Original Paper Characterization of high molecular weight multimeric ...

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Tatiana Pimenova1 Claudia P. Pereira2 Dominik J. Schaer2 Renato Zenobi1 1

Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland 2 Internal Medicine Research Unit, University of Zurich, Zurich, Switzerland

T. Pimenova et al.

J. Sep. Sci. 2009, 32, 1224 – 1230

Original Paper Characterization of high molecular weight multimeric states of human haptoglobin and hemoglobin-based oxygen carriers by high-mass MALDI MS High-mass MALDI-TOF mass spectrometry (MS) is a novel analytical approach to study large biomolecules and their interactions. It is a powerful alternative method to gel electrophoresis (GE) and size exclusion chromatography (SEC) for obtaining information on the molecular weights of macromolecules and for determining protein complexes. The precision of mass measurements (mass accuracy), high sensitivity, speed of the analysis, and tolerance toward sample heterogeneity are the major features of this MS-based approach. Remarkably, MS provides direct stoichiometric information of macromolecular protein complexes, when noncovalent interactions are stabilized during desorption/ionization by use of chemical cross-linking reagents. In this study, high-mass MALDI-TOF MS was applied to characterize the multimeric state of the human plasma protein haptoglobin (Hp), which is in the mass range of 150 – 300 kDa. Also, higher order structures of hemoglobin-based oxygen carriers (HBOCs) and their interactions with human haptoglobin were analyzed. These investigations are of clinical importance and contribute to the overall understanding of specific toxicity and clearance of HBOCs. Keywords: Cross-linking / Haptoglobin / Hemoglobin / Mass spectrometry / Protein-protein complexes / Received: November 4, 2008; revised: January 15, 2009; accepted: January 22, 2009 DOI 10.1002/jssc.200800625

1 Introduction Hemoglobin-based oxygen carriers (HBOCs) are blood substitutes based on chemically modified hemoglobin (Hb) of either bovine or human origin [1, 2]. HBOCs belong to a group of oxygen therapeutics, which are developed to mimic human blood's oxygen transport ability in patients with massive blood loss and when red blood cell transfusion is not an option. Modified Hbs exhibit different circulation times following transfusion as a result of specifically altered interactions with the major endogenous Hb scavenger systems. These interactions are intimately related to structural characteristics such as molecular size, sites of intramolecular cross-linking and/or surface modifications [3, 4]. Cell-free Hb can Correspondence: Professor Renato Zenobi, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland E-mail: [email protected] Fax: +41-44-632-1292 Abbreviations: DSS, disuccinimidyl suberate; HBOC, Hb, hemoglobin; hemoglobin-based oxygen carrier; Hp, haptoglobin

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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

be toxic to physiologic environments as a result of its interaction with various oxidants as well as with the vasodilator nitric oxide [5]. Extracellular Hb is bound and detoxified by the plasma protein haptoglobin (Hp) which also promotes clearance by the monocyte/macrophage Hb scavenger receptor pathway [6–8]. Hemoglobin–haptoglobin (Hb–Hp) is a very high affinity complex that attenuates Hb's participation in redox reactions and protects Hb structural stability in oxidant environments [7, 8]. Haptoglobin is found in three major phenotypes: Hp 1-1, 2-1, and 2-2, presenting diverse structural and functional properties with significant biological and clinical implications [9]. Gel electrophoresis under either native (BN-PAGE: “blue native” PAGE) [10] or denaturing conditions (SDSPAGE) [11] has generally been used for the identification and structural analysis of haptoglobin phenotypes [9, 12] and for Hp–Hb complexes. Electrophoresis is not always accurate for molecular weight determination, since the calibration is nonlinear and depends on the number of molecular weight markers in the range of the proteins analyzed. Correct localization of gel bands to correspond-

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ing proteins can be challenging, even for standard proteins of known molecular weight that are used as markers. Moreover, identification of the appropriate conditions for high-resolution separation while maintaining structural and functional integrity of proteins and protein complexes is quite labor-intensive [13]. Size exclusion chromatography (SEC) is an alternative analytical technique for the separation of macromolecules by size and for molecular weight evaluation as a structural determinant. SEC has also been applied for the characterization of several HBOCs [3, 14, 15]. The major limitations of SEC are that it is very difficult to accurately measure the molecular weight of large molecules and to resolve their oligomeric state. The available high-resolution molecular weight and structural information on Hp, Hp–Hb complexes, and HBOCs is thus very limited. Biophysical methods such as NMR analysis and X-ray crystallography were so far unsuccessful in obtaining high-resolution structures for Hp and Hp–Hb complexes, presumably due to the large size of Hp and heavy glycosylation [16]. MS is an alternative, powerful tool to study protein structures, to analyze protein interactions, and to characterize noncovalent protein–protein complexes [17–19]. The mass-resolving detection allows for label-free monitoring of reactants, products, and intermediate species in the assembly/disassembly of protein–protein complexes, without the need to separate these components prior to analysis [20]. Viewed differently, mass spectrometric analysis is a kind of high-performance separation in its own right. Usually, ESI [21] or nano-ESI-MS are used for studying noncovalent complexes. ESI is able to spray “native-like” aqueous solutions, it is very soft, and protein–protein and protein–ligand complexes are found to remain intact upon ionization. ESI-MS has been used to study the hemoglobin tetrameric structure [22–24]. However, special sample preparation procedures such as desalting, and the use of aqueous volatile buffers at pH L 7, are mandatory to keep noncovalent complexes intact when working with ESI [25]. The specificity of interactions measured by ESI MS can be an issue [26], and finding optimum instrumental conditions can be tricky [27]. Griffith and Kaltashov [16] made first attempts to analyze intact human Hp 1-1 and its complexes with human Hb by ESI MS. However, the spectra obtained were poorly resolved and strongly broadened due to insufficient sample clean-up and heavy glycosylations. Desalting and other purification steps are not generally necessary when using MALDI-MS [28, 29], due to its high salt tolerance. An additional advantage of MALDI MS is the spectra interpretation which is much simpler compared to ESI mass spectra, since mainly singly charged pseudomolecular ions are produced during the MALDI ionization process. Thus, overlap of multiply charged peaks in the low m/z region, typical for ESI MS when com-

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plex protein mixtures are analyzed, is absent. A difficulty with MALDI, however, is the need to detect the very high m/z ions that result when z = 1; also high laser powers are needed to produce gas-phase ions from high-mass analyte [30]. In the case of noncovalent assemblies, this can result in disruption of the complex. Recent developments in detector technology, such as the development of an ion conversion dynode (ICD) detector allows for the sensitive detection of ions with m/z above 100 kDa, and in some cases even exceeding 1 MDa [31, 32]. Possible dissociation of the noncovalent protein complexes during the MALDI sample preparation [33] and during the laser desorption/ionization step is avoided by chemical stabilization of the complexes [34]. In the present study, the two most popular phenotypes of haptoglobin, Hp 1-1 and Hp 2-2, and their interaction with Hb and HBOCs were analyzed directly by high-mass MALDI MS. The exact m/z value for each sample was measured and the oligomeric state of the proteins was characterized. The results obtained were compared with the earlier studies by GE and SEC. The main advantages of high-MALDI MS are the rapidity and high sensitivity of the analysis. The procedure is straightforward, requires only low amounts of sample, and the interpretation of the mass spectra obtained is simple.

2 Experimental 2.1 Materials Human Hp 1-1 and Hp 2-2 were purchased from Sigma– Aldrich Chemie (Buchs SG, Switzerland) as an essentially salt-free lyophilized powder and dissolved in 50 mM potassium phosphate buffer, pH 7.4, at a protein concentration of 1 mg/mL. Highly purified Hb (HbA0) was a gift from Hemosol (Missisauga, Ontario, Canada). Purified Hb cross-linked between a-globins (aXLHb) or b-globins (bXLHb), respectively, by bis(3,5-dibromosalicyl)fumarate was from the United States Army (Washington, DC). Oxyglobin is a gluteraldehyde (predominantly a-a) crosslinked and polymerized bovine Hb (aPolyBvHb) and it was from Biopure (Cambridge, MA, USA) [14]. The interactions of these modified Hbs with Hp have been extensively characterized by surface plasmon resonance (SPR) analysis [3]. The amine reactive homobifunctional crosslinking reagent disuccinimidyl suberate (DSS) was obtained from Pierce (Rockford, IL, USA). The MALDI matrix, sinapinic acid, was obtained from Fluka (Buchs SG, Switzerland). BSA (>99% purity) and immunoglobulin G (IgG) (>95% purity) from bovine serum for external MALDI-TOF MS calibration were purchased from Sigma– Aldrich Chemie (Buchs SG, Switzerland). Four to fifteen percent resolving gels Tris-HCl Criterion Precast Gel for SDS-PAGE and Silver Stain Plus were purchased from BioRad Laboratories AG (Reinach BL, Switzerland). www.jss-journal.com

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tein concentration of about 2–5 lM and to reduce the potassium phosphate salt content that can hamper MALDI MS analysis. The mass spectra shown in Fig. 1 were recorded with 30 ng (total protein amount) deposited onto the MALDI target in the case of Hp 1-1 and with 50 ng in the case of Hp 2-2. In order to keep noncovalent Hb–Hp protein complexes intact during desorption/ionization, they were stabilized by DSS prior to high-mass MALDI MS analysis. For this, 1 lL of 2 mg/mL DSS in dimethylformamide was added to 10 lL of a protein solution in the range of 2–5 lM, in 10 mM triethylammonium bicarbonate buffer at pH 9.2. Then, the samples were incubated at room temperature for about 2 h and analyzed by high-mass MALDI-TOF MS.

2.3 High-mass MALDI-TOF MS

Figure 1. High-mass MALDI-TOF mass spectra (A) of Hp 1-1 measured using 30 ng total protein deposited on the target and (B) of Hp 2-2 measured using 50 ng protein. The cartoons represent the multimeric structures of Hp 1-1 and Hp 2-2. Inset in (A): nonreducing SDS-PAGE of Hp 1-1 dimer and Hp 2-2 multimers (adapted with permission from Kristiansen, M., Graversen, J. H., Jacobsen, C., Sonne, O. et al., Nature 2001, 409, 198–201. Copyright i 2001 Nature Publishing Group).

A commercial MALDI-TOF mass spectrometer (Axima CFR, Shimadzu/Kratos Analytical, Manchester, UK) equipped with a high-mass detector (HM1, CovalX AG, Zurich, Switzerland) was employed. Analysis of highmass ions by MALDI is much more efficient with this HM1 than with standard detectors such as microchannel plates, because the conversion to secondary ions is much more efficient than generation of electrons following impact of the heavy ion on the detector surface. Also, saturation by low-mass species is largely avoided due to the discrete dynode architecture of the HM1. The instrument was operated in the positive linear ion mode, and the accelerating voltage was 20 kV. Mass spectra were acquired by averaging 100–150 laser shots at different locations within a sample. The laser pulse energy was adjusted slightly above threshold for ion production. The instrument was externally calibrated with a mixture of BSA and IgG from bovine serum, each at a concentration of 10 lM. BSA and IgG monomer and multimers at m/z = 66, 132, 150, 198, 264, and 300 kDa were used for calibration, due to the lack of suitable monodisperse calibrants in the 150–500 kDa range. The mass accuracy was around 500 Da at 200 kDa. The mass spectra of Hp 1-1 and Hp 2-2 were background subtracted and smoothed by a dedicated software package (Complex Tracker 1.1, CovalX AG). The rest of the mass spectra were smoothed using instrument specific software (Kratos, Shimadzu).

2.2 MALDI sample preparation All samples were prepared for MALDI MS analysis by mixing equal volumes of a protein solution with sinapinic acid (10 mg/mL in 50% ACN/0.1% TFA) in an Eppendorf tube. One microliter of the mixture was then spotted onto the MALDI plate and dried on air at room temperature. The covalent multimers of Hp were directly analyzed by high-mass MALDI-TOF MS, without the need for stabilization by a cross-linking reagent. Both Hp 1-1 and Hp 2-2 were diluted with ultrapure water to reach a pro-

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2.4 SDS-PAGE analysis SDS-PAGE was performed on 4–15% Tris-HCl Criterion Precast Gel in nonreducing conditions according to Laemmli's method [11]. Protein samples were loaded at 2 lg of total protein in a loading buffer. For molecular weight calibration in the range of 10–250 kDa, a commercial protein mixture was used. The samples were run for 55 min at 200 V constant and stained using commercial silver stain plus. www.jss-journal.com

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3 Results and discussion 3.1 High-mass MALDI MS analysis of human Hp Human haptoglobin (Hp) is a plasma glycoprotein constituted of two types of polypeptide chains, the a-chain and the b-chain, that are covalently associated by disulfide bonds [35]. In humans, the a-chain exists in two major allelic forms, a1 of about 9 kDa (Hp 1-1) and a2 of about 18 kDa (Hp 2-2), and the b-chain has a molecular weight of about 35 kDa [36]. Two phenotypes of haptoglobin consisting of different fractions of dimeric Hp (phenotype 1-1) and polymeric Hp (phenotype 2-2) were analyzed by high-mass MALDI-TOF MS, as described in Section 2. The analysis revealed different association of subunits in haptoglobins (Fig. 1). The corresponding schematic structures of Hp 1-1 which has the formula (a1b)2 and Hp 2-2 with the formula (a2b)n (n = 3, 4, 5, …) [35] are shown on the spectrum for each peak. The molecular weight of Hp 1-1 was found to be 89.1 kDa (Fig. 1A), in a close agreement with the published mass of Hp 1-1 dimer (a1b)2 which is about 86 kDa, as determined by gel electrophoresis [35]. The discrepancy in mass is due to the limited accuracy of the electrophoretic method; MS is expected to give much more accurate molecular weights. The signals observed at m/z 45 and 177 kDa correspond to [M + 2H]2+ and to [2M + H]+, respectively. These types of ions are commonly observed in MALDI MS experiments. When analyzing polymeric Hp 2-2 (Fig. 1B), high-mass species at m/z = 151.3, 199.1, 246.3, and 293.5 kDa were detected. The high-mass MALDI mass spectrum is very well resolved allowing for the correct assignment of Hp 2-2 subunit architectures that consist of 3, 4, 5, and 6 (a2b)-subunits. Since, the production of a high resolution and high quality gel is quite difficult and requires a lot of experience and optimization of the experimental conditions, our results were also compared with the state-ofthe-art in nonreducing SDS-PAGE analysis [6]. The inset in Fig. 1A shows a gel image of the same Hp 1-1 and Hp 2-2 samples which were analyzed by high-mass MALDITOF MS with their allocated multimeric states. Consistently with the assigned Hp structures on the mass spectra, Hp 1-1 was detected only in its dimeric and Hp 2-2 was in 3, 4, 5, and 6-meric forms. The most abundant band on the gel corresponds to the tetrameric Hp, which also gives the most prominent peak at m/z = 199.1 kDa in the MS. This suggests that Hp 2-2 tetrameric state is the major association pathway formation. A gel band that represents trimeric Hp 2-2 is on the other hand much less abundant that is in agreement with the MS of Hp 2-2 (Fig. 1B). This good agreement between SDS-PAGE and our method suggests that high-mass MALDI-TOF MS is capable to provide qualitative information on subunits association. Another attractive aspect of MS is the total time requirements for sample preparation and analysis. Typically, for MALDI MS analysis of one sample about 20–

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30 min is needed. GE analysis requires about 2–3 h, if precast gels are used, longer if the gel has to be prepared. In terms of sensitivity issue, silver staining is the most sensitive for visualizing proteins in polyacrylamide gels; it can detect about 5–10 ng of total protein [37]. While the MS technique detects proteins on a molar basis, therefore the higher the molecular weight, at the same mass, the higher the limit of detection (LOD). For example 100 ng of a 20 kDa protein is 5 pmol, while 100 ng of a 200 kDa protein is only 0.5 pmol. Both proteins will have similar stain intensities, but there is ten times less protein on a molar basis from the 200 kDa protein. The LOD in our MALDI-MS was estimated based on the peak intensities and the S/N ratio. For the peak at m/z = 199.1 (Fig. 1B), we estimate a LOD of 10 ng (S/N = 3), i.e., comparable with the silver staining method in gels! While SDS-PAGE is conventionally a technique of choice for the separation of proteins and associated complexes, accurate mass determination by SDS-PAGE is not practical since it requires a large number of reference measurements for a good calibration. Sometimes, even under reducing conditions, the 2-D/3-D protein structure as well as hydrophobic character can influence the mass determination. In contrast, the mass of proteins and protein complexes can be obtained directly by MS.

3.2 High-mass MALDI MS analysis of HBOCs Tetrameric and polymeric HBOCs with covalent modifications on b-globin subunits (bXLHb) and a-globin subunits (aXLHb and PolyHbBv) were also analyzed (Fig. 2). The most intense peaks on the mass spectra were assigned using cartoons corresponding to different aand b-globin associations. In order to prevent the fragmentation of the hemoglobin a/b-subunits during the MALDI desorption/ionization process and to stabilize their native structure for MALDI-TOF MS analysis, a chemical stabilization with DSS was carried out as described in Section 2. High-mass MALDI mass spectra revealed that after reaction with DSS, bXLHb is mostly in the dimeric and tetrameric form (Fig. 2A) and oxyglobin mainly consists of Hb-tetramers and octamers (Fig. 2B). In the MALDI mass spectra of nonstabilized HBOCs, a dominant peak occurs at m/z 15.2 kDa in the case of bXLHb and at m/z 16.4 kDa in the case of oxyglobin, corresponding to the singly charged ions of free a- and b-globin subunits, respectively. Since, free a- and b-subunits are the lightest species in this sample, it is very easy to get them ionized. The signals at m/z 47.2 and 63.2 kDa of the nonstabilized oxyglobin (Fig. 2B) correspond to trimers (a2b) and tetramers (a2b2) of a/b-globin subunits. There was no trimer and tetramer formation observed in the case of nonstabilized bXLHb (Fig. 2A, bottom). The major peaks at m/z 65.4 kDa (bXLHb) and 66.5 kDa (oxyglobin) correspond to the tetrameric Hb, and its detection was only www.jss-journal.com

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Figure 2. High-mass MALDI-TOF mass spectra of (A) bXLHb and (B) PolyHbBv (oxyglobin) measured before (lower traces) and after (upper traces) ab-stabilization with DSS. The cartoons represent the Hb-assemblies. Inset: silver-stained nonreducing SDS-PAGE of bXLHb and PolyHbBv. Molecular weight markers in kDa on the right side of the gel were from a commercial source.

possible in the case of bXLHb when a/b-globins were covalently stabilized by DSS. Generally, molecular weight of DSS-stabilized proteins and protein complexes increases by 2–5% due to DSS molecules covalently attached [32]. After a/b-globin stabilization, the peaks became slightly broadened. This is likely due to the binding of DSS molecules and also to unresolved doubly charged ions formed during MALDI desorption/ionization. For instance, the peak corresponding to the free a-subunit at 15.2 kDa in nonstabilized bXLHb (Fig. 2A, bottom) moved to 16.7 kDa after stabilization with DSS (Fig. 2A, top), and it very likely overlaps with the peak of the doubly charged b–bdimer at 33.4 kDa. The same was observed for the peaks at 65.4 kDa corresponding to the stabilized Hb-tetramer

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and 33.4 kDa (Fig. 2A, top); in Fig. 2B, the peaks at 30.9 kDa corresponding to a–a-dimer and 63.2 kDa corresponding to the Hb-tetramer (Fig. 2B, bottom) moved to 33.9 and 66.5 kDa (Fig. 2B, top), respectively, after stabilization by DSS. We have also performed nonreducing SDS-PAGE analysis of these HBOCs and the resulting gel image is displayed in the inset of Fig. 2. A distinct band of bXLHb migration was observed at about 30 € 5 kDa and it is consistent with the dimer of the cross-linked b–b-globin subunits. Two less abundant bands corresponding to l45 € 5 kDa and l60 € 5 kDa multimers were detected. These results are in a agreement with the high-mass MALDI MS analysis of the stabilized bXLHb. The SDSPAGE pattern of PolyHbBv does poorly represent the molecular composition of the Hb. It appears as only a smear on the gel which is a likely result of nonspecific protein–protein interactions under the nonreducing conditions applied. Therefore, it is not possible to make any assignment of its oligomeric state. Both bXLHb and PolyHbBv (oxyglobin) were previously characterized [3, 14] using size exclusion chromatography. The molecular weight of PolyHbBv was estimated to be in the range of 87–500 kDa. Standard MALDI MS was also applied to measure the masses of these HBOCs, but no species were detected above 47 kDa. This limitation was due to the use of conventional detector. It has been shown that the detection efficiency strongly decreases for masses above 30–50 kDa when standard detectors are used [38]. Therefore, high-mass MALDI MS is the method of choice for obtaining high-resolution and accuracy information in the high mass range. We detected peaks at m/z = 131.5 and 195.1 kDa, but no further signals in the higher mass range (Fig. 2B). The differences in the molecular weight obtained by high-mass MALDI MS and SEC could be due to an unspecific adsorption of protein molecules to the column material, causing a shift in the elution time of protein assemblies. Another reason could be due to the differences in the molecular shapes between calibrant proteins and macromolecules analyzed [39]. This might have led to the overestimation of the molecular size in SEC analysis. Again, MS is able to yield much more accurate data, provided that the highmass complexes can be detected.

3.3 Investigation of large Hp–Hb complexes High-mass MALDI-TOF MS analysis of Hp–Hb complexes revealed that natural HbA0 associates with Hp 1-1, forming complexes of different stoichiometries of hemoglobin a/b-subunits (Fig. 3A). They are schematically shown on the figure with the corresponding cartoons: Hp 1-1 bound with one Hb-dimer, with one Hb-dimer and one Hb-monomer chain, and with two Hb-dimers. It is not possible to identify which of the two subunits, a or b, parwww.jss-journal.com

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for Hp. (Fig. 3B). The predominant MALDI peaks in this reaction correspond to non-Hp bound dimeric and tetrameric Hb and unchanged Hp. A very likely explanation for this observation is that aXLHb does only very weekly interact with Hp and does not form the stable complexes required for efficient inter-molecular DSS reaction. This interpretation is in perfect agreement with our previous observation that unmodified a-chains of Hb are crucial for a high affinity interaction with Hp [3]. We also analyzed the interaction of Hp 1-1 with bXLHb and Hp 2-2 with HbA0 and the different HBOCs by highmass MALDI-TOF MS, but almost no complex formation was observed (data not shown). A reason for this observation could be the lower binding affinities of these complexes. In fact, Hp1-1-Hb is one of the strongest known noncovalent protein–protein interactions in biology with the dissociation constant of 10–15 mol/L [7, 12]. Recently, the relative binding affinities of Hp 1-1 and Hp 2-2 with Hb and the HBOCs were studied by SPR [3]. The following order of the reduction in Hp 1-1 binding affinity compared with HbA0 has been reported: HbA0 > bXLHb > aXLHb = aPolyBvHb, with 15–30% reduction for bb-cross-linked Hbs and with 50–95% for aa-cross-linked Hbs. Therefore, the binding affinity can be so low that it is not possible anymore to stabilize protein complexes for the subsequent MALDI MS analysis. It is currently an open question and an active research area to correlate the binding affinity with the ability of a cross-linker to specifically stabilize a protein complex.

4 Concluding remarks

Figure 3. High-mass MALDI-TOF mass spectra of (A) HbA0-Hp and (B) aXLHb-Hp before (lower traces) and after (upper traces) ab-stabilization with DSS. The cartoons represent the Hb- and Hb–Hp complexes formed.

ticipated in the complex giving rise to the peak at 138.9 kDa, because of the limited resolution at this high mass. In order to detect these protein complexes in their native stoichiometry, stabilization by DSS was applied. The peak at m/z = 154.5 kDa could correspond to a complex of Hp 1-1 with the Hb tetramer, but it is known that Hp binds the Hb-dimer and not the intact tetramer [40]. Since, HbA0 was present in excess compared to Hp 1-1 in order to complete the binding, some free a- and b-subunits of Hb remain and are observed in the spectrum after DSS-stabilization. The ability to detect covalently cross-linked Hb–Hp complexes after DSS reaction is significantly reduced when using aXLHb instead of HbA0 as a binding partner

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The multimeric states of human haptoglobin of two phenotypes were characterized by high-mass MALDI-TOF MS analysis for the first time. This is important because the different Hp phenotypes have different physiological effects regarding their protective activity against Hb toxicity [41, 42]. The intact Hp–Hb complexes were studied and the results provide valuable information on mass and stoichiometry of haptoglobin binding, which might help in rational design of HBOCs with limited Hb toxicity. High-mass MALDI MS in combination with chemical cross-linking was shown to be a powerful analytical method for Hp, Hp–Hb and large molecular size HBOCs investigation providing accurate and high resolution information. This MS-based approach offers excellent separation and accurate mass determination compared to electrophoretic separation or size exclusion chromatography requiring only a small amount of sample (2– 5 lM) for the analysis. This work was supported by the Swiss National Science Foundation, grant no. 200020-111831. The authors declared no conflict of interest. www.jss-journal.com

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