Bioconjugate Chem. 1997, 8, 400−406
400
Electrospray Mass Spectrometry of r and β Chains of Selected Hemoglobins and Their TNBA and TNB Conjugates Maciej Adamczyk* and John C. Gebler Department of Chemistry (D9NM), Abbott Laboratories, Diagnostics Division, Building AP-20, 100 Abbott Park Road, Abbott Park, Illinois 60064. Received December 4, 1996X
The molecular weights of R and β hemoglobin chains from 15 different vertebrate animal sources and 2 common human variants were determined by electrospray mass spectrometry and compared to the calculated masses based on published amino acid sequences. Conjugates were prepared for 14 of the globins using 2 traditional colorimetric derivatizing reagents, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and trinitrobenzenesulfonic acid (TNBS), and the mass of each conjugate was determined by mass spectrometry.
INTRODUCTION
(Hb)1
Hemoglobin is the major interplasmic protein of vertebrate red blood cells. Its primary function is O2 and CO2 transport (1). In its native state, Hb from vertebrates is a tightly folded tetrameric protein comprised of the noncovalent association of two similar chains (2R2β) and heme (2). Typical denaturing conditions such as heat, acid, or organic solvents will dissociate Hb into the individual components (heme, R and β globins). Due to its critical biological function, high abundance, and ease of isolation, Hb is one of the most intensely studied proteins. The most fundamental characteristic of Hb is its primary amino acid sequence which corresponds to a specific molecular weight. Primary sequences for many Hbs were determined by polypeptide degradation/peptide sequencing or DNA/RNA sequencing. Reported molecular weights for R and β Hb chains were calculated from the amino acid sequences. Traditionally, gel electrophoresis has been used to verify Hb molecular weight; however, its limited resolution (∼5%) is not suitable for primary sequence verification (3). Moreover, detailed studies of mutations, amino acid modifications, or protein homogeneity cannot be accurately examined by electrophoresis. Over the past several years, electrospray ionization mass spectrometry (ESI-MS) has become a powerful means of determining the molecular weight of peptides and proteins to ∼200 kDa with a precision e0.01%. ESIMS is a nondestructive ionization technique which typically gives only ions of the intact parent compound. The principle of ionization and mass analysis by ESI-MS is described in several excellent review articles (4). ESIMS along with other mass spectrometry techniques has been utilized for detailed studies of human Hbs and their variants (5). * Correspondence should be addressed to this author. Telephone: (847) 937-0225. Fax: (847) 938-8927. E-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, May 1, 1997. 1 Abbreviations: BSA, bovine serum albumin; CZE, capillary zone electrophoresis; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); ESI-MS, electrospray ionization mass spectrometry; Hb, hemoglobin; Hbs, hemoglobins; HPLC, high-performance liquid chromatography; LC, liquid chromatography; PPG, poly(propylene glycol); SDS, sodium salt of dodecyl sulfate; TNB, trinitrobenzene; TNBA, thionitrobenzoic acid; TNBS, trinitrobenzenesulfonic acid.
S1043-1802(97)00039-6 CCC: $14.00
In this work, we describe the use of ESI-MS for the characterization of R and β Hb chains from selected species. In addition, we demonstrate the usefulness of ESI-MS to assess the effects of two traditional derivatizing reagents, DTNB and TNBS, used for the colorimetric quantification of cysteines and lysines. We prepared thionitrobenzoic acid (TNBA) and trinitrobenzene (TNB) conjugates with 14 of the Hbs and measured the molecular weight of each new conjugate by ESI-MS. This information allowed us to determine the number of TNBA or TNB groups attached to each globin. MATERIALS AND METHODS
Hemoglobins from baboon, bovine, cat, dog, garter snake, goat, horse, human A0,2 human A2,3 human S,4 mouse, pig, pigeon, rabbit, rat, sheep, and turkey, and BSA and horse heart myoglobin were obtained from Sigma (St. Louis, MO). 5,5′-Dithiobis(2-nitrobenzoic acid) was purchased from Pierce (Rockford, IL), and 2,4,6trinitrobenzenesulfonic acid, sodium salt, was from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals and solvents were of the highest purity available. HPLC was carried out on a Microtech Scientific (Sunnyvale, CA) binary microbore LC system employing a Polymer Laboratories (Amherst, MA) PLRP-S 8 µm 1000 Å 50 × 2.1 mm column. Mass spectra were recorded on a Perkin Elmer Sciex API 100 single quadrupole mass spectrometer equipped with a heated pneumatically-assisted ion source (TurboIonSpray). The ESI-MS was calibrated just prior to analysis using the manufacturer’s protocols and calibration solutions (0.1 mM PPG 1000, 0.2 mM PPG 2000, 0.1% formic acid, and 2 mM ammonium acetate in 1:1 water/methanol). The calibration solutions provide ions throughout the mass range from 350 to 2400 amu. Accuracy and precision were determined using horse heart myoglobin and BSA by infusion of each protein (0.1 mg/mL in 0.1% formic acid, 2 mM ammonium formate, and 20% acetonitrile) directly into the instrument’s ion source at a flow rate of 50 µL/min. All spectra were collected using multi-scan-averaging (10 scans for myoglobin and 20 scans for BSA). Spectra for each Hb were obtained on 2 µL of a 1 mg/ mL solution (∼125 pmol) in 0.1% formic acid. Samples were injected into the HPLC column and eluted with a 2
Normal human Hb. Purified minor human Hb A2 comprised of (2R2δ). 4 Purified Hb from humans with sickle cell disease (β chain mutation A6T). 3
© 1997 American Chemical Society
Bioconjugate Chem., Vol. 8, No. 3, 1997 401
ESI-MS of Hb R and β Chains
Table 1. Calculated versus Observed Molecular Weights of r and β Globins of Various Hemoglobins calculateda source (references)
R globin
baboon (24, 25) bovine (26, 27) cat (21)
15435.7 15053.1 15305.4
dog (28)
(R-I) 15217.3 (R-II) 15247.3 nab
garter snake goat (29, 30) horse (31) human A0 (32) human A2 (33) human S (34) mouse (35)
(R-I) 15033.1 (R-II) 15060.1 15114.3 15126.3 15126.3 15126.3 14995.0
pig (36) pigeon (14, 15, 16)
15039.1 15123.2
rabbit (37, 38) rat (39) sheep (40, 41) turkey (17)
15457.6 15197.3 15047.1 (RA) 15309.7 (RD) 15649.8
observed by ESI-MS β globin
R globin
15895.2 15954.4 (β-A) 15926.2 (β-B) 15943.1 15996.3
15435.4 ( 1.0 15053.5 ( 1.1 15305.7 ( 1.9
na 16021.4 16008.3 15867.2 (δ) 15924.3 15837.2 (major) 15616.8 (minor) 15709.0 16035.3 16152.6 16001.3 15848.2 16073.4 16306.8
(R-I) 15217.8 ( 1.8 (R-II) 15247.2 ( 1.0 15445.4 ( 1.9 15802.6 ( 0.6 (R-I) 15033.3 ( 1.2 (R-II) 15060.0 ( 2.0 15115.1 ( 1.2 15126.7 ( 1.4 15126.8 ( 0.9 15126.0 ( 1.0 14995.9 ( 1.5 15040.2 ( 1.2 15117.1 ( 0.6 15150.2 ( 1.2 15458.5 ( 0.9 15197.6 ( 1.1 15047.0 ( 0.6 (RA) 15308.5 ( 2.1 (RD) 15767.0 ( 1.0
β globin 15894.1 ( 1.2 15954.9 ( 1.5 (β-A) 15924.9 ( 2.1 (β-B) 15941.6 ( 2.5 15995.8 ( 1.0 16028.3 ( 1.6 16021.8 ( 1.0 16008.2 ( 0.6 15867.4 ( 0.7 15922.8 ( 1.3 15836.4 ( 0.5 (major) 15617.2 ( 1.3 (minor) 15708.3 ( 1.7 16035.6 ( 1.3 16164.0 ( 0.3 16006.2 ( 1.6 15848.7 ( 1.3 16074.2 ( 1.0 16304.7 ( 1.3
a Molecular weights were determined from primary amino acid sequences downloaded from electronic databases (see footnotes 5 and 6). b na, not available.
Figure 1. Total ion count (TIC) chromatograph from LC/ESIMS of bovine Hb. Scheme 1
nebulizer air flow 1.5 L/min; ion spray 4500 V; counterelectrode (curtain plate) 1000 V; and orifice-to-skimmer potential difference 30 V. The instrument scanned a range of 650-1450 m/z in 0.1 amu steps for a total scan time of 8 s. The resulting spectra were “reconstructed” (deconvoluted) based on the Bayesian statistical analysis of the entire spectrum using the manufacturer’s software (BioMultiview version 1.2) which gave the results as true mass. No other data manipulation such as smoothing, noise filtering, or enhancement was employed. For presentation of spectra, files worked up in BioMultiview were imported as ASCII text files and then graphed and labeled using Axum (version 5.0 for Windows, Math Soft, Cambridge, MA). Conjugation with DTNB was carried out using a modified method of Ellman (6). To Hb solutions (50 µL of 1 mg/mL in 100 mM phosphate buffer, pH 8.0) was added a solution of DTNB (50 µL of 4 mg/mL in H2O). The reaction mixture was incubated for 15 min at ambient temperature and then directly analyzed by LCESI-MS using the protocol for unmodified Hbs described earlier. The number of attached TNBA groups was determined from the equation:
no. of TNBA groups in conjugate ) TNBA conjugate mol wt - native Hb mol wt mol wt of TNBA group
linear gradient of 10% acetonitrile in 0.1% TFA to 100% acetonitrile in 20 min at a flow rate of 100 µL/min. The column output was directly linked to the ESI-MS ion source. ESI-MS operating parameters were as follows: interface auxiliary air flow of 4-5 L/min at 350 °C and
Conjugation with TNBS was based on a modified method of Kakade et al. (7). To Hb solutions (100 µL of ∼1 mg/mL in 4% aqueous sodium bicarbonate) was added a solution of TNBS (100 µL of 1 mg/mL in water). The reaction mixture was incubated at 37 °C for 2 h and then treated with hydrochloric acid (100 µL, 1 N) and SDS solution (100 µL, 10% w/v). The reaction mixture was directly analyzed by LC/ESI-MS using the protocol for unmodified Hbs previously described. The number of attached TNB groups was determined from the equation:
no. of TNB groups in conjugate ) TNB conjugate mol wt - native Hb mol wt mol wt of TNB group
402 Bioconjugate Chem., Vol. 8, No. 3, 1997
Adamczyk and Gebler
Figure 2. (A) Mass spectra for bovine Hb R and β globins. (B) Deconvoluted mass spectra for bovine Hb R and β globins. RESULTS AND DISCUSSION
Molecular weight estimation was carried out on a single quadrupole electrospray mass spectrometer equipped with a heated pneumatically-assisted ion source interfaced with a microbore HPLC system. Typical ESIMS instrumentation has a precision of e0.01%. Adopting the method of Feng et al. (8), we calibrated the instrument with a mixture of two poly(propylene glycol) solutions (average molecular weight of 1000 and 2000) employing multichannel averaging to improve signal-tonoise and reduce minor random peak position drifting between scans. As a result, we routinely achieved a precision of
