cellular matrix,32 we were able to correlate the decrease of glutathione in our cells with the total number of spots revealed in the 2D map (Figure 6). It can be seen that corresponding to a decrease in glutathione there is an exponential increase in the total number of spots

The investigation reported here and carried out using proteomic analysis is the first overview of the changes that occur in the RBC cytoskeleton during blood storage. Only through the use of MS/MS has it been possible to recognize that some apparently new proteins are only truncated forms, fragments, or aggregates of native proteins. ROS produced prevalently from oxy-haemoglobin during storage, through an initial oxidation of amino acid residues with consequent protein fragmentation and/or aggregation phenomena, cause the wellknown morphologic, biochemical, and metabolic changes in RBCs. Consistent with this, most of the affected proteins investigated here are located in the cytoskeleton. In line with a recent paper,52 our results also showed that oxygen removal is a more effective way of limiting RBC storage lesions than any chemical addition. Although oxygen removal could be obtained by bubbling N2, which is less expensive than helium, the possible presence of some nitric oxide contamination, which reacts quickly with superoxide, giving peroxynitrite, could give the opposite effect. In fact, it has recently been reported that peroxynitrite51 can hijack human RBCs leading them toward senescence and apoptosis by inducing proteases. Moreover, since this study has made it apparent that a small number of changes are related to proteolytic cleavages, addition of chemical inhibitors during storage should be avoided, since as yet there is no information on RBC function or viability. In contrast, since the presence of leukocytes is well-known to have adverse affects on the quality of blood products and potentially contributes to the mechanisms involved in RBC storage lesions as well as in active oxygen species formation,10 pre-storage

Figure 6. Comparison of glutathione level and number of protein spots (refer to Figure 2 maps) during storage days. GSH was determined as given in Materials and Methods, and its concentration was expressed as micromoles per milliliter of RBCs. Journal of Proteome Research • Vol. 6, No. 8, 2007 3253

research articles leukocyte-depleted blood is advisable before removing oxygen for extended storage. Abbreviations: ASB 14, 3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate; CDB3, N-terminal cytoplasmic domain of band 3; CID, collision-induced dissociation; CPD, citrate phosphate dextrose; GSH, glutathione; GSH-PX, glutathione-peroxidase; Hb, haemoglobin; HMWA, high molecular weight aggregate; PMSF, phenylmethanesulfonyl fluoride; RBCs, red blood cells; ROS, reactive oxygen species; SAGM, saline-adenine-glucose-mannitol; TBP, tributylphosphine.

Acknowledgment. We thank Anna Maria Timperio and Marco Fagioni for their invaluable assistance. We also acknowledge Dr. Jaqueline Scarpa for manuscript revision. This work is supported by grants from PRIN 2006 (MURST), GENZOOT, and by CIB (Consorzio Interuniversitario Biotecnologie). Supporting Information Available: Table of the MS/ MS results obtained from control samples (fresh blood/day 0). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Chin-Yee, I.; Arya, N.; D’Almeida, M. S. The red cell storage lesion and its implication for transfusion. Transfus. Sci. 1997, 18, 447458. (2) Berezina, T. L.; Zaets, S. B.; Morgan, C.; Spillert, C. R.; Kamiyama, M.; Spolarics, Z.; Deitch, E. A.; Machiedo, G. W. Influence of storage on red blood cell rheological properties. J. Surg. Res. 2002, 102, 6-12. (3) Greenwalt, T. J. The how and why of exocytic vesicles. Transfusion 2006, 46, 143-152. (4) Tinmouth, A.; Chin-Yee, I. The clinical consequences of the red cell storage lesion. Transfus. Med. Rev. 2001, 15, 91-107. (5) Ho, J.; Sibbald, W. J.; Chin-Yee, I. H. Effects of storage on efficacy of red cell transfusion: when is it not safe? Crit. Care Med. 2003, 31, 687-697. (6) Hess, J. R.; Hil, I. H. R.; Oliver, C. K.; Lippert, L. E.; Rugg, N.; Joines, A. D.; Gormas, J. F.; Pratt, P. G.; Silverstein, E. B.; Greenwalt, T. J. Twelve-week RBC storage. Transfusion 2003, 43, 867-872. (7) Beppu, M.; Mizukami, A.; Nagoya, M.; Kikugawa, K. Binding of anti-band 3 autoantibody to oxidatively damaged erythrocytes. Formation of senescent antigen on erythrocyte surface by an oxidative mechanism. J. Biol. Chem. 1990, 265, 3226-3233. (8) Pradhan, D.; Weiser, M.; Lumley-Sapanski, K.; Frazier, D.; Kemper, S.; Williamson, P.; Schlegel, R. A. Peroxidation-induced perturbations of erythrocyte lipid organization. Biochim. Biophys. Acta 1990, 1023, 398-404. (9) Gutteridge, J. M. C. Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides. FEBS Lett. 1986, 201, 291-295. (10) Racek, J.; Herynkova´, R.; Holecˇek, V.; Faltysova´, J.; Krejcˇova´, I. What is the source of free radicals causing hemolysis in stored blood? Physiol. Res. 2001, 50, 383-388. (11) Dumaswala, U. J.; Zhuo, L.; Jacobsen, D. W.; Jain, S. K.; Sukalski, K. A. Protein and lipid oxidation of banked human erythrocytes: role of glutathione. Free Radical Biol. Med. 1999, 27, 1041-1049. (12) Dumaswala, U. J.; Zhuo, L.; Mahajan, S.; Nair, P. N.; Shertzer, H. G.; Dibello, P.; Jacobsen, D. W. Glutathione protects chemokinescavenging and antioxidative defense functions in human RBCs. Am. J. Physiol. Cell Physiol. 2001, 280, 867-873. (13) Dumaswala, U. J.; Wilson, M. J.; Wu, Y. L.; Wykle, J.; Zhuo, L.; Douglass, L. M.; Daleke, D. L. Glutathione loading prevents free radical injury in red blood cells after storage. Free Radical Res. 2000, 33, 517-529. (14) Mandal, D.; Baudin-Creuza, V.; Bhattacharyya, A.; Pathak, S.; Delaunay, J.; Kundu, M.; Basu, J. Caspase 3-mediated proteolysis of the N-terminal cytoplasmic domain of the human erythroid anion exchanger 1 (band 3). J. Biol. Chem. 2003, 278, 5255152558. (15) Boivin, P.; Galand, C.; Dhermy, D. In vitro digestion of spectrin, protein 4.1 and ankyrin by erythrocyte calcium dependent neutral protease (calpain I). Int. J. Biochem. 1990, 22, 1479-1489.

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PR070179D


Proteomic Analysis of RBC Membrane Protein Degradation during

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