You are on page 1of 5

Proteomics 2004, 4, 18251829

DOI 10.1002/pmic.200300690


Proteomic characterization of novel serum amyloid P

component variants from human plasma and urine
Urban A. Kiernan, Dobrin Nedelkov, Kemmons A. Tubbs, Eric E. Niederkofler
and Randall W. Nelson
Intrinsic Bioprobes Inc., Tempe, AZ, USA
Serum amyloid P component (SAP) is a human plasma protein that has been widely studied
for its influence on amyloid plaque formation and stabilization. SAP was characterized directly
from human plasma and urine samples via novel affinity mass spectrometry-based proteomic
technology that is able to readily discriminate between mass-altered protein variants. These
analyses were able to identify several variants of SAP that have not been previously reported.
These variants include microheterogeneity of the glycan structure, from the loss of one or both
terminal sialic acid residues, as well as the loss of the C-terminal valine residue. Moreover, the
analysis of urine allowed for the consistent identification of serum amyloid P component as a
normal constituent of the urine proteome.
Keywords: Human plasma / Mass spectrometry / Serum amyloid P component / Urine



1 Introduction
Serum amyloid P component (SAP) belongs to a superfamily of proteins known as the pentraxins. This protein
superfamily is highly conserved throughout nature and is
known for its calcium-dependent ligand binding and lectin properties [1]. Even though SAP was discovered nearly
four decades ago and has been intensely studied, the
exact function of this homopentameric protein still
remains elusive. However, the role serum amyloid P component plays in amyloid plaque formation and stability
has been well documented which has recently led to it
becoming the target of a new anti-Alzheimers therapy [2].
Previous characterization studies using electrospraymass spectrometry identified the mass of endogenous
SAP to be 25 462 Da, which corresponds well with its
known amino acid sequence and glycan structure
(Figs. 1A, B) [3, 4]. It has been determined that native
SAP contains a single N-glycosylation site, at Asn 32,
containing a typical complex biantennary oligosaccharide
chain [4]. Interestingly, previous oligosaccharide characterization studies were unable to identify any microheterCorrespondence: Dr. Urban A. Kiernan, Intrinsic Bioprobes Inc.,
625 S. Smith Rd., Ste Tempe, AZ, 85281 USA
Fax: 11-480-804-0778
Abbreviations: AP, amyloid P component; BRPTrp, bioreactive
probes-trypsin; ddH2O, doubly distilled H2O; HBS, HEPES-buffered saline; SAP, serum amyloid P component

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. (A) Primary amino acid sequence of human

serum amyloid P component. (B) Biantennary human
SAP N-linked glycan located on Asn 32.

ogeneity in the SAP glycan, which is atypical of mammalian glycoproteins [5]. The detection of serum amyloid P
component and/or amyloid P component (AP, SAPs
identical amyloid fibril counterpart) in healthy human
urine is still under debate. Studies into the metabolic
pathway of SAP, using human tissue biopsies and mouse
models, suggest that the only site of SAP catabolism and
clearance is through the liver [6]. However, a single publication by Maury et al. [7] details the development of a
radio immunoassay for the detection of urinary AP from
healthy humans and states that AP is released from amyloid plaques by a cyclic pyruvate acetal derivative of


U. A. Kiernan et al.

galactose. Nevertheless, the prevailing opinion is that

SAP/AP is not a normal constituent of healthy human
We present here the application of affinity-targeted proteomics technology to rapidly isolate, detect, and characterize human proteins from crude human biological fluids.
SAP, which will also include AP, was isolated directly from
human plasma and urine for matrix assisted laser desorption/ionization-time of flight-mass spectrometric (MALDITOF-MS) analysis. The isolated SAP was also subjected
to various enzymatic characterization methods, resulting
in full protein characterization allowing for the identification of structural variations of SAP that have not been previously reported.

2 Materials and methods

2.1 Study subjects, sample collection and
Plasma and urine samples were collected from three
healthy unrelated male subjects, ages ranging from 29
48. Samples were obtained following protocols approved
by Intrinsic Bioprobes Inc.s Institutional Review Board,
and after each individual had read and signed an informed
consent form. Whole blood (75 mL per individual) was
acquired under sterile conditions through a lancet punctured finger using a 75 mL microcolumn (Drummond Scientific, Broomall, PA, USA). Each whole-blood sample
was immediately combined with 400 mL HEPES-buffered
saline (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005%
v/v polysorbate 20, pH 7.4 (HBS)) containing 2 mL of a protease inhibitor cocktail 100 mM 4-(2-aminoethyl)-benzene-sulfonyl fluoride (AEBSF); 80 mM aprotinin; 5 mM
bestatin; 1.5 mM E-64; 2 mM leupeptin; 1 mM pepstatin A
added to prevent any enzymatic breakdown) and centrifuged for 2 min (at 7000 rpm) to pellet red blood cells. An
aliquot of supernatant (200 mL of diluted plasma) was
removed from each sample and dispersed into the individual wells of a 96-well microtiter plate and immediately
analyzed. Urine samples, 50 mL mid-stream voids, from
the same three individuals were also collected, directly
into sterile urine collection cups that were pretreated
with 50 mL of the same protease inhibitor cocktail used
previously, again to prevent any enzymatic breakdown or
modification. The pH of each individual urine sample was
adjusted by the addition of concentrated ammonium hydroxide to increase the urine pH to neutral conditions
(pH 7). The pH adjusted urine was then combined 1:1 v/v
with doubly distilled H2O (ddH2O) to dilute endogenous
urine salts that may interfere with mass spectrometric
immunoassay (MSIA) analysis or MS detection, and SAP
was immediately extracted for analysis.

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2004, 4, 18251829

2.2 In situ deglycosylation

Reactive glycan-sequencing utilizing in situ exoglycosidase
sample pretreatment for the removal of selected terminal
carbohydrate residues was employed to partially deglycosylate the protein. Plasma samples (25 mL) were treated
either 1 mL aliquots of sialidase (1 mg/mL; Sigma Chemical,
St. Louis, MO, USA), or a sialidase and b1, 4-galactosidase
(0.5 mU; Calbiochem, San Diego, CA, USA) mixture. The in
situ enzymatic treatments proceeded overnight at 407C.

2.3 Plasma and urine mass spectrometric

All plasma samples were addressed in parallel using an
eight-barrel multichannel pipettor outfitted with affinity pipettes (Intrinsic Bioprobes, Tempe, AZ, USA) derivatized
with anti-SAP polyclonal antibody (DakoCytomation, Carpinteria, CA, USA). Analysis protocols were identical for
both nontreated and pretreated samples. Sample incubation consisted of repeatedly cycling (aspiration and dispense; 50 cycles; 3 s/cycle; 150 mL/cycle) the samples
through individual affinity tips. After incubation, tips were
rinsed using HBS (10 cycles, 150 mL), ddH2O (5 cycles,
150 mL), 20% acetoniltrile/2 M ammonium acetate wash
(10 cycles, 150 mL), and finally with ddH2O (15 cycles,
150 mL). Retained proteins were eluted by drawing 6 mL of
MALDI matrix solution (saturated aqueous solution of sinapic acid (SA), in 33% v/v acetonitrile, 0.4% v/v trifluoroacetic acid (TFA)) into each tip and depositing directly onto a
96-well formatted hydrophobic/hydrophilic contrasting
MALDI-TOF target [8]. Samples were allowed to air-dry prior
to insertion into the mass spectrometer. The total time
required for the preparation of all three plasma samples
was approximately 10 min. Due to the lower protein concentration in urine, each urine sample was analyzed individually with the anti-SAP affinity pipettes. Sample incubation
consisted of 300 cycles, 150 mL, and the rinses used were
identical to those used in the plasma analyses, with the
exception of prolonged final ddH2O rinses (20 cycles).
Retained proteins were eluted by drawing 6 mL of the
MALDI matrix solution that was prepared in the same
fashion as used in plasma runs. Eluate was deposited directly onto a MALDI target and allowed to air dry. Because
of the larger sample volumes of urine and the number of
cycles used, the time spent to run the assay was , 7 min/

2.4 Peptide mapping

SAP was isolated from plasma samples using the protocols listed above. Retained SAP was eluted from the affinity pipettes using a MALDI-matrix solution (

Proteomics 2004, 4, 18251829

Proteomics characterization of serum amyloid P component


hydroxycinnamic acid (ACCA), in 33% v/v acetonitrile,

0.3% v/v TFA) and applied directly onto a trypsin-activated Bioreactive probe-trypsin (BRPTrp; Intrinsic Bioprobes) that was prepared as previously described [9].
The eluate was allowed to dry before the proteolytic
digestion was initiated by the addition of 10 mL of 25 mM
Tris (pH 9.1) and 1 mL of 1 mM dithiothreitol. Protein digestion was carried out at 507C in a humidified enclave. To
keep the sample solvated, one 10 mL aliquot of water
was added at ,15 min into the digestion. Digestion was
terminated after 25 min by air-drying the BRP. In preparation for MALDI-TOF-MS, the sample spot was rehydrated
with 10 mL aliquots of 0.8% TFA, and allowed to air-dry

2.5 Mass spectrometry

MS analysis was performed on a Bruker Biflex III MALDITOF mass spectrometer. Intact protein analysis was performed in linear delayed extraction mode with a 25.50 kV
full accelerative potential and draw-out pulses of 2.25 kV
(600 ns delay). MS were acquired manually by summing
100 laser shots from within the sample spot. Peptide
maps (200 laser shots) were acquired in linear and reflectron modes. The reflectron MS analysis used a full accelerating potential of 19.35 kV, an ion-mirror voltage of
20.00 kV and a draw-out voltage of 2.70 kV (600 ns delay)
while the linear used a 19.5 kV accelerating potential and
a draw-out voltage of 1.65 kV (400 ns delay).

Figure 2. Results of the anti-SAP affinity MS of human

plasma. Each trace contains the protein expression profile of parent SAP isolated from each individual. Multiple
SAP signals are observed. The most prominent is SAP
with an intact glycan but signals from monosialo- and
asialo-SAP are also observed. Strong consistency in the
SAP profiles is observed between all three individuals.

dase, an exoglycosidase that specifically targets terminal

galactose residues with a b1,4-linkage, resulted in the
generation of a new SAP peak from the enzymatic cleavage of the exposed galactose residues (data not shown).
This cleavage could only occur if one of the terminal sialic
acids was not present in the native plasma SAP.

The results of the plasma anti-SAP affinity MS analyses

are shown in Fig. 2. Each trace is the specific protein
expression profile of SAP characteristic to each individual. A high level of consistency is observed in the profiles
between all three samples analyzed. The major signal
observed in each trace has a mass of 25 463 6 3 Da,
which matches the theoretically predicted mass of SAP
and agrees with the MS results made by other groups [5].

The presence of a monosialo-SAP variant had been previously observed by Pepys et al. [1] but was dismissed as
the result of the harsh environment during protein purification. Conversely, even when SAP is purified directly
from its native environment, as done with in this work,
the monosialo-modification and minute amounts of
asialo-SAP (the loss of both sialic acid residues, observed
in Fig. 2A) are still readily and consistently observed. To
date, this is the first report of endogenous microheterogeneity in the human SAP glycan.

A second peak, with a mass shift of ,291 Da, was also

present. A loss of 291 Da corresponds precisely with the
loss of a single sialic acid (N-acetylneuraminic acid) residue. Mammalian glycoproteins frequently have glycans
that terminate with a sialic acid residue, which when
cleaved, is known to make glycoproteins targeted for
hepatic removal [10, 11]. As depicted in Fig. 1B, each
strand of the native SAP glycan terminates with a sialic
acid residue (MW 291). The presence of this novel monosialo-SAP variant in plasma was verified through the use
of in situ enzymatic glycan sequencing. The overnight
incubation (at 407C) of native plasma with b1,4-galactosi-

When the same analysis was applied to urine samples,

the SAP protein expression profiles observed (Fig. 3)
were almost identical to those obtained from plasma.
Again, signals corresponding to the intact SAP were present along with a monoasialo-variant. As with the plasma,
the urinary SAP profile is highly conserved between all
three individuals. No identifiable fragments of SAP were
identified in the lower mass region of the mass spectra.
The consistent detection of intact SAP conclusively
demonstrates that it is a normal constituent of the human
urine proteome (note: we have detected SAP in the urine
of , 25 different healthy individuals).

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3 Results and discussion


U. A. Kiernan et al.

Proteomics 2004, 4, 18251829

respectively. The signals from the SAP glyco-variants
were removed from the mass spectrum but the existence
of an other, potentially truncated SAP variant, was suggested by the presence of additional signals in the mass
spectrum. The observed mass difference of ,99 Da corresponds to the removal of the C-terminal Val residue
(MW 99.07). The existence of this truncated variant was
postulated from examination of the normal SAP protein
expression profile, Fig. 4A, but could not be clearly established due to the potential signal overlap by the monosialo-SAP matrix adduct.

Figure 3. Results of the anti-SAP affinity MS of human

urine. Strong consistency is observed between all three
samples as well as with the plasma MS. Each trace shows
intact SAP and the same associated variants that were
observed in Fig. 2.

To verify the existence of this truncated SAP variant, proteolytic digestion of affinity-purified SAP was employed.
Trypsin-bioreactive probe (BRPTrp) technology was utilized [9, 12]. The observed and matched peptide digest
fragments resulted in 100% sequence coverage of the
captured SAP biomolecule along with all retained variants
(Fig. 5). The majority of the peptides were identified using

Since the expression profile of SAP is somewhat convoluted from the presence of a glyco-variant, further in situ
exoglycosidase-reactive sequencing was performed to
simplify the parent MS spectrum. The results of the overnight plasma sample incubations with sialidase and sialidase with b1,4-galactosidase is shown in Figs. 4B and C,

Figure 4. Results of in situ reactive glycan-sequencing.

(A) Control SAP profile from native plasma. (B) Profile of
SAP captured from plasma digested overnight with sialidase, which resulted in the complete removal of both terminal sialic acid residues. (C) SAP profile from plasma
treated with sialidase and b1, 4-galactosidase. The
removal of the terminal sialic acid or both the sialic acid
and galactose residues resulted in a less convoluted
mass spectrum and prominently shows the presence of
a novel truncated form of SAP.

Figure 5. Results of the tryptic digest of affinity-captured

SAP using bioreactive probe technology. Digest map (top)
shows that 100% protein coverage was achieved with the
observed tryptic digest peptides. Corresponding peptides that were detected in reflectron MS mode are shown
in black while those obtained in linear mode are shown in
gray. The reflectron MS (bottom) of SAP tryptic peptide
digest profile. The linear MS of the SAP tryptic digest is
in the bottom-inset. The corresponding peptide fragment
number annotates each major peak in the trace.

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2004, 4, 18251829

Proteomics characterization of serum amyloid P component

reflectron MS but there were two major gaps in the protein coverage, which corresponded to large peptides
(outside the mass window of reflectron MS analysis) that
either contain the site of glycosylation (residues 1438;
MW 5117.27) or lack tryptic cleavage sites (residues
147193; MW 5322.62). Examination of the digest fragments with linear MS readily identified these two missing
fragments (Fig. 5, inset).
Moreover, further examinations of the signal from the linear MS clearly establish the presence of residues 1438
with monosialo- and asialo-variations of the glycopeptides. A Profound search using all peptide masses from
the reflectron and linear mass spectra returned SAP as
the best match, with a high probability and z-score. All
major peaks observed in the digest profile were matched
to human SAP. Finally, signals corresponding to the C-terminal digest fragment (residues 194204; MW 1285.78)
and a truncated variant (residues 194203; MW 1186.71)
were identified in the mass spectrum shown in Fig. 6, confirming the presence of this novel SAP truncated variant
initially observed in the parent protein mass spectrum.


nous variants may prove useful in determining the role of

SAP in vivo. Moreover, these data were able to specifically
identify SAP as a normal constituent of human urine. As
more components of the human urine proteome are identified, the superlative nature of this biofluid for protein
analyses will become more evident. Even though the
analysis of proteins from urine has historically been difficult, this next generation of protein profiling and characterization technology is readily capable of such demands
[13, 14]. These results, from both plasma and urine, could
only have been accomplished this efficiently via the use of
these affinity mass spectrometry-based proteomics technologies. This clearly illustrates the necessity for population based protein characterization studies, not only to
further investigate the role of SAP and its novel variants,
but any other protein biomarker suitable for population
We would like to thank Dr. Allan Bieber for the critical
reading of this manuscript. This publication was supported in part by contract No. N43-DK-1-2470 and N44ES-35511 from the National Institutes of Health. Its contents are solely the responsibility of the authors and do
not necessarily represent the official views of the National
Institute of Health.

5 References

Figure 6. Region of the SAP tryptic digest MS containing

the C-terminal cleavage peptide (residues 194204;
MW 1285.78). The peptide that corresponds to the SAPVal variant (residues 194203; MW 1186.71) was also present, confirming the presence of this novel form of SAP.

4 Concluding remarks
The analysis of SAP via affinity-based proteomic technology is a major advancement in direct protein analysis. Not
only were these SAP analyses performed in a rapid time
frame, within hours, but several endogenous variants also
were discovered in the process. Since SAP is a biomarker
for amyloid disease, the discoveries of these variants are
of great importance because protein structure ultimately
dictates its function. However, since the exact function of
SAP is still unknown, the identification of these endoge-

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

[1] Pepys, M. B., Booth, D. R., Hutchinson, W. L., Gallimore, J.

R. et al., Amyloid Int. J. Exp. Clin. Invest. 1997, 4, 274295.
[2] Pepys, M. B., Herbert, J., Hutchinson, W. L., Tennent, G. A.
et al., Nature 2002, 417, 254259.
[3] Prelli, F., Pras, M., Frangione, B., J. Biol. Chem. 1985, 260,
[4] Tennent, G. A., Pepys, M. B., Biochem. Soc. Trans. 1994, 22,
[5] Pepys, M. B., Rademacher, T. W., Amatayakul-Chantler, S.,
Williams, P. et al., Proc. Natl. Acad. Sci. USA 1994, 91, 5602
[6] Hutchinson, W. L., Noble, G. E., Hawkins, P. N., Pepys, M.
B., J. Clin. Invest. 1994, 94, 13901396.
[7] Maury, C. P. J., Teppo, A.-M., J. Lab. Clin. Med. 1985, 106,
[8] Niederkofler, E. E., Tubbs, K. A., Gruber, K., Nedelkov, D. et
al., Anal. Chem. 2001, 73, 32943299.
[9] Kiernan, U. A., Black, J. A., Williams, P., Nelson, R. W., Clin.
Chem. 2002, 48, 947949.
[10] Morell, A. G., Irvine, R. A., Sternlieb, I., Scheinberg, I. H.,
Ashwell, G., J. Biol. Chem. 1968, 243, 155159.
[11] Ashwell, G., Morell, A. G., Adv. Enzymol. Relat. Areas Mol.
Biol. 1974, 41, 99128.
[12] Nelson, R. W., Dogruel, D., Krone, J. R., Williams, P., Rapid
Commun. Mass Spectrom. 1995, 9, 13801385.
[13] Kiernan, U. A., Tubbs, K. A., Nedelkov, D., Niederkofler, E.
E., Nelson, R. W., Biochem. Biophys. Res. Commun. 2002,
297, 401405.
[14] Kiernan, U. A., Tubbs, K. A., Nedelkov, D., Niederkofler, E. E.
et al., J. Proteome Res. 2003, 2, 191197.