(Received for publication, May 8, 1995; and in revised form, June 15, 1995)
From the
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
The extracellular Hb ( 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) . 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) 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 A weakness of the ESI-MS method can be the formation of
``adducts'' of the proteins under examination. (i) Adducts
with Na 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.
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.
)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.
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)
- 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).
-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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.