Anal. Chem. 2005, 77, 3372-3378
N-Terminal Adducts of Bovine Hemoglobin with Glutaraldehyde in a Hemoglobin-Based Oxygen Carrier Maryline Gasthuys,†,‡ Sandra Alves,† and Jean-Claude Tabet*,†
UMR CNRS 7613, University Pierre et Marie Curie, Boite 45, 4 place Jussieu 75252 Paris Cedex 05, France, and Laboratoire National de De´ pistage du Dopage, 143 avenue Roger Salengro, 92490 Chaˆ tenay-Malabry, France
Hemoglobin-based oxygen carriers (HBOCs) are being developed for the medical field, but because they could increase an athlete’s performance, they are misapplied for doping purposes. We previously presented a screening method to detect Oxyglobin (Biopure Corp.) and PolyHeme (Northfield Laboratories Inc.) in serum samples using total acid hydrolysis followed by electrospray mass spectrometry analyses. An alternative mass spectrometric method involving enzymatic hydrolysis is here presented. Digestion of Oxyglobin by endoproteinase Glu-C and LC/ MS analyses of the mixture allowed the detection of unique peptidic fragments in comparison with a bovine hemoglobin digest. Tandem mass spectrometry experiments of these peptide ions were performed, and two specific species were actually identified as the N-terminal enzymatic fragment of the β chain carrying two different modifications. Sequential MS3 experiments using an ion trap mass spectrometer permitted us to locate the chemical modification by the glutaraldehyde on the NH2terminal group and to propose a structure for the modified peptides. In another set of experiments, screening of these two diagnostic ions into Oxyglobin-spiked serums using precursor ion scan mode in a triple quadrupole instrument allowed the detection of this HBOC with a detection limit of 2 g L-1. Cell-free hemoglobin tetrameric structure can dissociate in plasma into nephrotoxic R,β-dimers.1 To maintain molecular stability and improve oxygen off-loading, intra- and intermolecular cross-linking has been performed on ultrapurified hemoglobin.2 These hemoglobin-based oxygen carriers (HBOCs) are being developed as temporary blood substitutes for the medical field.3 They could, therefore, represent a safe alternative to blood transfusion and rectify the lack of available whole blood. Hemopure and Oxyglobin, both products developed by Biopure Corporation (Cambridge, MA), are solutions of glutaraldehydepolymerized bovine hemoglobin. Because they have shown the potential to increase oxygen-carrying capacity of circulating blood * Corresponding author. E-mail:
[email protected]. † University Pierre et Marie Curie. ‡ Laboratoire National de De´pistage du Dopage. (1) Debaene, B.; Barbot, A. Reanimation 2003, 12, 580-591. (2) Riess, J. G. Chem. Rev. 2001, 101, 2797-2919. (3) Re´my, B.; Deby-Dupont, G.; D’Ans, V.; Ernest, P.; Lamy, M. Ann. Fr. Anesth. Reanim. 1999, 18, 211-224.
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and to improve tissue oxygenation, they can be misapplied for doping purposes. As a consequence, artificial oxygen carriers have been added to the list of prohibited substances by the World AntiDoping Agency (WADA).4 Owing to this fact, the detection and identification of these molecules have become a priority for doping control. For the present, their analysis is commonly achieved using size-exclusion chromatography5,6 or electrophoresis.7 Recently, some efforts have been made in mass spectrometry to identify the presence of artificial blood transfer enhancers. Perfluorocarbons are easily detected in plasma using GC/MS.8 Mass spectrometry has also proved to be an efficient method to analyze high molecular mass compounds, such as hemoglobins, thanks to the advent of soft ionization techniques, such as MALDI9,10 and electrospray,11 and can be therefore utilized for the detection of HBOCs in serum samples.12,13 ESI produces multiply charge ions so that quadrupole analyzers with limited m/z range can be used. We previously reported a fast method based on electrospray mass spectrometry analysis of acid hydrolysis products to detect the presence of glutaraldehydepolymerized hemoglobins in human serum samples, whatever their origin, bovine or human. The detection has been achieved with good specificity and an acceptable sensitivity limit, in particular, using constant neutral loss experiments in a triple quadrupole mass spectrometer.14 Acid hydrolysis treatment coupled to mass spectrometry brought sensitive and reproducible results in this special case. However, the dominant approach for studying proteins is based on the characterization of peptide fragments from (4) The 2005 prohibited list, Internal Standard, The World Anti-Doping Code, http://www.wada-ama.org. (5) Varlet-Marie, E.; Ashenden, M.; Lasne, F.; Sicart, M. T.; Marion, B.; De Ce´aurriz, J.; Audran, M. Clin. Chem. 2004, 50, 723-731. (6) Trout, G. J.; Kazlaukas, R. Chem. Soc. Rev. 2004, 33, 1-13. (7) Lasne, F.; Crepin, N.; Ashenden, M.; Audran, M.; De Ceaurriz, J. Clin. Chem. 2004, 50, 410-415. (8) Mathurin, J. C.; de Ceaurriz, J.; Audran, M.; Kraft, M. P. Biomed. Chromatogr. 2001, 15, 443-451. (9) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (10) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (11) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (12) Guan, F.; Uboh, C. E.; Soma, L. R.; Luo, Y.; Jahr, J. S.; Driessen, B. Anal. Chem. 2004, 76, 5127-5135. (13) Guan, F.; Uboh, C. E.; Soma, L. R.; Luo, Y.; Driessen, B. Anal. Chem. 2004, 76, 5118-5126. (14) Gasthuys, M.; Alves, S.; Fenaille, F.; Tabet, J. C. Anal. Chem. 2004, 76, 6628-6634. 10.1021/ac048107i CCC: $30.25
© 2005 American Chemical Society Published on Web 04/15/2005
enzymatic digestion. “Bottom up” sequencing15 allowed the determination of primary structure as well as posttranslational modifications (phosphorylation,16,17 glycation,18 etc.) of proteins using tandem mass spectrometry (MS/MS) experiments.19 Moreover, on-line combination of electrospray with liquid chromatography (LC) facilitates the detection of low-abundance modified peptides in complex mixtures, and it has been widely used to characterize hemoglobin variants 20-22 as well as modified hemoglobins.23-25 LC/MS study of enzymatic digest of the polymerized hemoglobins can constitute a way to establish the presence of exogenous hemoglobin in human plasma12,13,26 by screening of specific bovine or equine peptidic fragments. To our knowledge, no glutaraldehyde-modified fragment that could be useful diagnostic species for artificial cross-links detection, has been detected after tryptic treatment and mass spectrometry analysis. It would then be impossible to differentiate a normal human hemoglobin from a polymerized human hemoglobin (PolyHeme, Northfield Laboratories Inc.) on the basis of their tryptic digest patterns. Enzymatic digestion with another protease with a different specificity can be an alternative way for the detection of modified peptides. Endoproteinase Glu-C has been successfully used for the detection of modified peptides in the case of glycated hemoglobin27 and minor component HbA1b28 of hemoglobin and was, therefore, used in the study here presented. We report here a method based on the comparison of peptidic fragments produced after enzymatic digestion by endoproteinase Glu-C of a glutaraldehyde-polymerized bovine hemoglobin (Oxyglobin) and natural bovine hemoglobin. Bovine hemoglobin and Oxyglobin first underwent endoproteinase Glu-C digestion, which specifically cleaved the C-terminal peptidic bonds of aspartate and glutamate residues. LC/ESI-MS analysis of the peptidic fragment mixtures led to the detection of specific peptides of the polymerized protein. Sequencing of two of these peptides by sequential MS2 and MS3 experiments using an ion trap mass spectrometer allowed the location of the modification on the N-terminal residue (Met) of the hemoglobin β chain. Because the mass-to-charge ratios (m/z) of the corresponding diagnostic ions differ by only 2 Th, we suggested that the peptidic fragment -β-NH2-terminal hexapeptide- was involved in similar chemical modifications but that one contained an additional unsaturation. Finally, low-energy (15) Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806-812. (16) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269-295. (17) Mann, M.; Org, S. E.; Gronborg, M.; Steen, H.; Jensen, O. N.; Pandey, A. Trends Biotechnol. 2002, 20, 261-267. (18) Fenaille, F.; Morgan, F.; Parisod, V.; Tabet, J.-C.; Guy, P. A. Rapid Commun. Mass Spectrom. 2003, 17, 1483-1492. (19) Roepstorff, P. Biomed. Mass Spectrom. 1984, 11, 601. (20) Wada, Y. J. Chromatogr., B 2002, 781, 291-301. (21) Friess, U.; Beck, A.; Kohne, E.; Lehmann, R.; Koch, S.; Haring, H. U.; Schmuelling, R. M.; Schleider, E. Clin. Chem. 2003, 49, 1412-1415. (22) Gatlin, C. L.; Eng, J. K.; Cross, S. T.; Detter, J. C.; Yates, J. R., III Anal. Chem. 2000, 72, 757-763. (23) Mason, D. E.; Liebler, D. C. J. Proteome Res. 2003, 2, 265-272. (24) Rai, D. K.; Landin, B.; Griffiths, W. J.; Alvelius, G. Rapid Commun. Mass Spectrom. 2002, 16, 1793-1796. (25) Bondarenko, P. V.; Chelius, D.; Shaler, T. A. Anal. Chem. 2002, 74, 47414749. (26) Thevis, M.; Orgozalek Loo, R. R.; Loo, J. A.; Scha¨nzer, W. Anal. Chem. 2003, 75, 3287-3293. (27) Nakanishi, T.; Shimizu, A. J. Chromatogr., B 2000, 746, 83-89. (28) Prome, D.; Blouquit, Y.; Ponthus, C.; Prome, J. C.; Rosa, J. J. Biol. Chem. 1991, 266, 13050-13054.
collision-induced dissociation experiments (CID) permitted postulation of a structure for the modified peptides and fragmentation mechanisms. The immonium ions of the modified methionins were then chosen to perform precursor ion scan experiments on Oxyglobin-spiked serum samples using a triple quadrupole mass spectrometer and showed the applicability of this sensitive screening method for Oxyglobin identification in biological matrixes at a concentration limit of 2 g‚L-1. MATERIAL AND METHODS Material and Chemicals. Lyophilyzed bovine hemoglobin and 10 mM phosphate buffer saline (PBS) were obtained from SigmaAldrich (Steinheim, Germany). Endoproteinase Glu-C was purchased from Fluka (Deseinhofen, Germany), and acetonitrile and formic acid from SDS (Marseille, France). All solutions and buffers were prepared using deionized water (MilliQ grade). Oxyglobin solution was a generous gift from Biopure Corp. (Cambridge, MA) by the intermediate of the French Laboratoire National de Depistage du Dopage (LNDD). The human serum samples were obtained by simple centrifugation of blood samples from healthy volunteers. Doping Human Serum Samples. For therapeutic purposes, a hemodilution of 20% is needed for an adult. This dose is supposed to be efficient for increasing an athlete’s performance. Considering a total product dilution in the whole blood volume, a concentration of Oxyglobin in the circulatory system of ∼16 g L-1 is expected.29 We chose to spike 25 µL of human serum sample aliquots with different amounts of Oxyglobin solution [13 g/dL], resulting in final concentrations from 0 to 50 g L-1 in serum samples. Digestion Step. In a first set of experiments, bovine hemoglobin and Oxyglobin were dissolved in 10 mM phosphatebuffered saline (PBS) at a concentration of 10 g L-1 (1.55 × 10-4 M) and mixed with endoproteinase Glu-C solution at a concentration of 1.14 × 10-5 M with a mass ratio enzyme/protein ) 1/30 to a final volume of 100 µL. In a second set of experiments, spiked 25-µL serum samples were mixed with 5 µL of endoproteinase Glu-C solution at a concentration of 25 g L-1. Volume was completed to 50 µL with PBS, 10 mM. The mixtures were then incubated overnight at 37 °C. The spiked serum samples were afterward diluted 500-fold before direct injection into the mass spectrometer. LC-MS and LC-MSn Analyses. Chromatographic separation was carried out using a HP1100 HPLC system equipped with a Waters Nova-Pak C18 column (3.9 × 150 mm, 4 µm, 60 Å). The injection volume of digestion product solution was 20 µL. The mobile phase consisted of the following: phase A, 0.1% HCOOH, and phase B, acetonitrile/0.1% HCOOH (70/30, v/v). Percentage of B was maintained at 0% for 2 min and then increased according to a linear gradient of from 0 to 100% B in 55 min before reequilibrating the column. The flow rate was set at 0.7 mL min-1 and split to 70 µL min-1 before entering into the ESI source. The analyses were performed in the positive ESI mode with an ion trap instrument (ESQUIRE 3000, Bruker Daltonics, Bremen, Germany) operating in the data-dependent mode. The analytical conditions were as follows: capillary voltage of 3.5 kV and source temperature of 350 °C. Heated nitrogen was used as both (29) Toussaint-Hacquard, M.; Devaux, Y.; Longrois, D.; Faivre-Fiorina, B.; Muller, S.; Stoltz, J. F.; Vigneron, C.; Menu, P. Life Sci. 2003, 72, 1143-1157.
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Figure 1. Total ion current chromatograms (ESI/ITMS) of the digestion products of (A) bovine hemoglobin and (B) Oxyglobin using endoproteinase Glu-C. The common peaks are noted from 1 to 14, and the additional peaks for bovine hemoglobin or Oxyglobin are noted from I to V (details in Table 1).
nebulizing gas (35 psi; 1 psi ) 6894.76 Pa) and drying gas (12 L min-1). Solution ran at atmospheric pressure through a grounded stainless steel capillary. The m/z ratio range (noted in Thomson unit30) used for the analytical scan was from 50 up to 1000 Th. Resonant ion ejection occurred at βz ) 2/3 (qz ) 0.78) within a ∆(m/z) value of 0.4 Th. All CID mass spectra were recorded with a low mass cutoff set to ∼60 Th. Direct Infusion Mass Spectrometry. Direct infusion mass spectrometry experiments were performed in the positive electrospray ionization mode on a triple quadrupole Quattro I (Micromass Inc., Manchester, England) mass spectrometer. Analyses were realized by infusing the solution at a flow rate of 6.7 µL min-1 using a KD scientific Inc. syringe pump (Holliston, USA). The spray voltage was set at 3.0 kV. Nitrogen was used both as nebulizing gas (20 L h-1) and disolvation gas (400 L h-1). The cone voltage was set at 30 V, and CID experiments were performed using argon in the collision cell at a pressure of 1.6 × 10-4 mbar. Precursor ion scan experiments were performed by scanning of the third quadrupole, and laboratory collision energy was optimized to 70 eV. Data acquisition and processing were carried out using the Micromass MassLynx software. RESULTS AND DISCUSSION LC/MS Chromatographic Peak Profiles of Peptides from Digestion by Endoproteinase Glu-C. The digestion products of bovine hemoglobin and Oxyglobin by endoproteinase Glu-C were analyzed by LC/MS/MS (Figure 1) using an ion trap mass spectrometer. Although the chromatographic profiles appeared very different at first sight, the reconstructed ion chromatograms (data not shown) of the corresponding ions revealed that most of the species were common to both protein digests. Some of the chromatographic peaks, however, strongly differed in relative (30) Cooks, R. G.; Rockwood, A. L. Rapid Commun. Mass Spectrom. 1991, 5, 93.
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intensities. The major chromatographic peaks were identified thanks to product ion scan experiments under collision-induced dissociation conditions. The expected peptidic fragments resulting from the endoproteinase Glu-C digestion were not all detected in the mixture, and some nonspecific cleavages and miscleavages were also observed (Table 1). For example, the peak 10 corresponded to a peptidic fragment resulting from a miscleavage at the C-terminal position of the Asp 46 of the β chain. Peak 11 corresponded to the same segment of the sequence but was produced by a nonspecific cleavage at the C-terminal position of the Ala 50 residue. The intensity of peak 10 relative to that of peak 11 (I10/I11) decreased from 13 to ∼1 in Oxyglobin digestion products. This relative decrease of the intensity then suggested that nonspecific peptides were more likely produced from the cross-linked hemoglobin. This observation may be explained by a steric hindrance, yielding a limited accessibility to the substrate residue when the protein was intermolecularly cross-linked. I6/I8 intensity ratio also decreased from 2 to 0.5. The contribution of N-terminal β1-6 peptide in the digestion product of Oxyglobin was then divided by 4 in comparison with the digestion product of natural bovine hemoglobin. This last finding suggested either that the access to the site of cleavage was hindered by the polymerization process, or that the NH2-terminal group of the protein was partly modified during the polymerization process and consequently, a smaller amount of intact peptide was liberated under digestion conditions. However, these observations could not be used to differentiate the cross-linked protein, because the relative difference of the chromatographic peak intensities could not be simply evaluated without any internal standard in the mixture. A precise comparison of these LC profiles has revealed that some chromatographic peaks were unique either to bovine hemoglobin or to Oxyglobin digestion products. For the bovine hemoglobin digest, the chromatographic peak at retention time (tR) of 16 min (peak II) was only observed for the native hemoglobin digest and resulted from a nonspecific cleavage of
Table 1. Chromatographic Peaks of the Peptides Obtained after Proteolytic Digestion of Bovine Hemoglobin and Oxyglobin by Endoproteinase Glu-C and Their Amino Acid Sequences peaks
tR,a min ((0.2)
ions,b m/z
1
8.6
2 I 3 4 5 6 7 II 8 III 9 10 11 12 13
9.8 11 11.3 12.3 13.2 14.2 15.5 16 16.3 18 18.3 19.2 19.6 21.1 24.5
221 439 207 648 575 709 564 693 743 802 752 761 686 912 797 890 1167
14 IV V
24.9 26.5 31.3
1038 933 819 748 689 594 892 759
residue
sequencec
dipeptide SD, DS, or TT R24-27 (E) YGAE dipeptide TS, ST, or GM ? ? R1-6 (-) VLSAAD ? ? β1-5 (-) MLTAE β1-6 (-) MLTAEE β122-128 (F) TPVLQAD R64-71 (A) AALTKAVE R24-30 (E) YGAEALE *β1-6 (-) dMLTAEE R 76-82 (D) LPGALSE β43-51 (E) SFGDLSTAD β 43-50 (E) SFGDLSTA β121-128 (E) FTPVLQAD nonelucidated peptidic fragments.
β 32-38 eβ 1-6
(L) VVYPWTQ (-) eMLTAEE
a LC retention time of digest from bovine hemoglobin, except for the specific peaks of the Oxyglobin digest (I, III, IV, V). The same peaks are observed for Oxyglobin digest with an acceptable shift of retention times (( 0.2 min). b Observed nominal mass-to-charge ratios. c Elucidated from the observed mass-to-charge ratios and product ion mass spectra. CID conditions were optimized for each ion to provide useful fragments. The amino acid in parentheses at the beginning of the sequence stands before it in the protein. d +68 u. e +66 u.
the peptidic bond between Ala 63 and Ala 64 residues of the R chain of bovine hemoglobin. We, thus, could detect Oxyglobin thanks to one missing peptidic fragment. This type of result has already been observed after digestion by trypsin.31 But the search of one missing peptidesand more likely, if this peptide resulted from a nonspecific cleavage by endoproteinase Glu-Cscould not constitute an applicable means of detection of Oxyglobin in biological matrixes, especially in plasma, where numerous proteins could interfere with the test. A more sensitive and specific means of detection was to focus on the specific species from the Oxyglobin digestion mixture. The chromatographic peaks noted I, III, IV, and V were unique to Oxyglobin at tR of 11, 18, 26.5, and 31.3 min, respectively. The corresponding ESI mass spectra displayed the single charge m/z 648, 761, and 759 ions. These ions did not correspond to conventional expected peptides from natural bovine hemoglobin digestion. We suggested that they contained a chemical modification produced during the reaction of cross-linking of bovine hemoglobin by glutaraldehyde. These specific peptides were, thus, selected as target compounds for differentiation of Oxyglobin from a normal bovine hemoglobin using LC/MS. To enhance structural information, these protonated ions were further studied under lowenergy CID conditions to perform product ion scans. (31) Alma, C.; Trout, G.; Woodland, N.; Kazlauskas, R. Proc. Manfred-Donike Workshop, Cologne 2002, Sport & Buch Strauss, 2002, pp 169-178.
Figure 2. CID spectra of the (a) m/z 693 (peak 6 at tr ) 14.2 min), (b) m/z 761 (peak III tr ) 18 min) and (c) m/z 759 (peak V at tr ) 31.3 min) ions The “°” symbol designates the product ions, according to the Roepstorff nomenclature, which have lost a molecule of water either from the threonin backbone chain, for b and a fragment ions, or from glutamate residue, for the y fragment ion. The modified m/z 761 and 759 ion species yield common product ions of the b series which differ by 2 Th when comparing the two CID spectra. These experiments were performed using an ion trap mass spectrometer. The resonant excitation was applied with an amplitude of 0.70 Vp-p for recording all the provided CID spectra.
Sequential MS/MS and MS3 study of Unique Oxyglobin Ions. Among the various peptides obtained from the Glu-C digestion, the large number of the ions has been attributed to expected peptides of the bovine hemoglobin sequence. However, the Oxyglobin-specific peaks I, III, IV, and V have been herein scrutinized in order to get more information on their structure and that of potential cross-links present in the exogenous protein. The chromatographic peak I displayed mainly the m/z 648 ion. From its product ion spectrum, its sequence could not be elucidated directly under CID conditions because of the low number of product ions. Moreover, sequential MSn experiments were not possible because of the precursor m/z 648 ion’s low abundance. On the contrary, the product ion spectra of the m/z 759 and 761 ions mentioned above (peaks III, IV, and V in Figure 1 and Table 1) provided important information that was essential to elucidate their amino acid sequences. The ESI mass spectra corresponding to peaks IV and V displayed a common m/z 759 ion. The CID experiments on the m/z 759 ion conducted at both retention times revealed that the two m/z 759 ions were characterized by similar structure and could, thus, be considered as isomers. The product ion spectrum of the selected m/z 759 ion mainly displayed the b ion series in Roepstorff19 nomenclature (Figure 2c). This fragmentation pattern is characteristic of the partial -TAEE amino acid sequence and only differs by 2 Th when compared to the product ion spectrum of the precursor m/z 761 ion (Figure 2b). The 2-Th difference which persisted overall the product bi series until the b2 and a2 product ions indicated that the modification of both species was located at the N-terminus residues of the sequence. This partial sequence was analogous to that of the β1-6 fragment (m/z 693) for both ions (Figure 2a). The Oxyglobin-specific m/z 761 and 759 ions would then be the NH2-terminal fragment of the β chain which carried two different modifications, involving a mass-shift of either +68 u or +66 u when compared to the nonmodified β1-6 peptidic fragment (peak 6 on Figure 1 and Table 1). The product ion spectra of m/z 693 Analytical Chemistry, Vol. 77, No. 10, May 15, 2005
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Scheme 1. Proposed Mechanism for the Formation of Glutaraldehyde-Modified β1-6 Peptidic Fragments (MLTAEE) in Slightly Basic Solution (pH 7.8)32,a
Figure 3. Sequential MS3 spectra of (a) m/z 285 (a2 from m/z 761) and (b) m/z 283 (a2 from m/z 759) recorded with an ion trap mass spectrometer. The resonant excitation was applied with an amplitude of 0.72 Vp-p.
(nonmodified β1-6), m/z 759 (β1-6 +66 u) and m/z 761 (β1-6 +68 u) presented in Figure 2 displayed also bi ions, which yield a common loss of an 18-u neutral, that is, a molecule of water. This loss can be attributed to the presence of either the carboxyl groups of the two glutamate residues or the hydroxyl group of the threonin residue of the sequence. The abundance of these product ions was high enough to perform sequential MS3 experiments in order to get more accurate information on the modification sites of these two characteristic precursor ions. Furthermore, the resulting spectra may provide a better understanding of the structure of the N-terminal modifications. On-line LC-sequential MS3 experiments were then performed on the a2 ions (m/z 283 and 285, respectively) from the m/z 759 and 761 precursor ions. The obtained spectra displayed the a1 ions, m/z 170 and m/z 172, respectively (Figure 3). This result is consistent with a modification located at the methionin NH2-terminal residue. It has previously been shown that many chemical agents reacted with the amino group of the NH2-terminal valine residues of human hemoglobin.32,33 Chemical agents and, here, glutaraldehyde could display a similar reactivity toward the NH2-terminal group (Schiff base formation) of the methionin residue of the β chain of bovine hemoglobin during the synthesis of Oxyglobin to produce either cross-links or, in this more particular case, a simple modification causing a mass shift of 68 or 66 u. Synthesis of Oxyglobin consists of the reaction of glutaraldehyde on ultrapure bovine hemoglobin to produce cross-links through imine group formation. Because these functional groups are not stable in solution and are easily hydrolyzed into reactants, a reduction step using NaBH4 is required for maintaining the cohesion between the different moieties of the cross-links.34 To explain the 2-Th shift observed between our two diagnostic ions, an incomplete reduction could be proposed. This statement has been confirmed by reducing the peptide mixture prior to the analysis by LC/MS/MS. The two IV and V chromatographic peaks at retention times of 26.5 and 31.3 min, respectively, corresponding
to the m/z 759 ion vanished, whereas the peak at tR ) 18 min corresponding to the m/z 761 ion was more abundant (data not shown). Considering all these experimental data, a structure for the modified peptides and mechanisms for the fragmentation of the a1 ions as well as hypothesis concerning the formation of the modified species during synthesis can be proposed (Scheme 1). The N-terminal modification of the β chain of hemoglobin by glutaraldehyde is thought to happen via the formation of a sixmembered ring.35,36 According to this assumption, the terminal primary amino group should be modified into a tertiary amino group, resulting in enhanced gas-phase basicity.37 This could be a possible reason, although the same laboratory energy is employed for the three sequential MS2 experiments (Figure 2), the product ion spectrum of the nonmodified peptide (Figure 2a) displayed b and y ions in competition, whereas the product ion spectra of modified species, m/z 759 and 761, specifically displayed bi ions38,39 and corresponding bi - H2O and ai ions (Figure 2b,c). This could also explain the enhanced stability of the quasimolecular ion when the N-terminal amino group is modified.
(32) Boogard, P. J. J. Chromatogr., B 2002, 778, 309-322. (33) Tareke, E.; Rydberg, P.; Karlsson, P.; Eriksson, S.; To ¨rnqvist, M. Chem. Res. Toxicol. 2000, 13, 517-522. (34) Rausch, C. W.; Gawryl, M. S.; Houtchens, R. A.; Laccetti, A. J.; Light, W. R. U.S. Patent no. 5,840,852, 1998.
(35) Schilling, B.; Row, H. R.; Gibson, B. W.; Guo, X.; Young, M. M. J. Am. Soc. Mass Spectrom. 2003, 14, 834-850. (36) Falick, A. M.; Hines, W. M.; Medzihradszky, K. F.; Baldwin, M. A.; Gibson, B. W. J. Am. Soc. Mass Spectrom. 1993, 4, 882-893. (37) Hunter, E. P. L.; Lias, S. G. J. Phys. Chem. Ref. Data 1998, 27, 413-656.
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a The position of insaturation of the partially reduced peptide (m/z 759) can be located at other positions of the six-membered heterocycle.
Scheme 2. Possible Mechanisms of Fragmentation of the a2 Ions, m/z 285 and 283, Respectively, from m/z 761 and m/z 759 Precursor Ions
Moreover, the sequential MS3 spectra of the a2 ions (m/z 283 and 285) displayed fragment ions other than a1 ions (m/z 170 and 172). The m/z 122 and 124 fragment ions are produced by the consecutive loss of a CH3SH molecule from the methionin side chain. Depending on the charge location on the m/z 283 ion, another specific fragmentation pathway initiated by the loss of the N-terminal six-membered ring was proposed for the formation of the m/z 200 and 172 fragment ions (Scheme 2). With the experimental data thus providing confidence in the hypothetical structure proposed in this scheme, these specific ions were afterward tested in spiked human serum samples. Detection into Human Serum Samples. CID spectra of the Oxyglobin-specific ions using a triple quadrupole analyzer did not display as many fragment ions as using an ion trap mass spectrometer. The low-mass cutoff40 (LMCO) which limited the m/z ratio range of the CID spectrum in the ion trap did not allow analyzing product ions below m/z 200 from the precursor ions of m/z values larger than 750 Th. With the triple quadrupole instrument, abundant diagnostic ions appeared at either m/z 170 or 172 corresponding to the a1 fragment ions from the m/z 759 and 761 precursor ions, respectively (data not shown). We thus used this property to detect the specific peptides into Oxyglobinspiked human serum samples after digestion by endoproteinase Glu-C using the precursor ion scan mode (Figure 4). These (38) Dongre´, A. R.; Jones, J. L.; Somogyi, A.; Wysocki, V. H. J. Am. Chem. Soc. 1996, 118, 8365-8374. (39) Vaisar, T.; Urban, J. J. Mass Spectrom. 1998, 33, 505-524. (40) March, R. E. J. Mass Spectrom. 1997, 32, 351-369.
experiments were also performed on a blank serum sample which underwent the same treatment by endoproteinase Glu-C. The detection of m/z 761 and 759 on the precursor ion mass spectra of m/z 172 and 170, respectively, was specific to Oxyglobin-spiked samples. It must also be pointed out that the precursor m/z 170 and 172 ions spectra from a native bovine hemoglobin digest did not display the two diagnostic m/z 759 and 761 ions. The latter experiment thus confirmed the specificity of the diagnostic ions for Oxyglobin. The detection of the two Oxyglobin-specific ions in spiked serum samples was possible down to 2 g L-1, which is ∼10-fold lower than the concentration obtained just after the administration of the product. Moreover, the linearity of the method was also tested (Figure 5) and showed good results in a concentration range from 0 to 10 g L-1. However, at higher concentrations of Oxyglobin in serum samples (20 and 50 g L-1), the relation between ion intensity and Oxyglobin concentration did not remain linear. This may be due to an enzyme saturation at a larger concentration of enzyme or a decrease in electrospray ionization efficiency at higher sample concentrations. As long as the measure is performed in the lower concentration range, the method proposed here could be perfectly applicable for further quantification. Audran and co-workers5 studied the kinetics of Hemopure clearance in healthy individuals and showed that for an injection of 30 g of polymerized hemoglobin, it remained a product concentration of 3.7 g L-1 in plasma after 24 h. The method presented here would then be applicable 24-48 h after the product was administered and could thus be particularly useful for a delayed doping control. Precursor ion scanning of a1 ions is a particularly rapid, sensitive, and robust Analytical Chemistry, Vol. 77, No. 10, May 15, 2005
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Figure 4. Precursor ion mass spectra of m/z 170 and 172 ions in blank serum (a, b) and in serum spiked with Oxyglobin at 4 g L-1 (c, d) obtained using a triple quadrupole instrument. The peak resolution was lowered to obtain signal-to-noise ratios respectively of 9.2 (c) and 25 (d).
Figure 5. Graphical representation of the evolution of the intensity of the signals produced in electrospray by the diagnostic m/z 759 and 761 ions on precursor m/z 170 and 172 ion mass spectra, respectively, vs the concentration of Oxyglobin in serum samples. The relation was linear in a concentration range from 0 to 10 g L-1 and tended to a polynomial law for higher concentration range.
method to identify Oxyglobin-specific peptides among a complex mixture with an easy sample preparation and without any chromatographic separation. CONCLUSION Both native bovine hemoglobin and glutaraldehyde polymerized bovine hemoglobin (Oxyglobin) underwent endoproteinase Glu-C proteolysis. Specific modified peptides for Oxyglobin were found by comparing LC/MS chromatographic peak profiles of the obtained peptide mixtures. Multiple-stage CID experiments permitted the elucidation of their amino acid sequence. The specific m/z 759 and 761 ions corresponded to the β NH2-terminal MLTAEE proteolytic peptide carrying two types of glutaraldehyde modification. Sequential MS3 experiments performed using an ion 3378 Analytical Chemistry, Vol. 77, No. 10, May 15, 2005
trap mass spectrometer allowed us to propose a six-membered ring structure around the NH2-terminal group. This modification could be either totally or partially reduced, involving a shift of mass respectively of +68 u (for m/z 761) or +66 u (for m/z 759). CID spectra of the two specific m/z 759 and 761 ions performed with a triple quadrupole instrument displayed mainly the a1 fragment ions, m/z 170 and 172, respectively. These product ions did not correspond to immonium fragments from natural amino acids. This characteristic was thus exploited for the detection of these specific modified peptides in biological matrixes. Serum samples spiked with different amounts of Oxyglobin were thus submitted to endoproteinase Glu-C digestion. The obtained peptide mixtures were analyzed using the triple quadrupole instrument after direct injection into an ESI source. Precursor m/z 170 and 172 ion spectra of spiked serum samples allowed the detection of Oxyglobin-specific ions at a low concentrations. This mass spectrometry method has been successfully applied for the detection of Oxyglobin in serum samples of any origin without any complex sample preparation or chromatographic separation. This method showed an excellent specificity and a detection limit of 2 g L-1, which is far lower than the dose administered. It could be further improved for quantification in mass spectrometry, for example, using MRM scan mode. Further investigations should concern the detection of several oxygen therapeutics (either polymerized with glutaraldehyde or not) in natural matrixes using this type of rapid and sensitive mass spectrometry method. ACKNOWLEDGMENT We thank French Laboratoire de Depistage du Dopage for financial support and for providing hemoglobin-based products and human serum samples. Received for review December 22, 2004. Accepted March 15, 2005. AC048107I
