CLIN.CHEM.38/8, 1444-1448 (1992)
Characterizationof Hemoglobin Lepore Variants by AdvancedMass-Spectrometric
Procedures
Maurizio De Caterina, Pasquale Esposito, Ernesto Grunaldl, Giosu#{233}
Di Maro, Franco Scopacasa, Pasquale
Ferranti,1 Anna Parlaplano, Antonio Malorni,’ Pletro Puccl,”3 and Gennaro Marlno”2
We describe
(9, 10) and in other Mediterranean
populations
(6).
However, little is known about the relative incidence of
the different Hb Lepore molecular types, given that the
structural definition of the specific Lepore variants
needs careful investigation at the molecular level. This
characterization
is at present accomplished by DNA
restriction
polymorphism analysis (11, 12) or by DNA
sequencing after amplification of the appropriate Lepore-specific gene fragment
through polymerase chain
reaction (13).
We have explored an alternative
approach: the use of
advanced mass-spectrometric
procedures. In the last
decade, fast atom bombardment
mass spectrometry
(FAB/MS) has been successfully applied to the analysis
for hemoglobin variants by several authors (14-18). The
AddItIonal Keyphraus:
electrospray mass spectromeby
fast atom bombardment mass spectmmefty
hybrid globins
peptide mixture generated by the proteolytic digest of
peptkles - genetic variations
the abnormal globin chain can be directly analyzed by
FAB/MS, and the anomalous mass value(s) recorded in
Hemoglobin Lepore (Hb Lepore) is a class of structurthe spectra can be used to identify the structural abnorally abnormal hemoglobins composed of normal a
mality.
Recently,
electrospray
mass spectrometry
chains and abnormal non-a chains, the latter being
(ESMS) has been used to accurately
determine
the
hybrid proteins that contain the N-terminal sequence of
molecular mass of large biomolecules (19-21). Green et
a 8 chain and the C-terminal sequence of a (3 chain.4
al. (22,23) have already shown that ESMS is suitable
This &-$ fused gene originated from a nonhomologous
for rapid identification of globin chain variants, even in
crossing-over between misaligned 8 and (3 genes (1).
the presence of the corresponding
normal chain, proThree different molecular types of Hb Lepore have
vided that their masses are 14 mass units (amu) apart.
been described so far, in which the transition
from #{244}to Here we describe an analytical protocol for the rapid
(3 sequences occurs at different positions along the
purification,
identification,
and structural characterizapolypeptide chain. Hb Boston (887-5116) and Hb Baltition of the various molecular types of Lepore and Leporemore (850-586) are the two most common molecular
like variants,
making
use of these advanced
masstypes; Hb Hollandia (822-550) represents a very rare
spectrometric
procedures. The abnormal chains from
variant (2-4).
the most common Lepore hemoglobins,
Hb Boston and
Hb Lepore is produced at a markedly low rate; in the
Hb Baltimore, are directly analyzed by ESMS to idenheterozygous
state, it accounts for only 6% to 15% of the
tify the variant globin through accurate determination
total hemoglobin. The Hb Lepore syndrome can easily
of its molecular mass. Alternatively,
the anomalous
be recognized by detection with standard hemoglobin
globins are digested with trypsin and their nature is
electrophoresis procedures, in which Hb Lepore shows
determined by FABIMS analysis of the resulting peptide
about the same mobility as Hb S (5). In some cases, Hb
mixture.
Lepore is associated with (3-thalassemia
(6) or with
other hemoglobin variants such as Hb Beograd (6), Hb S
Materials and Methods
(7), and Hb E (8). The relative incidence of Hb Lepore
The variant globin chains from Hb Lepore Boston and
variants
is particularly high in Southern Italy (-5.4%)
Hb Lepore Baltimore were purified from the blood of
heterozygous
individuals
by a two-step chromatographic procedure. The abnormal Lepore hemoglobins
Servizio Speciale di Ematologia di Laboratorio (II Facolt#{224}
di
Medicina e Chirurgia), Universit#{226}
di Napoli, Napoli, Italy.
were separated
from the normal Hb A by ion-exchange
‘ICMB and Servizio di Spettrometria di Massa del CNR, Via
chromatography
with “F’pT” apparatus
(Pharmacia,
Pansini 5,80131 Napoli, Italy.
Uppeala, Sweden). The hemolysate was diluted to an Hb
2CEfl’GE, Centro di Ingegneria Genetica,Napoli, Italy.
3Author for correspondence.
concentration of -4 g/L with 50 mmol/L ‘l’ris HC1
4Nonstandard abbreviations: Hb, hemoglobin; ESMS, electrobuffer (pH 8.5) containing KCN (0.1 g/L), and 500 zL
spray mass apectrometry and FAB/MS, fast atom bombardment
was loaded onto a Mono Q column (Pharmacia) previmass spectrometry.
ously equilibrated in the same buffer. The protein samReceived January 29, 1992; accepted March 31, 1992.
an analytical protocol for characterizing the
of hemoglobin (Hb) Lepore variants
by using two different mass-spectrometnc
approaches.
The first method consists of direct examination of the
chromatographically
separated hybrid globins by electrospray mass spectrometry; the variant Lepore globin is
Identified through the accurate determination of its molecular mass. Alternatively, the anomalous globins are digested with trypsin and their structures are determined by
fast atom bombardment mass-spectrometric analysis of
the peptide mixture. The application of this procedure to
the identification of Hb Lepore Boston and Hb Lepore
Baltimore is described.
molecular
structure
-
.
1444
CLINICALCHEMISTRY,Vol.38, No.8, 1992
ples were eluted with a gradient elution system consisting of 50 mmolJL Ths HC1, pH 8.5 (buffer A) and 50
mmol/L Tris HC1, pH 6.5 (buffer B), each containing
KCN, 0.1 g/L; in the linear gradient, buffer B was
increased from 0% to 75% of the elution buffer over 40
mm. The Lepore hemoglobins,
which were eluted before
the normal Hb A, were manually collected. The hemoglobin solutions were desalted and concentrated
to a
minimum volume by use of CS/10 cartridges (Amicon,
Danvers, MA). The abnormal hybrid chains were obtained from the Lepore hemoglobins
by reversed-phase
HPLC with a 25 x 0.46 cm Vydac 04 column (5-tm
particles; Vydac, Hesperia, CA), essentially as described
by Shelton et al. (24) with slight modifications. The
Lepore globin chains were collected and either directly
analyzed by ESMS or dried down in a Speedy Vac
centrifuge (Savant Instruments,
Farmingdale, NY) and
submitted to hydrolysis with trypsin.
The trypsin
(L-1-tosylamide-2-phenylethylchloromethyl ketone-treated,
cat. no. T8642; Sigma Chemical
Co., St. Louis, MO) digestion of the variant globins was
carried out in 4 g/L ammonium bicarbonate, pH 8.5, at
37 #{176}C
for 4 h.
ESMS analysis for the intact Lepore globins was
performed with a BIO-Q triple-quadrupole
mass spectrometer (VG, Manchester, UK). HPLC-purifled globin
samples (10 L, 25-50 pmol) were injected into the ion
source at a flow rate of 2 41mm; the spectra were
scanned from m/z 1600 to 600 at 10 s/scan. Mass-scale
calibration
was carried out by using the multiple
charged ions of a separate introduction of myoglobin; all
ESMS molecular
masses are reported
as average
masses.
FAB/MS spectra were recorded with a VG ZAB 2 SE
double-focusing
mass spectrometer
equipped with a cesium gun operating at 22 kV (2 MA). Samples (1-3 nmol)
were dissolved in 0.1 mol/L HC1 and loaded onto a
glycerol-coated
probe tip; thioglycerol was added just
before insertion into the ion source. Spectra were recorded on ultraviolet sensitive paper and counted manually; the mass signals were assigned to the corresponding peptides along the Lepore globin sequences on the
basis of their molecular
masses. All the mass values
recorded in FAB mode are shown as monoisotopic
masses.
Results and DIscussion
Figure 1 shows the FPLC elution profile of the hemolysate from an individual heterozygote for Hb Lepore
Boston. The Lepore Hb was eluted from the Mono Q
column well before the normal Hb A peak by use ofa
decreasing pH gradient. The third peak that was eluted
after Hb A was identified as fetal hemoglobin (Hb F),
the concentration of which increases in the Lepore
heterozygous state.
Once the Lepore hemoglobins
were separated from
the normal Hb A, the hybrid globin chains were easily
obtained by reversed-phase
HPLC with a Vydac C4
column by the procedure described elsewhere (24).
The molecular definition of the particular type of Hb
0.I
E
C
0
C
HbF
008
Fig. 1. FPLC elution profile of Hb separation
Hemolysatefroman individualheterozygous for Hb LeporeBostonwas loaded
onto a Mono 0 column; the hemoglobinsamples were eluted with a descendlog pH gradient Hb Lepore Boston was eluted from the column after 13 rThn,
well separated from the normal Hb A
Table 1. Measured and Predicted Molecular Messes of
Globin Chains from Normal and Lepore Hemoglobins
Molscutsrmess, OS
GlobIn typo
Normala
Normalp
Boston
p BaltImore
p Hollandla
Prodsd
Measured
15 126.6
±
0.7
15866.7 ±0.3
15864.6
15822.2±
±
0.9
0.5
15126.4
15867.2
15865.2
15822.1
15837.2
-0.7
-0.7
-0.6
+0.1
Lepore variants was performed with the two massspectrometric approaches, each of which is based on the
structural differences
amongst the various Lepore
globin chains. The following structural analyses were
carried out on the hybrid globin chains from Hb Boston
and Hb Baltimore (no samples of Jib Hollandia were
available).
In the first procedure, we determined the accurate
molecular mass of the hybrid globins by ESMS. In fact,
because the molecular masses predicted for all the
Lepore globin chains differ uniquely as shown in Table
1, the exact measurement
of their molecular
masses
provides a straightforward
identification of the particular Jib Lepore variant.
Subjecting
20-50 pmol of the Lepore globins to ESMS
analysis gave rise to the characteristic
bell-shaped distribution of multiply charged ions with between 13 and
19 charges, from which the molecular masses of the two
globins were easily calculated (Figure 2). The globin
from Hb Lepore Boston showed a measured molecular
mass of 15 864.6 (±0.9) Da, whereas the mass determined from the globin from Hb Lepore Baltimore was
15822.2 (±0.5) Da. Both values are in excellent agreement with those predicted on the basis of the amino acid
sequences of the two Lepore globins (Table 1).
The multiply charged ion spectra were transformed to
CLINICALCHEMISTRY,Vol. 38, No. 8, 1992 1445
Fig. 2. Electrospray mass-spectrometrlc analysis for Lepore globin
chains
The hybrid chains from Hb Lepore Boston (A) and Hb Lepore Baltimore (B)
were purified by HPt.C and dIrectly submitted to ESMS analysis. A characteristic bell-shaped distribution of multiply charged Ions was produced; the
calculated molecular masses of the two Lepore globins are shown
a real mass scale to allow easy identification of all the
present in the globin samples; the results
are shown in Figure 3.
The transformed spectrum of the Jib Lepore Boston
globin (Figure 3A) shows the presence of two components; the major peak corresponds to the molecular mass
of the Lepore globin, whereas the minor one, exhibiting a
higher mass value, was interpreted as an artifact adduct
formed during the mass analysis, in agreement
with the
findings of Chowdhury et al. (25). These authors, in fact,
demonstrated that protein and peptide ions often show a
strong propensity for attachment to sulfuric acid and (or)
phosphoric acid moieties (both have a molecular mass of
98 Da), possibly present in traces in the original sample
or in the reagents and solvents used during purification.
These unwanted adduct ions practically disappeared after treatment
of the globin solution with dilute HC1
before ESMS (data not shown).
Several components were detected in the transformed
spectrum of Jib Lepore Baltimore globin (Figure 3B).
The major component
corresponds
to the abnormal
globin chain, whereas most of the remAining components originate from the addition of sodium ions to the
globin chain. We have often observed such adducts
during ESMS analysis of proteins, niziinly when the
protein samples are stored in glass tubes for a long time
components
1446 CLINICAL CHEMISTRY, Vol. 38, No. 8, 1992
Fig. 3. Transformed electrospray mass spectra oftheLeporeglobins
The multiplychargedIon spectra of the Lepore globin chains from Hb Boston
(A) and Hb Baltimore (B) are transformed to a real mass scale. The results
Indicate the presence of protein adducts (see text)
before analysis. Adding a small amount of HCI to the
protein solution greatly decreases the amount of adducts
formed.
An alternative
and complementary
approach to an
unambiguous
and straightforward
identification of the
particular type of Lepore globin involves the analysis of
the tryptic digest of the variant globin by FAB/MS (26,
27). The hydrolysis of the Lepore globin chains with
trypsin yields a mixture of fragments specific for S chain
or chain or common to both globin chains. Given the
dissimilarity
of the primary
structure of the various
Lepore globins, because of the different points of transition from S to 9 chain, the respective tryptic digests
contain several peptides specific for each Lepore molecular type. Consequently FAB/MS analysis of the tryptic
peptide mixtures shows the occurrence of the specific
sets of mass signals that are used to identify the particular Lepore variant.
Table 2 shows the FAB/MS mass signals diagnostic
for the definition of the various Lepore variants and the
corresponding
peptides along the hybrid globin sequences; the occurrence of a particular
set of mass
signals in the FAB/MS spectra unambiguously
identifies the molecular type of Lepore globin. As an example,
Figure 4 shows the FAB/MS spectrum of the tryptic
digest from the Jib Boston globin. When the recorded
mass signals were compared with those listed in Table
2, the occurrence of the specific set of masses 959, 1256,
Ills
‘I,.
1133142)
I,-’,)
1114
III’,,’
!V0
(5,-OS.
Ill
Il-S
IH’lO4
‘VI
(“-‘U)
1310
5200
‘no
lI7
1400
1750
1750
1100
SOlO
2550
Fig. 4. FAB/MS spectrum of thetrypticdigest of Hb LeporeBoston
Purified LeporeglobIn from Hb Boston was digested with hypein and the
resultingpeptidemixture was directlyanalyzed by FAB/MS;each mass signal
Is correlated to the corresponding peptlde along the globin sequence on the
basis of Its molecular mass
Table 2. Mass Signals DIagnostic for the identification
of the Specific $ Lepore ChaIn In the FAB/MS Analysis
of the Abnormal Chains Tryptlc Digest.
Puptid.
Normal p
0 Boston
0 Baltimore
fi Hollandla
9-17
932
18-30
41-59
83-95
83-104
1314
2058
1421
2528
any Lepore hemoglobin as well as any anti-Lepore
hemoglobin. ESMS, in particular, seems to offer a
unique method of analysis for Hb Lepore through its
ability to directly determine the exact molecular mass of
the hybrid
globins.
Moreover,
this technique
can be
applied to direct examination of hemolysate without the
need for globin variant purification, even it in this case
only globin chains with molecular mass 14 amu apart
can be identified (22,23). Direct hemolysate
analysis,
for example, would have meant failure to detect and
characterize Hb Lepore Boston in a heterozygous individual because its molecular mass is only 2 amu less
than that of the normal $ chain. Therefore, despite the
outstanding performance of ESMS, identification of the
molecular type of Hb Lepore variants is greatly facilitated when the two mass-spectrometric
approaches are
used in combination-with
ESMS analysis providing a
preliminary characterization
and FABIMS mapping exposing the finer details of the abnormal globin structure.
This work was supported in part by CNR grants to P.P. and G.M.
The skillful assistance of Ms. M. E. Lisboa is gratefully acknowledg
959
1256
959
959
1256
1256
References
2044
1464
2571
2044
1421
2528
2058
1421
2528
1. Bunn HF, Forget BG. Hemoglobin: molecular, genetic and
clinical aspects, 2nd ed. Philadelphia: WB Saunders Co., 1986:
297-417.
2. Baglioni C. The fusion of two peptide chains in hemoglobin
Lepore and its interpretation as a genetic deletion. Proc Nati Aced
Sci USA 1962;48:1880-6.
3. Barnabas J, Muller CJ. Haemoglobin-Lepore
Hollandia. Nature 1962;194:931-6
4. Ostertag W, Smith EW. Haemoglobin.Lepore Baltimore, a
third type of Sp crossover (o#{176},
).
Eur J Biochem 1969;10:371-6.
5. De Caterina M, Esposito P. A simplified procedure for Rb
Lepore detection by means of “Sickle-thal”
microcoluxnn. J Lab
Med 1985;12:99-103.
6. Efremov GD. Beta-delta beta-thalassemia
and Rb Lepore
among Yugoalav, Bulgarian, Turkish and Albanian. Haematologica 1990;75:31-41.
7. Weasels RA, Rogers BB, Ou CN, Alcorn R., Buffone GL Liquid
chromatography used in diagnosis of a rare hemoglobin combination: hemoglobin S/Lepore Boston. Clin Chem 1986;32:903-6.
8. Boontrakoonpoontawee
P, SvastiJ, Fucharoen 8, Winichagoon
P. Double heterozygoeity for HbE and a Lepore-typehemoglobin
found in a Thai woman. Birth Defects 1987;23:269-74.
9. Silvestroni E, Bianco I. Screening for microcytemia in Italy:
analysis of data collected in the past 30 years. Am J Hum Genet
2044, and 2571 was easily recognized, immediately
indicating the molecular nature of the Lepore globin
chain.
The peptide 83-104 listed in Table 2 is generated by
an incomplete cleavage at Lys#{176}5.
This protein region is
located within the globin “core,” which shows a strong
resistance to proteolysis and gives rise to the incomplete
cleavage
mentioned above; we have always seen fragment 83-104 in the tryptic digests of both normal and
variant p chains. The existence of this peptide is instrumental for the identification of Hb Lepore Boston; in
fact, the globin region 83-104 contains the junction
point between the3 and $ chains, which gives rise to the
Jib Lepore Boston variant (587-$116). The tryptic fragment diagnostic for this Lepore globin is peptide 83-95,
which always shows a weak signal at m/z 1126 in the
FABIMS map-partly
because its concentration
in the
mixture
is lower than that of the other fragments
(because of the incomplete cleavage mentioned above)
and partly because of its structural characteristics
(28,
29). The presence of another peptide encompassing this
particular
globin region, which gives rise to a strong
mass signal in the FAB/MS spectrum, is extremely
useful to distinguish
between Hb Boston and Hb Baltimore (Table 2).
Although the results discussed above are restricted to
the analysis of only two Lepore variants, the logic of the
approach can be extended to the structural definition of
197527:198-212.
10. Tentori L, Marinucci M, Massa A, Giuliani A, Mavilio F.
Hemoglobinopathies
in Italy: geographic distribution
and criteria
for their screening. Rec Prog Med 1981;71:148-69.
11. Camaschella
C, Serra A, Bertero M, et al. Molecular characterization
of Italian chromosomes
carrying the Lepore Boston
gene. Acta Hematol 1989;81:136-9.
12. Camaschella C, Alfarano A, GottardiE, et al. Prenatal diag-
of fetal hemoglobin
Lepore-Boston
disease on maternal
peripheral blood. Blood 1990;75:2102-.6.
13. Lindeman R, Volpato F, Trent RJ. Detection and characterization of the Hb Lepore (Boston) defect by the polymerase chain
nosis
reaction. Br J Haematol 1989;73:566-8.
14. Pucci P, Carestia C, Fioretti G, Mastrobuoni AM, Pagano L.
Protein fingerprint by fast atom bombardment mass spectrometry:
characterization
of normal and variant human hemoglobins. Biothem Biophys Res Commun 1985;130:84-90.
15. Rahbar 5, Lee TI), Baker JA, et al. Reversed-phase
high
performance liquid chromatography and secondary ion mass spec-
CLINICALCHEMISTRY,Vol.38, No. 8, 1992 1447
trometry.
variants.
A strategy
Hemoglobin
for identification
of 10 human
hemoglobin
1986;10:379-400.
16. Prome D, Prome JC, Prathernon F, et al. Identification
abnormal haemoglobin
etry and fast atom
of some
by fast atom bombardment mass spectrom-
bombardment tandem
mass
spectrometry.
Biomed Environ Mass Spectrom 1988;16:41-4.
17. WadaY,InkolaE,
Imai K, etal. Structure and function ofa
new hemoglobin variant, Rb Meilahti (a p 36 (C2) Pro-oThr)
characterized
13.
by mass spectrometry.
Acta Haematol 1987;78:109-
18. MarshG,MarinoG,PucciP,etal.Athirdinstanceofthehigh
oxygen affinity hemoglobin, Rb Heathrow (p103 Phe-oLeu):
iden-
tification of the mutation by mass spectrometry
and by DNA
analysis. Haemoglobin 1991;15:43-51.
19. Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse
CM.
Electrospray ionization for mass spectrometry of large biomolecules. Science 1989246:64-71.
20. Smith ED, Leo JA. Edmonds CG, Barinaga C,!, Udseth HR.
New developments
in biochemical mass spectrometry:
electrospray ionization. Anal Chem 1990;62:882-99.
21. Loo JA, Edmonds CG, Smith RD. Primary sequence information from intact proteins by electroapray ionization tandem mass
spectrometry. Science 1990248:201-4.
22. Green BN, Oliver RWA, Falick AM, Shackleton CHL, Roitman E, Witkowska HE. Electrospray MS, LSIMS and MS/MS for
the rapid detection and characterization of variant hemoglobins.
1448 CLINICALCHEMISTRY,Vol.38, No.8, 1992
AL, McCloskey JA, eds. Biological mass spectrometry. Amsterdam: Elsevier, 1990:129-46.
23. Shackleton
CHL, Falick AM, Green BN, Witkowska
HE.
Electrospray mass spectrometry in the clinical diagnosis of variant
hemoglobins. J Chromatogr
1991;562:175-90.
24. Shelton JB, Shelton JR. Schroeder WA. High performance
liquid chromatographicseparationofglobin chains on a large-pore
C4 column. J Liq Chromatogr 1984;7:1969-77.
25. Chowdhury SK, Katta V, Beavis RC, Chait BT. Origin and
In: Burlingame
removal of adducta (molecular mass = 98 u) attached to peptide
and protein ions in electrospray ionization mass spectra. JAm Soc
Mass Spectrom 1990;1:382-8.
26. Pucci P, Ferranti P, Marino G, Malorni A. Characterization
ol
abnormal human hemoglobins by FAB/MS. Biomed Environ Mass
Spectrom 1989;18:20-6.
27. Pucci P, Ferranti P, Malorni A, Marino G. Fast atom bombardment mass spectrometric analysis of haemoglobin variants:
use of V-S protease in the identification of Rb M Hyde Park and Rb
San Jose. Biomed Environ Mass Spectrom 1990;19:568-.72.
28. Naylor 5, Findeis AF, Gibson BW, Williams DR. An approach
towards the complete FAB analysis of enzymic digest of peptides
and proteins. J Am Chem Soc 1986;108:6359-64.
29. Pucci P, Sepe C, Marino G. Factors affecting the fast atom
bombardment mass spectrometric analysis of proteolytic digests ol
proteins. Biol Mass Spectrom 199221:22-6.