©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Electrospray Ionization Mass Spectrometric Determination of the Complete Polypeptide Chain Composition of Tylorrhynchus heterochaetus Hemoglobin (*)

(Received for publication, May 8, 1995; and in revised form, June 15, 1995)

Brian N. Green (1) Tomohiko Suzuki (2) Toshio Gotoh (3) Askar R. Kuchumov (4) Serge N. Vinogradov (4)(§)

From the  (1)From VG Organic, Altrincham, Cheshire WA14 5RZ, United Kingdom, the (2)Department of Biology, Kochi University, Kochi 780, Japan, the (3)Department of Biology, University of Tokushima, Tokushima 770, Japan, and the (4)Biochemistry Department, School of Medicine, Wayne State University, Detroit, Michigan 48201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Electrospray ionization mass spectrometry (ESI-MS) of the native, reduced, and carbamidomethylated forms of the extracellular, 3.38-MDa hemoglobin from the marine polychaete Tylorrhynchus heterochaetus, when combined with a maximum entropy (MaxEnt) analysis, provided a complete description of the polypeptide chain composition. This hemoglobin, a hetero-multimeric complex of approximately 180 polypeptide chains, consisting of globin and linker subunits in an 3:1 mass ratio, is among the largest protein complexes investigated by ESI-MS. The globin subunits consist of a monomer subunit (chain I, 15575.4 Da) and a disulfide-bonded trimer subunit, 50068.4 Da, consisting of globin chains IIA (16601.9 Da), IIB (16680.4 Da), and IIC (16,794.0 Da). Linker subunits L1-L5, 23233.8, 24835.4, 25326.9, 28202.2, and 26317.2 Da, respectively, were found together with a disulfide-bonded dimer of L2, 52609.4 Da. Using the exact masses of the subunits, a plausible model of the hemoglobin consisting of 144 globin chains (36 monomers and 36 trimers) and 36 linker chains provides a calculated mass of 3.42 MDa.


INTRODUCTION

The extracellular Hb (^1)from the marine polychaete Tylorrhynchus heterochaetus is a typical HBL Hb found in annelids and vestimentiferans(1, 2, 3) . Its molecular mass of 3.38 MDa (4) and molecular dimensions obtained by a scanning transmission electron microscope and small angle x-ray scattering (5) are similar to other HBL Hbs. Suzuki and Gotoh and their collaborators have isolated and sequenced four globin chains (I, IIA, IIB, and IIC) (4, 6, 7, 8) and two linker chains L1 and L2 (9) of this Hb. Because of the amount of structural information already available, an ESI-MS study of this Hb was undertaken in order to both validate the known sequences and determine whether additional components were present in this giant complex of globin and linker chains.


MATERIALS AND METHODS

The Hb was prepared as described previously(7) , dialyzed against distilled, deionized water, lyophilized, and stored at -70 °C. Carbamidomethylation with iodoacetamide was carried out under the same conditions as carboxymethylation(10) ; upon termination of the reaction, the protein was thoroughly dialyzed against distilled, deionized water and lyophilized. ESI-MS data were acquired on a VG Quattro II electrospray mass spectrometer (VG Organic, Altrincham, Cheshire, UK), using sample concentrations of 0.5 µg/µl in 50% aqueous acetonitrile containing 0.2% formic acid. The sample flow rate into the electrospray source was 5 µl/min, and data were typically acquired for 5-10 min while scanning over the m/z range 600- 2500 at 10 s/scan. The cone voltage (sample orifice to skimmer potential) ramp was employed to optimize sensitivity over the m/z range scanned.

ESI-MS produces a series of multiply charged ions on the m/z scale from each protein in the sample. On this scale, m/z = (M + nH)/n, where M is the mass of the protein, H is the mass of the proton, and n is a series of consecutive integers. Since the Hb data arise from several proteins, each producing a series of 5-10 multiply charged ions, they were processed in order to condense each series into a single peak on a true molecular mass scale. Processing used a maximum entropy (MaxEnt)-based approach (11, 12) employing the MemSys5 program (MaxEnt Solutions Ltd., Cambridge, UK) incorporated as part of the VG MassLynx software suite on a 60-MHz Pentium PC supplied with the spectrometer. The multiply charged series from horse heart Mb (Sigma), mass of 16,951.5 Da(13) , was used for mass scale calibration. The masses are based on the following atomic weights of the elements: C = 12.011, H = 1.00794, n = 14.00674, O = 15.9994, and S = 32.066(14) .


RESULTS AND DISCUSSION

ESI-MS analyses were made on the native Hb (three determinations), the reduced and carbamidomethylated Hb (two determinations), and on the native Hb after exposure to DTT for 10, 30, and 60 min (one determination each). Fig. 1A shows the raw, multiply charged spectrum of the native Hb and Fig. 1B the corresponding MaxEnt processed spectrum. Fig. 1, C and D, shows the MaxEnt processed spectra of the native Hb partially reduced with DTT (after 30 min) and of the reduced and carbamidomethylated Hb, respectively. Table 1and Table 2present the measured and calculated masses of the constituent chains and subunits of Tylorrhynchus Hb and compare the experimental values with those calculated from the known amino acid sequences.


Figure 1: A, raw ESI-MS of native Tylorrhynchus Hb. The labels on the peaks denote the polypeptide chain or subunit and the total positive charge: M, monomer; T, trimer; D, dimer; and L, linker. B, the MaxEnt processed data shown in A. C, the MaxEnt processed data for Hb reduced with 5 mM DTT for 30 min. D, the MaxEnt processed data for the reduced and carbamidomethylated Hb.







There are four globin chains; of these IIA, IIB, and IIC have measured reduced masses (Table 1) that are within experimental error (±1.0 Da) of the reduced masses calculated from their known sequences(4, 7, 8) . Chain I, representing the monomer subunit, has an ESI-MS mass of 15,575.4 Da, 127.5 Da higher than the sequence mass (6) assuming one disulfide bond. The simplest explanation for the discrepancy is that the sequence should contain an extra Gln (128.1 Da) or Lys (128.2 Da). Surprisingly, chain I is observed in the native (Fig. 1B) and reduced native Hb (Fig. 1C) but not in the carbamidomethylated Hb (Fig. 1D). An additional monomer component, 15592 Da, is observed in the native Hb (Fig. 1B) and reduced Hb (Fig. 1C). The 16.5-Da difference is within the range found for many protein adducts in ESI-MS (see discussion below).

The ESI-MS results exhibit good agreement between the measured mass of the trimer subunit (chains IIA + IIB + IIC), 50068.4 ± 1.7 Da (mean ± S.D. of 3 determinations), and the sum of the masses of its component chains, 50066.2 Da (Table 2). Furthermore, the results confirm the number of Cys residues found in the three chains and the finding that the trimer contains 3 intrachain (one within each chain) and 2 interchain disulfide bonds IIA-IIC-IIB(15) .

The linker chains L1 and L2, whose sequences have been determined(9) , are not detected as such in the ESI-MS of native Hb. Only a disulfide-bonded dimer (L2)(2) is found at 52609.4 Da (S.D. ± 0.2 Da; estimated maximum error ± 4.0 Da). The measured reduced mass of L2 derived from the carbamidomethylated Hb data is 26317.2 Da (27001.8 - 12 57.052) agrees, within experimental error, with the reduced sequence mass (26316.2 Da) (Table 1). Furthermore, using the measured value and assuming that, of the 12 Cys present, only 1 is involved in interchain disulfide bonding, the calculated mass of the dimer, (L2)(2) - 22H = 52612.2 Da, is 2.8 Da higher than the mass of the dimer measured directly, 52609.4 Da, within the estimated experimental error of ±4.0 Da (Table 2). The measured reduced mass of linker L1 derived from the carbamidomethylated Hb data is 28886.8 - 12 57.052 = 28202.2 Da (estimated maximum error ± 2.0 Da) in agreement with the sequence mass (28200.5 Da) within the experimental error. However, a dimer of L1 was not detected in the ESI-MS of native Hb. Comparison of the results obtained with DTT-reduced Hb and with carbamidomethylated Hb, confirms that L1 and L2 each contain 12 Cys (Table 1).

In addition to the foregoing linkers, the ESI-MS results provide evidence for the possible existence of three, hitherto undetected linker chains, L3, 25326.9 Da, L4 (minor), 24835.4 Da, and L5, 23233.8 Da, with L3 and L5 containing 10 Cys (Table 1). However, the masses of these putative linkers and the number of Cys residues therein are consistent with their being degradation products of L1 and L2. Thus, cleavage of L1 at Arg-Ser and Asp-Pro would provide fragments 29-253 (25325.3 Da) and 34-253 (24833.8 Da) whose masses are within experimental error of the masses of L3 and L4, 25326.9 Da and 24835.4 Da, respectively (Table 1). Cleavage of L2 at Ala-Gly would give a fragment of 23232.7 Da to be compared with L5 23233.8 Da (Table 1). It should be noted that the Asp-Pro peptide bond is known to be labile in acid solution (16) and in the gas phase(17) . A referee has pointed out that although cleavage at the Arg-Ser bond is also possible, the Ala-Gly cleavage of L2 is highly improbable.

A weakness of the ESI-MS method can be the formation of ``adducts'' of the proteins under examination. (i) Adducts with Na and K (22 and 38 Da higher in mass) occur widely(18) . (ii) Adducts of protein with heme (616.5 Da), such as heme-apomyoglobin, have been observed(19) . (iii) Adducts with an increase in mass of 16-18 Da, which may be products of oxidation or hydroxylation are also encountered. Such a mass difference between two forms of bovine seminal RNase monomer, 13,733 Da and 13,749 Da, was ascribed recently to the oxidation of a Cys sulfhydryl to a sulfinic acid (-SOH)(20) . A mass difference of +16-18 Da will not be resolved from +22 Da; hence, depending on the relative intensities of its component(s), the peak(s) around +16-22 Da may be of indeterminate mass and multiple origins. (iv) Components of +98 Da have been observed with many proteins from natural sources(21) : they are believed to be adducts of either phosphoric or sulfuric acid. Examples of these adducts can be seen among many of the lesser peaks in Fig. 1. These phenomena make it imperative to examine a given protein in more than one form.

The results described above provide what appears to be a complete and self-consistent description of the subunit and polypeptide chain composition of Tylorrhynchus Hb. In addition to validating the sequences of three of the four globin chains and two linker chains, it demonstrates the presence of three additional linkers and provides a semiquantitative estimate of the relative proportions of the component chains. Based on the latter, an updated and plausible version of the quaternary structure proposed earlier (22) can be calculated as shown in Table 3; the resulting calculated mass of 3.42 MDa is very close to the sedimentation equilibrium mass of 3.38 MDa(4) .



The foregoing results present a striking illustration of MaxEnt processing of ESI-MS data to obtain the complete resolution of all the chains/subunits of a large, heteromultimeric protein complex (similar in size to a ribosome), generally one of the more intractable problems in biochemistry. ESI-MS provides complete mass information in unprecedented detail and accuracy, coupled with a relatively short time required for the analyses.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DK38674 (to S. N. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, Wayne State University, 540 East Canfield, Detroit, MI 48201. Tel.: 313-577-1511; Fax: 313-577-2765; svinogr{at}cms.cc.wayne.edu.

^1
The abbreviations used are: Hb, hemoglobin; Mb, myoglobin; HBL, hexagonal bilayer; ESI-MS, electrospray ionization mass spectrometry; DTT, dithiothreitol.


REFERENCES

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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.