Mass determination of major plasma proteins by matrix-assisted laser desorption/ionization time-of

13 Pages·2006·0.42 MB·English

08_Hortin.qxd 11/21/06 3:20 PM Page 103
Clinical Proteomics
Copyright © 2006 Humana Press Inc.
All rights of any nature whatsoever are reserved.
ISSN 1542-6416/06/02:103–116/$30.00 (Online)
Original Article
Mass Determination of Major Plasma Proteins
by Matrix-Assisted Laser Desorption/Ionization
Time-of-Flight Mass Spectrometry
Glen L. Hortin1,* and Alan T. Remaley1,2
1Department of Laboratory Medicine,National Institutes of Health,Bethesda,MD;and2National Heart
Lung and Blood Institute,National Institutes of Health,Bethesda,MD
and doubly charged monomer, and provided
Abstract
measured masses for these proteins. A
Matrix-assisted laser desorption/ionization number of proteins, including albumin, trans-
time-of-flight mass spectrometry (MALDI-TOF/ ferrin, apolipoproteins A-I, A-II, C-I, C-II, and
MS) serves as a rapid and accurate means C-III, and prealbumin, could be analyzed
todetermine masses of proteins independent directly in serum with appropriate dilution.
of their shapes or interactions with other Measured values for masses of major plasma
molecules. It provides one of the most funda- proteins will assist in analysis of serum and
mental characterizations of major plasma pro- plasma. It is possible to analyze a number of
teins. Purified proteins in saline or serum components by MALDI-TOF/MS directly in
specimens were prepared for analysis by dilu- diluted serum. Extremely simple sample
tion, mixing with a solution of sinapinic acid, preparation techniques may be useful in ana-
and drying on a target plate. Specimens were lyzing structural variation of several major
analyzed in a linear TOF mode with external plasma proteins, particularly those with
calibration. Analyses of 24 purified plasma masses <30 kDa, including a number of
proteins showed predominance of singly apolipoproteins and markers of nutritional
charged ions with lesser amounts of dimer status or acute phase responses.
*Author to whom all correspondence and reprint requests should be addressed:
Glen L. Hortin, Department of Laboratory Medicine, National Institutes of Health, Building 10, Room
2C-407, Bethesda, MD 20892.
E-mail: [email protected]
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104 ________________________________________________________________________Hortin and Remaley
Introduction might be applicable for routine in the clinical
laboratory and that would avoid modification
Matrix-assisted laser desorption/ionization
or selective losses of components during
time-of-flight mass spectrometry (MALDI-
preparation.
TOF/MS), invented in the late 1980s (1,2),
serves as a technique for analyzing peptides Materials and Methods
and proteins in relatively complex samples. It
has even been employed for the direct analysis Peptides and proteins used as calibrators and
of tissue specimens (3). In this technique, speci- sinapinic acid used as the specimen matrix were
mens containing peptides and protein are dried purchased from Bruker Daltonics (Billerica,
on a target plate together with a light-absorbing MA). Masses of calibrators were: (bovine
matrix molecule. Vaporization of the mixture of insulin)+, 5734.61; (equine cytochrome c)+,
protein and matrix by a laser releases ionized 12,361.20; (equine myoglobin)+, 16,952.62;
protein molecules, often with a single charge, (bovine serum albumin)2+, 33,216; (bovine
which can be analyzed in a TOF mass spec- serum albumin)+, 66,431; (bovine serum albu-
trometer. It serves as a simple and rapid analyt- min dimer)+, 132,859. Purified proteins were
ical technique for determining the molecular obtained from the following sources: human
weights and heterogeneity of small amounts of holo-transferrin, prealbumin, immunoglobulin
α α
protein. In general, intact molecular ions are G (reagent grade), 1-antitrypsin, and 1-acid
formed from individual peptide chains, allow- glycoprotein, from Sigma Chemical (St. Louis,
ing accurate determination of polypeptide mass MO); human haptoglobin (mixed types),
α α
(4–6). However, there may be some technique- 2HS-glycoprotein, Gc globulin, 2-macroglob-
dependent variation in measured mass related ulin, immunoglobulin A, neutrophil lysozyme,
to variable loss of labile groups, such as sialic and C-reactive protein from Calbiochem (La
β
acids or association of sodium or matrix with Jolla, CA); 2-glycoprotein I, Glu-plasminogen,
protein molecular ions (4–10). and antithrombin III from Haematologic Tech-
Sample preparation is a key step in the nologies, Inc. (Essex Junction, VT). Lipopro-
analysis of complex specimens by MALDI- teins including apolipoproteins A-I, A-II, C-I,
TOF/MS. Variables, such as protein con- C-II, C-III, and E were purified by standard
centration, salt concentration, or specimen published procedures (17). Purified proteins
fractionation, may strongly influence the were generally in stock solutions from 1 to
observed spectra. Usual approaches for sample 10g/L in concentration. Serum specimens
preparation for MALDI-TOF/MS involve were remnants of human specimens submitted
desalting and cleanup of specimens by tech- to the clinical laboratory used in accordance
niques such as dialysis, solid-phase extrac- with a protocol approved by an Institutional
tions, or fractionation through absorption to Review Board.
a specialized target surface (in a variant of Calculated masses of proteins were based
MALDI-TOF/MS that has been termed sur- on amino acid sequences in well-validated
face-enhanced laser desorption/ionization or databases, usually the Swiss-Prot database
SELDI) or to magnetic beads (5–16). These (www.ebi.ac.uk/swissprot). Calculations of
preparation steps involve significant time, polypeptide masses were performed with
expense, and potential losses of selected com- software offered by the National Institute
ponents. In the present study, we examined of Diabetes and Digestive and Kidney Dis-
direct analysis of serum and purified serum eases mass spectrometry group (sx.102a.
components as a very simple approach that niddk.nih.gov/peptide). Calculated peptide
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Direct Analysis of Serum Proteins _____________________________________________________________105
masses did not include the masses of A–C, respectively, or for apolipoprotein A-I
oligosaccharide components. and transthyretin in Fig. 2. These analyses
Analyses were performed with an Ultraflex served as a relatively rapid method to deter-
TOF mass spectrometer (Bruker Daltonics) in a mine the mass of specific components and to
linear positive ion mode similar to previously evaluate the tendency to form dimers and
described analyses (12). Specimens were multiply charged ions.
analyzed with two programs of separation Table 1 lists the measured masses of 23
parameters. To examine an m/z range of plasma proteins that were available as puri-
5000 to 30,000, pulsed extraction had a delay fied components, plus serum amyloid A,
of 300 ns. To examine m/z > 30,000, there was which was observed in serum from a patient
a delay of 550 ns. Specimens were prepared by with an acute phase response as characterized
dilution with 10 mmol/L ammonium acetate in other studies (18). The broader peaks of
and then by mixing 1 vol of diluted specimen high molecular weight proteins, probably
with 2 vol of matrix solution containing 10 g/L reflecting both lower resolution and increased
sinapinic acid in 40% acetonitrile/10% microheterogeneity from variable glycosyla-
ethanol/50% water containing 0.1% trifluo- tion, led to greater imprecision in the mea-
roacetic acid. Specimens were applied twice to surement of masses for these components.
a 384-position sample plate as 1-µL aliquots Comparison of measured values in Table 1 vs
and allowed to air-dry. Specimens were ana- older published values based on techniques
lyzed with manual laser positioning and such as compositional analysis, electrophoresis,
adjustment of laser power. All figures shown and ultracentrifugation generally showed
represent data collected from 10 positions, fairly good agreement for proteins lacking
summing a total of 300 laser pulses. Measure- oligosaccharides. MALDI-TOF/MS provided
ments of m/z were performed using external lower estimates of masses for most glycopro-
calibration. Masses of proteins were measured teins than the classical textbook values. Mea-
at the peak apex. sured values for several proteins with high
α
carbohydrate content such as -acid glyco-
Results 1
α
protein, antithrombin III, -HS-glycoprotein,
2
α
Most serum proteins are globulins that and -antichymotrypsin were more than 10%
1
require the presence of some salt to maintain lower than older published values. Measured
solubility, but salts tend to suppress ionization values for glycoprotein I and plasminogen
of proteins in MALDI-TOF/MS. Therefore, were about 10% higher than published values.
there is a compromise between physiological MALDI-TOF/MS should provide highly
salt conditions that maintain protein solubility accurate of proteins without posttransla-
and the efficiency of ionization. We examined tional modifications, and, in most cases,
the ability to analyze purified proteins in there is good agreement for these proteins
saline solutions by MALDI-TOF/MS, using with calculated masses based on amino acid
simple dilution of the proteins with matrix sequence databases. For proteins bearing com-
solution. Generally, these analyses yielded a plex oligosaccharides and other substantial
pattern in which the singly charged molecular posttranslational modifications, the sequence
ion predominated, but lesser amounts of databases are not very useful for estimating
dimer, trimer, or doubly charged monomer masses. Values measured by MALDI-TOF/MS
often were observed, as shown for the exam- should represent accurate estimates with the
α
ples of -acid glycoprotein, transferrin, and caveat that there may be some underestima-
1
immunoglobulin G as shown in Fig. 1 panels tion of mass as a result of loss of some groups
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106 ________________________________________________________________________Hortin and Remaley
Fig.1.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of α -acid glycoprotein (A),
1
transferrin (B),and immunoglobulin G (C).
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Direct Analysis of Serum Proteins _____________________________________________________________107
any detectable spectra. The high protein con-
tent of serum probably did not allow a high
enough ratio of matrix to protein to provide
for efficient ionization of undiluted specimens.
Matrix usually needs to be in more than
1000-fold molar excess relative to protein to
yield good spectra (6). Several diluents were
examined such as 10 mM ammonium acetate,
10 mM ammonium bicarbonate, normal
saline, and other solutions with varying ionic
strength. Diluents with an ionic strength above
that of normal saline suppressed ionization.
The solutions of 10 mM ammonium acetate
and 10 mM ammonium bicarbonate were sub-
jectively considered to yield similar results
with greatest peak intensities (not shown). A
solution of 10 mM ammonium acetate was
used as diluent for subsequent experiments
described here. Dilution of serum with 19 vol
of 10 mM ammonium acetate plus 40 vol of
matrix solution yielded spectra as shown in
Fig. 3. In the top panel, the m/z range from
Fig. 2. Matrix-assisted laser desorption/ionization 5000 to 30,000 is shown and, in the bottom
time-of-flight mass spectrometry of apolipoprotein panel, m/z from 30,000 to 100,000 is shown.
A-I (Top) and transthyretin also known as pre- For m/z > 30,000, singly and doubly charged
albumin (Bottom).
albumin is the dominant component. A peak
corresponding to expected position of transfer-
such as sialic acids during desorption and ion- rin generally was visible and a number of
ization (6–10). Losses of sialic acid may vary other minor unidentified peaks were observed.
depending on technique of analysis, leading If the analysis was extended beyond m/z of
to differences in measured mass; as an exam- 150,000, peaks were observed in the expected
ple, measurements of the mass of α -acid positions for albumin dimer and IgG. In the
1
glycoprotein by MALDI-TOF/MS vary from m/z range from 5000 to 30,000, there were a
about 32,000 to 36,000 in different studies number of sharper peaks, corresponding to the
(5,9,21). In general, the ionization efficiency expected positions of apolipoproteins A-I, A-II,
appeared to be poorer for high molecular C-I, C-II, C-III, and transthyretin. Although
weight and highly glycosylated components. some of these proteins, such as apolipoprotein
Haptoglobin and α -macroglobulin were not C-I, C-II, and C-III, are often considered
2
included in Table 1 as they yielded very weak relatively minor serum components, they are
and diffuse peaks at m/z of about 96,000 and among the 20 most abundant components on a
124,000 for haptoglobin (mixed types) and molar basis (20). In general, ion intensities
178,000 for α -macroglobulin. forindividual proteins in MALDI-TOF/MS
2
Serum required an initial dilution by a factor should relate to the molar concentration of a
of about fivefold with aqueous diluent before protein, and there is a strong bias for detection
further threefold dilution with matrix to obtain of low molecular weight proteins (20,21).
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asey 6802 47028702520209023400 6602 6502 5602 5402
br 7/ 6/7/6/0/6/ 7/ 7/ 6/ 6/
ataent P02/15 P02/15P02/15P19/15P01/15001/31 P02/15 P02/15 P02/15 P02/15
D 6 6 6 6 6P0 6 6 6 6
N1
al
r, n
ported Values aMass Peptide modifications.ercentage carbohydrate(CHO) None or cysteinylated,0% CHO3% CHO see None (text for possible)6% CHO 45% CHO 12% CHO N-terminal pyroGlumonomer ordisulfide-linked dime0% CHO None or cysteinylated, 0% CHO Phosphorylation X 2, 13% CHO No CHO orGalNAcGal* orGalNAcGalSAorGalNAcGalSA28% CHO None or loss of N-termiThrPro, 0% CHO
ee P
Rd
vs pti
ble 1d by MALDI-TOF/MS ques and Calculated Pe Calculated peptide mass/length in amino acids 6,438/585 aa or 66,557/586 aaNot calculated variable sequence 8,079/243 aa 5,144/679 aa 1,631/183 aa 4,325/394 aa 690.9 (monomer)/77 aa 17,379.8 (dimer)/2 X 77 aa 3,761.5 (subunit)/127 aa 2,944/A-chain 282 aa +B-chain 27 aa764.7 or 9130.0 or9421.3 or 9712.6/each 79 aa Not calculated, variable sequence630.6 or 6432.4/57or 55 aa
Tareni 6 2 7 2 4 8 1 3 8 6
uh
sc
ae
Masses Menalytical T Reportedmass (Ref) (38)6,000 (38)0,000 (39)8,016 (38)7,000 (38)0,000 (38)5,000 (39)7,414 4,400 4 X(38)3,600 (40)0,000 (39)8800 (38)0,000 (39)6630
A 6 6 2 7 4 5 1 51 5 7
ein al 1 1
tn
oo
Prti
of adi D 00 80 8 34 50 30 312 64 30 33 40 11
n Tr S 2 1 1 3 1 1 13
risofor
pa d
Com Measure(m/z) 66,400 148,420 28,077 79,110 35,290 50,300 869617,393 13,82613,862 42,420 91329427 160,820 66306431
I
I n II
A- )ei C-
st
Protein Albumin IgG Apolipoprotein Transferrin α-Acid 1glycoproteinα-Proteinase 1inhibitorApolipoprotein A-II Transthyretin (prealbumin)(four subunitαHS-Glyco-pro2 Apolipoprotein IgA Apolipoprotein C-I
108
08_Hortin.qxd 11/21/06 3:20 PM Page 109
s
4212925282729252124003 e
7010405000404030408030 c
7/0/7/6/0/7/6/7/7/8/2/ n
P026/15P016/15P026/15P0212/8P016/15P006/15P026/15P026/15P026/15P019/15NP001/17 refere
e
h
t
m, ns, om
4% CHO 27% CHO 19% CHO None, 0% CHO 15% CHO Phosphorylation X 1, 3% CHOSequence polymorphis0% CHONone or truncated chai0% CHON-terminal pyroGlu,0% CHONone, 0% CHO None, 0% CHO hydrate contents were fr
o
b
r
a
6 (E2)9 aa 6 aa ated cntries.
89 0 me
6/458 aa 6/400 aa 3/326 aa 0/79 aa 3/432 aa 6/791 aa 7 (E3) or 34,134,288 (E4)/22.8/104 aa or526.6/103 aa9 (subunit)/2 9.2/99 aa 2.7/130 aa ngle run. Estiated database alic acid.
51,21 45,26 36,23 8915. 49,03 88,38 34,23or 11,6811,23,02 11,72 14,69 n a siindicnd si
withid on se, a
(41)51,000 (41)68,000 (41)40,000 (39)8900 (41)65,000 (41)81,000 (39)34,145 (37)11,685 118,000 5 X (38)23,600(38)11,800 (38)14,500 asurements es were basemine, galacto
meassosa
160 510 160 23270 500 170 1115 4 7 d by 10 ptide mylgalact
eet
npe
mid ac
250 330 940 916342150 100 420 683528006 727 690 deterculateate N-
51, 52, 44, 810,57, 90, 34, 11,11,23, 11, 14, D) was ses. CalAindic
Gc globulin α-Antichymo-1trypsinβ-Glyco-protein I2 Apolipoprotein C-II Antithrombin III Plasminogen Apolipoprotein E Serum amyloid A C-reactive protein (X 5 subunits)β-microglobulin2 Lysozyme aThe imprecision (Scited for reported masGalNAc, Gal, and S
109
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110 ________________________________________________________________________Hortin and Remaley
Fig. 3. Matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry of diluted serum.
(Top) m/z range from 5000 to 30,000 with tentative
identification of major peaks. (Bottom) m/z range
from 30,000 to 100,000.
Fig. 4. Peak intensities (peak heights) with varying
Apeak corresponding to the expected position dilution of serum.Listed dilutions reflect dilution with
=
(m/z 6630) of apolipoprotein C-I was one of 10 mM ammonium acetate prior to further three-
the strongest peaks, suggesting unusually effi- fold dilution with matrix solution.
cient ionization of this protein, whereas the
ionization of apolipoprotein A-II appeared owing to their much lower abundance relative
relatively inefficient considering its higher to proteins.
abundance relative to apolipoproteins C-I, C-II, Most peaks in the spectra appeared to
and C-III. Different peptides and proteins are decrease in signal intensity with increasing
recognized to have highly variable ionization dilution with 10 mM ammonium acetate as
in MALDI-TOF/MS with content of arginine shown in Fig. 4 (prior to further threefold
residues a significant factor in ionization of dilution with matrix solution). Most compo-
peptides (22). Although there are reported to nents had decreasing intensities for dilutions
be a large number of small peptide compo- from about 10- to 300-fold; however, peaks
nents in serum (23–26), components with related to albumin increased in intensity over
m/z < 5000 were not observed clearly under this range of dilutions and began to decrease
the conditions of analysis used here and this in intensity only at dilutions more than 100-
region of the spectrum is not shown. Detection fold. Apparently the concentrations of albumin
of small peptides probably is suppressed were saturating at low dilutions. Absolute
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Direct Analysis of Serum Proteins _____________________________________________________________111
Table 2
Major Peaks in MALDI-TOF Mass Spectrometric Analysis
of Two Serum Specimens and Tentative Assignmentsa
Measured m/z Assignment Expected mass
High m/z region (>30,000)
147,860 ±490/147,170 ±700 [IgG +H]+ ~148,000
133,320 ±410/132,960 ±270 [Albumin +H]+ 132,855
2
79,180 ±240/79,360 ±310 [Transferrin +H]+ 79,110
66,540 ±90/66,550 ±140 [Albumin +H]+ 66,428
33,290 ±130/33,240 ±170 [Albumin +2H]+2 33,216
Low m/z region (5000–30,000)
28,015 ±15/28,038 ±12 [Apo A-I +H]+ 28,078
13,876 ±2/13,868 ±1 [Prealbumin +Cys +H] 13,946
13,756 ±2/13,747 ±2 [Prealbumin +H]+ 13,827
9424 ±1/9417 ±3 [Apo C-III +H]+ 9422.3
1
8914 ±1/8911 ±1 [Apo C-II +H]+ 8917
8809 ±1/8806 ±1 [Apo A-II +Cys +H]+ 8811
8686 ±3/8680 ±1 [Apo A-II (monomer) +H]+ 8692
6630 ±1/6631 ±1 [Apo C-I +H]+ 6631
6433 ±1/6432 ±1 [Apo C-I (–ThrPro-) +H]+ 6433
aValues for m/z are means ±SD of six measurements.
intensities of signals in MALDI-TOF/MS usu- components. Data for repeated analysis of two
ally are subject to substantial variation from specimens are shown in Table 2. Most peaks
one laser shot to the next. The fairly dense corresponded to expected values within the
application of matrix with serum in this study, precision of measurement. An exception was
however, yielded fairly consistent signals, and transthyretin, which is known to be polymor-
variation was reduced somewhat by summing phic and to undergo a variety of modifications
signals from 300 laser shots for each analysis. that may affect it mass (27,28). The mass mea-
Because peaks were compared within the surements indicated that it was possible to
same analysis, other peaks within an analysis obtain accurate mass measurements of several
served as controls for consistency of the results. of the most abundant components in the com-
Comparison of peak ratios between compo- plex mixture of serum proteins without exten-
nents might be considered a more valid sive fractionation or removal of salts. For the
approach for this analytical technique, but it smaller proteins, the pairing of sodium ions or
leads to similar results. These dilution studies matrix molecules with the protein would have
led to a conclusion that there was an optimal been apparent in mass measurements.
dilution for analysis of most components other
Discussion
than albumin that was about a 10- or 20-fold
dilution with ammonium acetate prior to Significant information about molecular
further dilution with matrix. masses and heterogeneity of concentrated
The measured m/z for a number of peaks solutions of purified proteins or even the
in serum samples appeared to correspond to complex mixture represented by serum was
the expected values for several major serum obtained by MALDI-TOF/MS simply by
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112 ________________________________________________________________________Hortin and Remaley
dilution of specimens together with matrix helpful in examining other protein compo-
and drying on a target plate. Measured mass nents in this region. This may be a practical
values in Table 1 serve as a database for the procedure considering that less than 1 µL of
expected positions of peaks for a number serum is required per analysis. For ions with
of serum components when analyzed by m/z < 30,000, only several of the most abun-
MALDI-TOF/MS. These data may be of value dant components are observed. Specimen frac-
in helping to identify which proteins are form- tionation would be required to see lower
ing peaks and in identifying peaks that may abundance components. Consequences of the
be useful for internal calibration of masses general suppression of ionization by other
during an analyses. Of course, masses of some ions and proteins are relatively weak signals,
proteins may vary owing to sequence poly- detection only of high abundance compoents,
morphism, variable posttranslational modifi- and the need to spend more time with data
cation, or degradation during processing or acquisition to sum data from a large number
storage. Measured masses of highly sialylated of laser shots. Use of relatively high laser
proteins by MALDI-TOF/MS also may vary power may be necessary to obtain measurable
depending on several variables, as previously peaks, and this may lower resolution and pro-
noted. Also, it must be considered that there mote fragmentation of proteins. Low peak
is structural variation in glycosylation of pro- intensities and resolution also can be a limit-
teins in different physiological states that may ing factor in obtaining precise and accurate
lead to changes in glycoprotein mass (27,28). mass measurements.
Direct analysis of diluted serum specimens The method of sample preparation
by MALDI-TOF/MS may offer a rapid and described here offers a simple approach to
inexpensive approach to analyze several major analyze a number of major serum compo-
serum components with minimal opportunities nents. In particular, it appears best suited to
for modification or selective loss of compo- analyze components with a mass less than
nents. For proteins lacking labile posttransla- 30,000 where there is better sensitivity, resolu-
tional modifications, the mass measurements tion, and less interference from albumin. This
determined by MALDI-TOF/MS should be technique would be useful for identifying
accurate measures of protein mass. For several polymorphisms or modifications of compo-
of the most abundant proteins with masses less nents, such as by cysteinylation in the exam-
than 30,000, mass measurements can be made ple of transthyretin (29,30). A significant
with sufficient precision and accuracy to allow portion of apolipoprotein A-I, appeared as a
detection of most chemical modifications of hump following the major expected peak, sug-
proteins or sequence polymorphisms. gesting a modification increasing its mass by
Two drawbacks of the analysis of a complex about 150 Da, which merits further investiga-
mixture, such as serum, without fractionation tion; this protein may undergo fatty acylation
are the detection of only a few of the most and is susceptible to nitration and chlorination
abundant components and the general sup- (31,32). Alternatively, this may represent adduct
pression of signals by the high protein and salt formation with a matrix molecule (6). Variants
concentrations. Dominance of the albumin of a number of other apolipoproteins have
peaks for monomer, doubly charged monomer, been described including a truncated form of
and singly charged monomer interfere with apolipoprotein C-I, which has lost two amino
the ability to detect other peaks with m/z > acids from its N-terminus (33,34). The latter
30,000. Selective immunodepletion of albumin truncated form of apolipoprotein C-I was
or albumin plus immunoglobulin G may be observed consistently in our analyses.
Clinical Proteomics________________________________________________________________Volume 2, 2006