Anal. Chem. 2004, 76, 5118-5126
Unique Tryptic Peptides Specific for Bovine and Human Hemoglobin in the Detection and Confirmation of Hemoglobin-Based Oxygen Carriers Fuyu Guan,† Cornelius Uboh,*,†,‡ Lawrence Soma,† Yi Luo,† and Bernd Driessen†
School of Veterinary Medicine, Department of Clinical Studies, University of Pennsylvania, New Bolton Center Campus, Kennett Square, Pennsylvania 19348, and PA Equine Toxicology and Research Laboratory, Department of Chemistry, West Chester University, 220 East Rosedale Avenue, West Chester, Pennsylvania 19382
Hemoglobin-based oxygen carriers (HBOCs) of bovine hemoglobin (Hb) or human Hb origin were developed for replacement or augmentation of blood during transfusion and have the potential to increase oxygen-carrying capacity of circulating blood and thus improve tissue oxygen delivery. Due to their potential for increasing oxygencarrying capacity of circulating blood, they are excellent candidates for abuse in human and equine athletes. To deter athletes from blood doping with HBOCs such as Hemopure and Oxyglobin (OXY), a method for detection, confirmation, quantification, and distinguishing of HBOCs from native hemoglobin in test samples is needed. The purpose of this study was to identify unique peptides specific for bovine Hb and human Hb that are useful in the detection and confirmation of HBOCs in test samples. The LC-MS chromatographic peak profiles of tryptic digests from OXY, bovine Hb, human Hb, and equine Hb were compared, and unique tryptic peptides specific for bovine Hb, human Hb, and equine Hb were identified. The peptides specific for bovine Hb and OXY are related to bovine Hb r chain residues 69-90 and β chain residues 40-58. The peptides specific for human Hb are related to human Hb r chain residues 63-91 and β chain residues 42-60 and 68-83. The amino acid sequences of these unique tryptic peptides were confirmed by their characteristic MS/MS spectra. MS/MS spectra, b-ion series and y-ion series, and LC retention time of the tryptic peptides are essential pieces of information for the unequivocal identification, detection, and confirmation of HBOCs. The results of this study provide useful and defensible data on identification, detection, and confirmation of HBOCs of bovine Hb or human Hb origin. In addition, in-ESI-source fragmentation of tryptic peptides was observed in this study. The fragmentation was undesired since it decreased intensities of the trypic peptide ions, but it was helpful to elucidating sequences of the * Corresponding author. Tel: +01-610-436-3501. Fax: +01-610-436-3504. E-mail:
[email protected]. † University of Pennsylvania. ‡ West Chester University.
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tryptic peptides thanks to the fragment peptide ions produced from the fragmentation. Allogeneic and xenogeneic, stroma-free solutions of hemoglobin (Hb) have been developed to overcome problems associated with blood transfusion.1,2 Among the lead products are hemoglobin glutamer-200 (Oxyglobin (OXY), Biopure, Cambridge, MA) and hemoglobin glutamer-250 (Hemopure (HMP), Biopure), solutions of glutaraldehyde-polymerized bovine Hb, and hemoglobin raffimer (Hemolink (HML), Hemosol, Toronto, Canada), a solution of human Hb cross-linked and polymerized with o-raffinose. Though hemoglobin-based oxygen carriers (HBOCs) were developed primarily to replace or augment blood during transfusion in patients with anemia and acute blood loss, the potential of these agents to increase oxygen-carrying capacity of circulating blood and to improve tissue oxygen delivery make them excellent candidates for abuse in human and equine athletes.3 To deter athletes from blood doping, international human and equine sports authorities such as the International Olympic Committee (IOC) and the Association of Racing Commissioners International have banned the administration of any products that enhance the uptake, transport, or delivery of oxygen. However, the use and abuse of HBOCs for doping purposes is still one of the challenges the international human sports community, horse racing and horse show industries, and equine forensic chemists are facing, because currently available methods such as bed-side hemometers (HemoCue)4 or cooximeters cannot distinguish between native Hb (from hemolyzed red blood cells) and HBOCs and between HBOCs originated from Hb of different species. Specific and unequivocal methods for detection, quantification, and confirmation of HBOCs are in urgent need to discourage blood doping in sports. Soon after this study was completed, an independent study on LC-MS of HBOCs in human plasma was published.5 (1) Winslow, R. M. Annu. Rev. Med. 1999, 50, 337-353. (2) Klein, H. G. N. Engl. J. Med. 2000, 342, 1666-1668. (3) Schumacher, Y. O.; Schmid, A.; Dinkelmann, S.; Berg, A.; Northoff, H. Int. J. Sports Med. 2001, 22, 566-571. (4) Lurie, F.; Jahr, J. S.; Driessen, B. Anesth. Analg. 2002, 95, 870-873. (5) Thevis, M.; Loo, R. R. O.; Loo, J. A.; Schanzer, W. Anal. Chem. 2003, 75, 3287-3293. 10.1021/ac035425t CCC: $27.50
© 2004 American Chemical Society Published on Web 07/24/2004
HBOCs are large protein molecules, and thus, they pose challenges to current analytical techniques established for small drug molecules. Advances in proteomics and related fields have provided much needed information for the understanding and determination of proteins. Large protein molecules can be hydrolyzed by an enzyme into segments of small peptides, and the peptides can be studied by current analytical techniques such as liquid chromatography coupled with mass spectrometry (LCMS). With this strategy at hand, bovine, equine, and human Hbs have been studied with respect to their amino acid sequences. Digestions of these proteins by trypsin were reported for screening of Hb variants,6-9 for detection of hemoglobin adducts with organic compounds,10,11 and for determination of linkage sites of crosslinked hemoglobins.12,13 The purpose of this study was to identify unique tryptic peptides specific for bovine, human, and equine Hbs in the detection and confirmation of HBOCs of bovine or human origin, such as OXY, HMP, and HML. EXPERIMENTAL SECTION Reagents. OXY solution (13 g/dL, 125 mL per bag) was purchased from Biopure (Cambridge, MA). Lyophilized powders of bovine Hb, equine Hb, human Hb, horse heart myoglobin, and trypsin from bovine pancreas (TPCK treated, Sigma Catalog No. T1426) were purchased from Sigma (St. Louis, MO). Acetonitrile (HPLC grade) and ammonium bicarbonate (certified) were purchased from Fisher Scientific (Pittsburgh, PA), formic acid was obtained from EM Science (Gibbstown, N.J), and HPLC grade methanol and water were purchased from J. T. Baker (Phillipsburg, NJ). Preparation of Reagents. Ammonium bicarbonate stock solution (1.0 M) was prepared by dissolving 7.9 g of ammonium bicarbonate in 100 mL of H2O, stored at 4 °C, and discarded after 3 months. Ammonium bicarbonate buffer (50 mM) was freshly prepared by dilution of the stock solution (1.0 M) with water. Formic acid (10%, v/v) was prepared by adding 1.0 mL of formic acid to 9.0 mL of water. OXY (400 µg/mL in ammonium bicarbonate buffer) were freshly prepared by dilution of OXY solution with the ammonium bicarbonate buffer. Solutions of bovine Hb, equine Hb, and human Hb (400 µg/mL in ammonium bicarbonate buffer) were freshly prepared by weighing the lyophilized powder of each standard and dissolving it in the buffer. Trypsin solution (400 µg/mL in the buffer) was similarly prepared. All protein solutions mentioned above were freshly prepared shortly before use, and any unused portion was discarded. Horse heart myoglobin stock solution (200 µg/mL) was prepared by weighing the lyophilized powder of the standard and dissolving (6) Turpeinen, U.; Sipila, I.; Anttila, P.; Karjalainen, U.; Kuronen, B.; Kalkkinen, N.; Ahola, T.; Stenman, U. H. Clin. Chem. 1995, 41, 532-536. (7) Wajcman, H.; Bardakdjian, J.; Ducrocq, R. Ann. Biol. Clin. 1993, 51, 867870. (8) Landin, B.; Jeppsson, J. O. Hemoglobin 1993, 17, 303-318. (9) Zeng, Y. T.; Ren, Z. R.; Chen, M. J.; Zhao, J. Q.; Qiu, X. K.; Huang, S. Z. Br. J. Haematol. 1987, 67, 221-223. (10) Moll, T. S.; Harms, A. C.; Elfarra, A. A. Chem.-Biol. Interact. 2001, 135136, 667-674. (11) Moll, T. S.; Harms, A. C.; Elfarra, A. A. Chem. Res. Toxicol. 2000, 13, 11031113. (12) Yang, T.; Horejsh, D. R.; Mahan, K. J.; Zaluzec, E. J.; Watson, T. J.; Gage, D. A. Anal. Biochem. 1996, 242, 55-63. (13) Marta, M.; Patamia, M.; Lupi, A.; Antenucci, M.; Di Iorio, M.; Romeo, S.; Petruzzelli, R.; Pomponi, M.; Giardina, B. J. Biol. Chem. 1996, 271, 74737478.
Table 1. LC Gradient time (min) B%a flow rate (mL/min)
0 15 0.2
0.5 15 0.2
25 40 0.2
28 80 0.2
29 80 0.2
29.5 15 0.2
30 15 0.4
34 15 0.4
34.5 15 0.2
35 15 0.2
a LC mobile phase A: acetonitrile/H O/formic acid (5/95/0.2, 2 v/v/v). LC mobile phase B: acetonitrile/H2O/formic acid (95/5/0.2, v/v/v).
it in acetonitrile/H2O/formic acid (50/50/0.2, v/v/v) and was stored at 4 °C for 30 days before discarding. Horse heart myoglobin solution (10 µg/mL) for calibration of the Q-TOF mass spectrometer was freshly prepared by dilution of the stock solution with acetonitrile/H2O/formic acid (50/50/0.2, v/v/v). Digestion of Hbs and HBOCs by Trypsin. Digestion of bovine, human, and equine Hbs and OXY was performed by modification of an existing protocol.14 A 25-µL volume of trypsin (400 µg/mL) was added to 0.5 mL of ammonium bicarbonate buffer (50 mM, pH 7.8) containing bovine Hb, equine Hb, human Hb, or OXY at 400 µg/mL and mixed. The mixture was incubated in a water bath (model 1245PC, VWR Scientific, Bridgeport, NJ) at 37 °C for 24 h. The digestion was stopped by adding 20 µL of formic acid (10%, v/v) and mixing. An aliquot of 100 µL of the digestion solution was transferred to a 250-µL vial insert for LCMS analysis. Instrumentation and Operating Parameters. Analysis of protein digests was performed on an LC-MS instrument, comprising a Hewlett-Packard 1100 LC binary pump with an on-line vacuum degasser and an autosampler (Agilent, Wilmington, DE), and a Q-TOF mass spectrometer equipped with a z-spray electrospray ionization (ESI) source (Micromass, Manchester, U.K.). LC separation was performed on a Zorbax 300SB-C3 column (2.1 × 150 mm, 5 µm) with a Zorbax 300 SB-C3 guard column (2.1 × 12.5 mm, Agilent, Wilmington, DE) maintained at 27 °C. An LC mobile-phase gradient in composition and flow rate used for resolution of tryptic peptides is shown in Table 1. Q-TOF mass spectrometry was operated in positive ion mode. The mass spectrometer was calibrated over mass range of m/z 600-1500 using horse heart myoglobin at concentration of 10 µg/mL in acetonitrile/H2O/formic acid (50/50/0.2, v/v/v). The ESI source parameters used were as collows: capillary, 3000 kV; cone, 35 V; extractor, 0 V; source block temperature, 120 °C; desolvation temperature, 400 °C. For MS/MS experiments, the collision gas (argon) pressure was adjusted so that the analyzer vacuum readback was 2.0 × 10-5 mbar. Data acquisition and analysis were accomplished by Masslynx software version 3.5 (Micromass). Generation of candidate peptides from mass-to-charge ratios (m/z) and charge states was carried out using Biolynx version 3.5 (Micromass). Simulation of digestion of proteins was accomplished using the same software. Fasta search of peptide sequences for sequence similarity was performed using the web site of the European Bioinformatics Institute (http://www.ebi.ac.uk/fasta33/). (14) Kinter, M.; Sherman, N. E. Protein Sequencing and Identification using Tandem Mass Spectrometry; Wiley-Interscience: New York, 2000; pp 160163.
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Figure 1. LC-MS chromatograms of tryptic digests of OXY (a), bovine Hb (b), human Hb (c), equine Hb (d), and trypsin blank (e) showing the different chromatographic peak profiles of the tryptic digests.
RESULTS AND DISCUSION LC-MS Chromatographic Peak Profiles of Tryptic Peptides from OXY, Bovine Hb, Human Hb, and Equine Hb. Since HBOCs such as OXY, HMP, and HML are of bovine Hb or human Hb origin, distinction between bovine Hb and human Hb and among other Hbs such as equine Hb is essential for the identification of HBOCs. A strategy for differentiation and identification of HBOCs is to identify unique peptides specific for the identity of each HBOC after digestion of HBOCs into tryptic peptides, since peptides are smaller than proteins and easier to determine by ESI mass spectrometry. The unique tryptic peptides are then used for identification of a specific HBOC. In this study, OXY, bovine Hb, human Hb, and equine Hb were digested with trypsin, and the tryptic peptide profiles of the digests were compared using LC-MS (Figure 1). The digest of OXY has the same profile of chromatographic peaks as that of bovine Hb, as expected, but the digests of human Hb and equine Hb have different profiles. Comparisons among these profiles have revealed the major chromatographic peaks unique to each of OXY, bovine Hb, human Hb, and equine Hb. For OXY and bovine Hb, the chromatographic peaks at retention time (tR) of 11.4 and 13.7 min in Figure 1a and b, respectively, are specific, so are the peaks at tR of 4.30, 11.85, 14.1, 15.6, and 16.6 min in Figure 1c for human Hb, and the peaks at tR of 13.9, 14.8, and 17.9 min in Figure 1d for equine Hb. Mass Spectra of the Tryptic Peptides Specific for OXY, Bovine Hb, Human Hb, and Equine Hb. The mass spectra of the specific chromatographic peaks mentioned above contain important information such as monoisotopic mass and charge state of the protonated peptide ions that are essential to elucidating 5120
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amino acid sequence of the respective peptides (Figure 2). Examination of each mass spectrometric peak in the mass spectra revealed the charge state of the protonated peptide ion that gave rise to the MS peak. For example, the peak at m/z 790.11 in Figure 2a is actually a cluster of peaks at m/z 789.81, 790.11, 790.41, and 790.77. From the mass difference between adjacent peak pairs in the cluster, the charge state of the protonated peptide ion was determined.15 For this particular case, the charge state of the protonated peptide ion was 3+. Once the charge state and the monoisotopic mass of the ion (for example, m/z 789.81 in the case above) were determined from the mass spectra, the amino acid sequence of the peptide was elucidated with the aid of Biolynx software. To achieve reasonable elucidation of the amino acid sequence for a tryptic peptide from the charge state and monoisotopic mass, the following criteria were applied to the elucidation processing: (1) the sequence of a peptide is a segment of the sequence of an Hb from which the peptide was derived; (2) a tryptic peptide has either lysine or arginine terminus in its C-terminal, or a peptide is produced from reasonable fragmentation of a tryptic peptide; (3) the N-terminal residue of a tryptic peptide is next to either a lysine or an arginine terminus of another tryptic peptide from the same Hb chain; (4) the mass difference between theoretical and measured m/z of the protonated peptide ion is within 0.5 mass unit, with consideration of the better mass accuracy and resolution with Q-TOF instrument compared with those of a triple quadrupole or an ion trap mass spectrometer. The MS peaks in Figure 2 for the unique chromatographic peaks of OXY and bovine Hb digests were manually examined regarding (15) Kinter, M.; Sherman, N. E. Protein sequencing and indentification using tandem mass spectrometry; Wiley-Interscience: New York, 2000; p 41.
Figure 2. Mass spectra for the chromatographic peaks shown in Figure 1a and b at tR of 11.4 min for OXY (a) and bovine Hb (b) and at tR of 13.72 min for OXY (c) and 13.71 min for bovine Hb (d). RT, retention time (tR).
the charge state and monoisotopic mass of protonated peptide ions, amino acid sequence of the respective peptides were then deduced with the aid of Biolynx software, and the results are presented in Table 2. The results indicated that the chromatographic peaks specific for OXY at tR of 11.44 min in Figure 1a and specific for bovine Hb at tR of 11.40 min in Figure 1b are related to a tryptic peptide with the amino acid sequence of ′AVEHLDDLPGALSELSDLHAHK′ that is from bovine Hb R chain residues 69-90 (bovine R T9). The sequence is in accordance with theoretical prediction from simulation of digestion of bovine Hb R chain by trypsin. Actually, it corresponds to the T9 segment from the simulated digestion (where T designates trypsin as the enzyme used for digestion). The chromatographic peaks specific for OXY at tR of 13.72 min in Figure 1a and specific for bovine Hb at tR of 13.71 min in Figure 1b are related to a tryptic peptide with the amino acid sequence of ′FFESFGDLSTADAVMNNPK′ that is from bovine Hb β chain residues 40-58 (bovine Hb β T6). The two abundant ions at m/z 780.4 and 893.5 in Figure 2a, which are not interpreted in Table 2, are b7 and b8 ions, respectively, of the triply charged ion at m/z 790 (see Table 3). The doubly charged ion at m/z 738 and the singly charged ion at m/z 893.5 were chosen for detection and confirmation of OXY in the accompanying paper because their intensities are higher than that of the ion at m/z 790 (Figure 2a). The MS peaks for the unique chromatographic peaks of human Hb digest and equine Hb digest are presented in Figure 3 and Figure 4, respectively. The MS peaks were manually examined regarding the charge state and monoisotopic mass of protonated peptide ions, and amino acid sequences of the respective peptides were then deduced with the aid of Biolynx software, as described
above. The results are presented in Table 2. The unique chromatographic peaks of human Hb digest at tR of 4.30, 11.85, 14.13, 15.61 and 16.65 min in Figure 1c are related to five tryptic peptides. They are ′SAVTALWGK′ from human Hb β chain residues 1018 (T2), ′VLGAFSDGLAHLDNLK′ from human Hb β chain residues 68-83 (T9), ′FFESFGDLSTPDAVMGNPK′ from human Hb β chain residues 42-60 (T5), ′KVADALTNAVAHVDDMPNALSALSDLHAHK′ from human Hb R chain residues 62-91 (T8-9), and ′VADALTNAVAHVDDMPNALSALSDLHAHK′ from human Hb R chain residues 63-91 (T9), respectively. The unique chromatographic peaks of equine Hb digest at tR of 13.91, 14.83, and 17.94 min in Figure 1d are related to three tryptic peptides, and they are ′FFDSFGDLSNPGAVMGNPK′ from equine Hb β chain residues 41-59 (T5), ′AAVLALWDKVNEEEVGGEALGR′ from equine Hb β chain residues 9-30 (T2-3), and ′VGDALTLAVGHLDDLPGALSNLSDLHAHK′ from equine Hb R chain residues 63-91 (T9). In-Source Fragmentation of Tryptic Peptides. The results shown in Table 2 indicate that there are a few fragment peptides accompanying each tryptic peptide and the sequences of the fragment peptides are a part of the sequence of the tryptic peptide. These fragment peptides were not generated from trypsin digestion. They have the same retention time as that of the tryptic peptide, and this means that they came out the LC column at the same time as that of the tryptic peptide. A reasonable explanation is that the fragment peptides resulted from in-source (in ESI source) fragmentation of the tryptic peptide. In-source fragmentation of the tryptic peptides was not intended and was not desired since it decreased the intensity of the tryptic peptides studied. The fragmentation was believed to be related to an ESI source Analytical Chemistry, Vol. 76, No. 17, September 1, 2004
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Table 2. MS Peaks of the Tryptic Peptides from Oxyglobin, Bovine Hb, Human Hb, Equine Hb, and Their Amino Acid Sequences tRa, min
m/zb
chargec
chain
11.44 11.44 11.44 11.44 13.72 13.72 13.72 13.72
789.81 737.95 689.40 625.38 1045.47 888.52 702.42 603.32
3+ 2+ 2+ 2+ 2+ 1+ 1+ 1+
R R R R β β β β
4.30 4.30 4.30 11.85 11.85 11.85 11.85 11.85 14.13 14.13 14.13 15.61
932.55 774.49 675.42 835.50 785.95 729.42 700.93 665.40 1030.06 928.53 645.39 781.93
1+ 1+ 1+ 2+ 2+ 2+ 2+ 2+ 2+ 1+ 1+ 4+
β β β β β β β β β β β R
Human Hb 10-18 931.51 12-18 773.44 13-18 674.38 68-83 1668.88 69-83 1569.82 70-83 1456.73 71-83 1399.71 72-83 1328.67 42-60 2057.94 52-60 927.45 55-60 644.33 62-91 3123.58
15.61 15.61 15.61 16.65
737.44 688.93 645.36 999.56
2+ 2+ 3+ 3+
R R R R
78-91 79-91 74-91 63-91
1472.77 1375.72 1932.94 2995.48
16.65 16.65 16.65 16.65
942.88 843.13 809.50 749.97
3+ 3+ 3+ 4+
R R R R
65-91 68-91 69-91 63-91
2825.38 2526.23 2425.18 2995.48
16.65 16.65 16.65
737.53 688.93 631.89
2+ 2+ 2+
R R R
78-91 79-91 80-91
1472.77 1375.78 1261.68
13.91 13.91 13.91 13.91 13.91 14.83 14.83 14.83 14.83 17.94 17.94 17.94 17.94 17.94 17.94
1000.49 773.44 716.42 661.71 645.39 1163.64 776.13 758.47 659.38 983.94 967.56 738.23 730.43 689.40 681.91
2+ 1+ 1+ 3+ 1+ 2+ 3+ 1+ 1+ 3+ 2+ 4+ 2+ 2+ 2+
β β β β β β β β β R R R R R R
41-59 52-59 53-59 41-59 54-59 9-30 9-30 23-30 24-30 63-91 63-82 63-91 78-91 69-82 79-91
Equine Hb 1998.91 772.39 715.37 1998.91 644.33 2325.20 2325.20 757.41 658.34 2948.54 1933.02 2948.54 1458.76 1376.73 1361.71
residue
massd
Oxyglobin and Bovine Hb 69-90 2366.19 77-90 1473.76 78-90 1376.70 80-90 1248.65 40-58 2088.95 51-58 887.42 53-58 701.41 54-58 602.28
sequencee (K) AVEHLDDLPGALSELSDLHAHK (L) (L) PGALSELSDLHAHK (L) (P) GALSELSDLHAHK (L) (A) LSELSDLHAHK (L) (R) FFESFGDLSTADAVMNNPK (V) (A) DAVMNNPK (V) (A) VMNNPK (V) (V) MNNPK (V) (K) SAVTALWGK (V) (A) VTALWGK (V) (V) TALWGK (V) (K) VLGAFSDGLAHLDNLK (G) (V) LGAFSDGLAHLDNLK (G) (L) GAFSDGLAHLDNLK (G) (G) AFSDGLAHLDNLK (G) (A) FSDGLAHLDNLK (G) (R) FFESFGDLSTPDAVMGNPK (V) (T) PDAVMGNPK (V) (A) VMGNPK (V) (K) KVADALTNAVAHVDDMPNALSALSDLHAHK (L) (M) PNALSALSDLHAHK (L) (P) NALSALSDLHAHK (L) (H) VDDMPNALSALSDLHAHK (L) (K) VADALTNAVAHVDDMPNALSALSDLHAHK (L) (A) DALTNAVAHVDDMPNALSALSDLHAHK (L) (L) TNAVAHVDDMPNALSALSDLHAHK (L) (T) NAVAHVDDMPNALSALSDLHAHK (L) (K) VADALTNAVAHVDDMPNALSALSDLHAHK (L) (M) PNALSALSDLHAHK (L) (P) NALSALSDLHAHK (L) (N) ALSALSDLHAHK (L) (R) FFDSFGDLSNPGAVMGNPK (V) (P) GAVMGNPK (V) (G) AVMGNPK (V) (R) FFDSFGDLSNPGAVMGNPK (V) (A) VMGNPK (V) (K) AAVLALWDKVNEEEVGGEALGR (L) (K) AAVLALWDKVNEEEVGGEALGR (L) (E) VGGEALGR (L) (V) GGEALGR (L) (K) VGDALTLAVGHLDDLPGALSNLSDLHAHK (L) (K) VGDALTLAVGHLDDLPGALS (N) (K) VGDALTLAVGHLDDLPGALSNLSDLHAHK (L) (L) PGALSNLSDLHAHK (L) (T) LAVGHLDDLPGALS (N) (P) GALSNLSDLHAHK (L)
aLC retention time. b Monoisotopic mass. c Determined from the mass difference between adjacent isotopic peak pair. d Theoretical value for mass of an uncharged peptide. e Elucidated from the monoisotopic mass and charge state. The two amino acids in parentheses at the two ends of each sequence are not part of the sequence but next to the sequence in the intact protein.
parameter called “cone voltage”. For the tryptic peptide from bovine Hb R chain residues 69-90, low cone voltage (20 V) resulted in low absolute intensity of the tryptic peptide and fragment peptides though intensity of the tryptic peptide relative to that of the fragment peptides was high (see Figure S-3). High cone voltage (35 V) led to high absolute intensity of the fragment peptides and tryptic peptide though intensity of the tryptic peptide relative to that of the fragment peptides was low. In this study, a cone voltage of 35 V was chosen and used. In-source fragmentation of tryptic peptides is dependent on sequences of the peptides. For example, the triply charged peptide 5122 Analytical Chemistry, Vol. 76, No. 17, September 1, 2004
ion at m/z 790 from bovine Hb R chain residues 69-90 is susceptible to in-source fragmentation, as evidenced by the presence of abundant fragment peptide ions at m/z 738 (an y14 ion) and 893.5 (a b8 ion). The easy fragmentation of the ion at m/z 790 is due to “preferential fragmentation at a proline and poor fragmentation at a glycine” effect16 (a proline at residue 77 is adjacent to a glycine at residue 78 in bovine Hb R chain). Though undesired, in-source fragmentation would be helpful for elucidating sequences of tryptic peptides. Fragment peptides (16) Kinter, M.; Sherman, N. E. Protein Sequencing and Identification using Tandem Mass Spectrometry; Wiley-Interscience: New York 2000; p 100.
Table 3. Interpretation of the MS/MS Spectra of the Triply Charged Ion at m/z 790.0 from Bovine Hb r Chain Residues 69-90 (T9) and the Doubly Charged Ion at m/z 737.9 from Bovine Hb r Chain Residues 77-90, the Triply Charged Ion at m/z 999.9 from Human Hb r Chain Residues 63-91 (T9), and the Triply Charged Ion at m/z 984.0 from Equine Hb r Chain Residues 63-91 (T9)
b-ion series massa sequenceb massa y-ion series sequenceb massa y-ion series b-ion series massa sequence massa y-ion series b-ion series massa sequence b-ion series massa sequence b-ion series massa sequenceb sequenceb massa y-ion series b-ion series massa sequenceb sequenceb massa y-ion series
m/z 790.0 b7 b8 780 893 D L
A
b2 171 V
b3 300 E
b4 437 H
b5 550 L
b6 665 D
S 807 y7
D 720 y6
L 605 y5
H 492 y4
A 355 y3
H 284 y2
P
b2 155 G 1377 y13
b3 226 A 1320 y12
b4 339 L 1249 y11
b5 426 S 1136 y10
b6 555 E 1049 y9
m/z 737.9 b7 b8 668 755 L S 920 807 y8 y7
b2
b3
A
V b2′
b5 550 L
b6 665 D
m/z 893.5 b7 780 D L
H
L
E b3′ 366 D
b4 437 H b4′ 481 D
V P 1473 y14
b2 171 A N 1376 y13
b3 286 D A 1262 y12
b4 357 A L 1191 y11
b5 470 L S 1078 y10
b6 571 T A 991 y9
V P 1459 y14
b2 157 G G 1362 y13
b3 272 D A
b4 343 A L 1234 y11
b5 456 L S 1121 y10
b6 557 T N 1034 y9
y12
b9 P 1474c y14
b10 1047 G
b11 1118 A y12
b12 1231 L 1249 y11
S 1136 y10
E 1049 y9
y13
b9 870 D 720 y6
b10 983 L 605 y5
b11 1120 H 492 y4
b12 1191 A 355 y3
1328 H 284 y2
K 147 y1
m/z 999.9 b7 b8 685 756 N A L S 920 807 y8 y7
b9 855 V D 720 y6
b10
b11 1063 H H 492 y4
b12
b13
V A 355 y3
m/z 984.0 b7 b8 670 741 L A L S 920 807 y8 y7
b9 840 V D 720 y6
b11 1034 H H 492 y4
b12 1147 L A 355 y3
L 920 y8
K 147 y1
594 L
A L 605 y5 b10 G L 605 y5
D H 284 y2
b14 1392 D K 147 y1
b15 1523 M
b13 1262 D H 284 y2
b14 1377 D K 147 y1
b15 1490 L
a Nominal mass for the b-ion series and y-ion series observed in the MS/MS spectra shown in Figure 5. b The entire sequence of the peptide is presented in two rows. c Nominal mass for the doubly charged ion at m/z 737.9.
resulting from in-source fragmentation of tryptic peptides are actually some of the “y-ion series” and “b-ion series”17,18 of the precursor tryptic peptides, and they provide further evidence for sequences of the precursor tryptic peptides. Sequence Confirmation of the Unique Tryptic Peptides by MS/MS. The sequences of triply charged peptide ion at m/z 790 from bovine Hb R chain residues 69-90, the doubly charged ion at m/z 738 from bovine Hb R chain residues 77-90, the singly charged ion at m/z 893.5 (the b8 ion of the ion at m/z 790), the triply charged ion at m/z 1000 from human Hb R chain residues 63-91, and the triply charged ion at m/z 984 from equine Hb R chain residues 63-90 were further confirmed by MS/MS. In the MS/MS spectra of these peptide ions shown in Figure 5, the product ions are mainly b-ion series and y-ion series. The b-ion series and y-ion series are quite informative of the sequence of a precursor peptide. Interpretation of the product ion spectra are summarized in Table 3. The MS/MS spectra as well as the b-ion (17) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601. (18) Kinter, M.; Sherman, N. E. Protein Sequencing and Identification using Tandem Mass Spectrometry; Wiley-Interscience: New York, 2000; pp 6877.
series and y-ion series are “fingerprints” of the specific tryptic peptides mentioned above and, thus, are very useful for confirmation of the peptides and the Hbs as well. Specificity of the Unique Tryptic Peptides to Identities of the Hbs and HBOCs. Hbs are a subgroup of the large protein family, and they consist of amino acids in certain order. The tryptic peptides from trypsin digestion of Hbs and HBOCs are fingerprints of the Hbs and HBOCs. The longer the tryptic peptide sequence, the more specific the peptide is for an Hb or HBOC from which the peptide is derived. The more specific the peptide is for an Hb or HBOC, the more confident the confirmation of it by the peptide. To evaluate specificity of the unique tryptic peptides mentioned above to the identity of Hb or HBOC, a sequence similarity search was conducted using the Fasta Program against Swiss-Prot Protein Sequence Database. Fasta search results are summarized in Table 4. As indicated by the search results, the unique tryptic peptide from bovine Hb R chain residues 69-90 (T9) has not been found in other known proteins except in yak (a wild, shaggy-haired ox of the mountains of central Asia) Hb R chain, the fragment peptide from bovine Hb R chain residues 77-90 has been found only in Analytical Chemistry, Vol. 76, No. 17, September 1, 2004
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Figure 3. Mass spectra for the chromatographic peaks of human Hb digest shown in Figure 1c at tR of 4.30 (a), 11.85 (b), 14.13 (c), 15.62 (d), and 16.65 min (e). RT, retention time (tR).
Figure 4. Mass spectra for the chromatographic peaks of equine Hb digest shown in Figure 1d at tR of 13.91 (a), 14.83 (b), and 17.94 min (c). RT, retention time (tR).
the Hb R chain of domestic water buffalo, yak, and gayal (a domesticated bovine mammal of India and Burma), and the tryptic peptide from bovine Hb β chain residues 40-58 (T6) has been 5124 Analytical Chemistry, Vol. 76, No. 17, September 1, 2004
found only in the Hb β chain of camel, antelope, and species related to ox. For the three unique tryptic peptides specific for human Hb shown in Table 4, they have been found only in Hb of
Figure 5. MS/MS spectra of the triply charged peptide ion at m/z 790 (bovine Hb R chain residues 69-90, T9) (a), the doubly charged y14 ion at m/z 738 (b) and the singly charged b8 ion at m/z 893.5 (c) of the triply charged bovine Hb R T9 at m/z 790, the triply charged peptide ion at m/z 999.9 (human Hb R chain residues 63-90, T9) (d), and the triply charged peptide ion at m/z 984 (equine Hb R chain residues 63-90, T9) (e). The collision energy used was 30 × 3 eV for CID of m/z 790 in (a), 30 × 2 eV for CID of m/z 738 in (b), 35 × 1 eV for CID of m/z 893.5 in (c), 40 × 3 eV for CID of m/z 999.9 in (d), and 40 × 3 eV for CID of m/z 984 in (e). Table 4. Specificity of the Tryptic Peptides to Identities of Bovine, Human, and Equine Hbs and HBOCsa matched sequences found in other proteinsb Hb chain
residue
original sequence
bovine Hb R bovine Hb R bovine Hb β
69-90 (T9c) 77-90 40-58 (T6c)
AVEHLDDLPGALSELSDLHAHK PGALSELSDLHAHK FFESFGDLSTADAVMNNPK
human Hb R
63-91 (T9c)
VADALTNAVAHVDDMPNALSALSDLHAHK
human Hb β human Hb β
42-60 (T5c) 68-83 (T9c)
FFESFGDLSTPDAVMGNPK VLGAFSDGLAHLDNLK
equine Hb R equine Hb β equine Hb β
63-91 (T9c) 9-30 (T2-3c) 41-59 (T5c)
VGDALTLAVGHLDDLPGALSNLSDLHAHK AAVLALWDKVNEEEVGGEALGR FFDSFGDLSNPGAVMGNPK
species
Hb chain
yak domestic water buffalo, yak, gayal greater kudu antelope, gayal, domestic water buffalo, wild banteng, camel, llama gorilla, orangutan, black-handed spider monkey, common gibbon, black-tailed marmoset gorilla, lion, common gibbon silvery marmoset,tamarin, gorilla, Presbytis entellus, orangutan, gibbon, chimpanzee, monkey, lion, kulan, donkey, plains zebra kulan, mountain zebra not found
R R β R β β R β
a Fasta search results conducted against Swiss-Prot database as of October 15, 2003. b 100% ungapped match. c Designation of a tryptic peptide predicted from simulated digestion of an Hb by trypsin.
a few species related to monkey, ape, and lion. Another specific tryptic peptide from human Hb β chain residues 10-18 is not listed in Table 4 since its sequence (SAVTALWGK) is too short to be useful for confirmation of human Hb. For the three unique tryptic peptides specific for equine Hb, they have been found only in Hb of a few species related to donkey and zebra. The Fasta search results indicated that these unique tryptic peptides are quite specific for bovine, human, and equine Hbs, respectively.
CONCLUSION Tryptic peptides specific for bovine, human, and equine Hbs were found by comparing LC-MS chromatographic peak profiles of tryptic digests of OXY, bovine Hb, human Hb, and equine Hb. The amino acid sequences of the peptides were elucidated from ESI MS spectra and confirmed by MS/MS spectra. Unique specificity of the peptides to the identities of OXY and the Hbs was demonstrated by Fasta search results of amino acid sequence Analytical Chemistry, Vol. 76, No. 17, September 1, 2004
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similarity. The results from this study are very useful for the detection and confirmation of the presence of bovine, human, and equine Hbs as well as HBOCs of bovine or human Hb origin in a test sample. Based on these results, a novel method for the detection, identification, confirmation, and quantification of OXY, HMP, and HML in equine and human plasma was developed and is published in another report.19
ACKNOWLEDGMENT This study was funded by the Pennsylvania Horse and Harness Racing Commissions for which we are very thankful. The authors also express their appreciation to Thoroughbred Horse Association at Philadelphia Park, The Horsemen Benevolent of Pennsylvania Association at Penn National, and The Horsemen Association at Pocono Downs for their cooperation, continuing support. and encouragement. The authors are equally thankful to the National Center for Bioinformatics (NCBI) for providing free access to its databases, to European Bioinformatics Institute for similar access to its Fasta search engine, and to Swiss-Prot Protein Sequence Database Library. (19) Guan, F.; Uboh, C.; Soma, L.; Luo, Y.; Jahr, J. S.; Driessen, B. Anal. Chem. 2004, 76, 5127-5135.
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SUPPORTING INFORMATION AVAILABLE (1) Amino acid sequences of bovine, human, and equine hemoglobins. (2) Enlarged portion of MS spectra of the chromatographic peak of OXY digest at tR of 11.44 min in Figure 1a showing the cluster of MS peaks around m/z 790. (3) The effect of cone voltage setting on intensity of the triply charged ion m/z 790 and the doubly charged ion m/z 738 showing that the intensity of m/z 738 is higher than that of m/z 790 even at low cone voltage (a) and the intensity of m/z 738 increases with increase in cone voltage. (4) MS spectra of the chromatographic peak (a) of Oxyglobin digest at retention time of 18.03 min shown in Figure 1 and that (b) of bovine Hb digest at retention time of 18.21 min showing the only difference found between Oxyglobin and bovine Hb digests. (5) MS/MS spectra of the doubly charged peptide ions at m/z 1046 (bovine Hb β chain residues 42-58) and at m/z 1030 (human Hb β chain residues 42-60). (6) MS peaks and their interpretations for all the chromatographic peaks of Oxyglobin, bovine Hb, human Hb, and equine Hb digests shown in Figure 1. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 4, 2003. Accepted June 10, 2004. AC035425T
