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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

Received 16/10/03

Revised 17/11/03 Accepted 18/11/03

Proteomics 2004, 4, 1825–1829 1825

1 Introduction

Serum amyloid P component (SAP) belongs to a super-

family of proteins known as the pentraxins. This protein

superfamily is highly conserved throughout nature and is

known for its calcium-dependent ligand binding and lec-

tin properties [1]. Even though SAP was discovered nearly

four decades ago and has been intensely studied, theexact function of this homopentameric protein still

remains elusive. However, the role serum amyloid P com-

ponent plays in amyloid plaque formation and stability

has been well documented which has recently led to it

becoming the target of a new anti-Alzheimer’s therapy [2].

Previous characterization studies using electrospray-

mass 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 charac-

terization studies were unable to identify any microheter-

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 mamma-

lian glycoproteins [5]. The detection of serum amyloid P

component and/or amyloid P component (AP, SAP’s

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 pub-

lication 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 amy-

loid plaques by a cyclic pyruvate acetal derivative of

Correspondence: Dr. Urban A. Kiernan, Intrinsic Bioprobes Inc.,

625 S. Smith Rd., Ste Tempe, AZ, 85281 USA

E-mail: [email protected]

Fax: 11-480-804-0778

Abbreviations: AP, amyloid P component; BRPTrp, bioreactive

probes-trypsin; ddH2O, doubly distilled H2O; HBS, HEPES-buf-

fered saline; SAP, serum amyloid P component

© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de

DOI 10.1002/pmic.200300690



1826 U. A. Kiernan et al. Proteomics 2004, 4, 1825–1829

galactose. Nevertheless, the prevailing opinion is that

SAP/AP is not a normal constituent of healthy human

urine.

We present here the application of affinity-targeted prote-

omics technology to rapidly isolate, detect, and charac-

terize 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 desorp-

tion/ionization-time of flight-mass spectrometric (MALDI-

TOF-MS) analysis. The isolated SAP was also subjected

to various enzymatic characterization methods, resulting

in full protein characterization allowing for the identifica-

tion of structural variations of SAP that have not been pre-

viously reported.

2 Materials and methods

2.1 Study subjects, sample collection and

preparation

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 punc-

tured finger using a 75 mL microcolumn (Drummond Sci-

entific, Broomall, PA, USA). Each whole-blood sample

was immediately combined with 400 mL HEPES-bufferedsaline (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005%

v/v polysorbate 20, pH 7.4 (HBS)) containing 2 mL ofa pro-

tease inhibitor cocktail 100 mM 4-(2-aminoethyl)-ben-

zene-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 centri-

fuged 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 indi-

vidual 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 hy-

droxide 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.

2.2 In situ deglycosylation

Reactiveglycan-sequencing utilizing in situ exoglycosidase

sample pretreatment for the removal of selected terminal

carbohydrate residues was employed to partially deglyco-

sylate the protein. Plasma samples (25 mL) were treated

either 1 mL aliquotsof 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

immunoassays

All plasma samples were addressed in parallel using an

eight-barrel multichannel pipettor outfitted with affinity pi-

pettes (Intrinsic Bioprobes, Tempe, AZ, USA) derivatized

with anti-SAP polyclonal antibody (DakoCytomation, Car-

pinteria, CA, USA). Analysis protocols were identical for

both nontreated and pretreated samples. Sample incuba-

tion consisted of repeatedly cycling (aspiration and dis-

pense; 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 sina-

pic acid (SA), in 33% v/v acetonitrile, 0.4% v/v trifluoroace-

tic acid (TFA)) into each tip and depositing directly onto a

96-well formatted hydrophobic/hydrophilic contrastingMALDI-TOF target [8]. Sampleswere allowedto air-dryprior

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 con-

centration in urine, each urine sample was analyzed individ-

ually 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 di-

rectly 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/

sample.

2.4 Peptide mapping

SAP was isolated from plasma samples using the proto-

cols listed above. Retained SAP was eluted from the affin-

ity pipettes using a MALDI-matrix solution ( a-cyano-4-




Proteomics 2004, 4, 1825–1829 Proteomics characterization of serum amyloid P component 1827

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

0.3% v/v TFA) and applied directly onto a trypsin-acti-

vated Bioreactive probe-trypsin (BRPTrp; Intrinsic Bio-

probes) 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 diges-

tion 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 prepara-

tion for MALDI-TOF-MS, the sample spot was rehydrated

with 10 mL aliquots of 0.8% TFA, and allowed to air-dry

again.

2.5 Mass spectrometry

MS analysis was performed on a Bruker Biflex III MALDI-TOF mass spectrometer. Intact protein analysis was per-

formed 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 reflec-

tron modes. The reflectron MS analysis used a full accel-

erating 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).

3 Results and discussion

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 individu-

al. 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].

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) resi-

due. 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 mono-

sialo-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-

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

plasma. Each trace contains the protein expression pro-

file of parent SAP isolated from each individual. MultipleSAP 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 cleav-

age 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 presence of a monosialo-SAP variant had been pre-

viously observed by Pepys et al. [1] but was dismissed as

the result of the harsh environment during protein purifi-

cation. 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 microhetero-

geneity in the human SAP glycan.

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 pre-

sent 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).




1828 U. A. Kiernan et al. Proteomics 2004, 4, 1825–1829

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

urine. Strong consistency is observed between all three

samples as well as with theplasma MS. Each trace shows

intact SAP and the same associated variants that wereobserved in Fig. 2.

Since the expression profile of SAP is somewhat convo-

luted 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 over-

night plasma sample incubations with sialidase and siali-

dase 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 siali-

dase, which resulted in the complete removal of both ter-

minal 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.

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 sug-

gested by the presence of additional signals in the mass

spectrum. The observed mass difference of,–99 Da cor-

responds 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 estab-

lished due to the potential signal overlap by the mono-

sialo-SAP matrix adduct.

To verify the existence of this truncated SAP variant, pro-

teolytic digestion of affinity-purified SAP was employed.

Trypsin-bioreactive probe (BRPTrp ) technology was util-

ized [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

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 pep-

tides 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.




Proteomics 2004, 4, 1825–1829 Proteomics characterization of serum amyloid P component 1829

reflectron MS but there were two major gaps in the pro-

tein coverage, which corresponded to large peptides

(outside the mass window of reflectron MS analysis) that

either contain the site of glycosylation (residues 14–38;

MW 5117.27) or lack tryptic cleavage sites (residues

147–193; MW 5322.62). Examination of the digest frag-

ments with linear MS readily identified these two missing

fragments (Fig. 5, inset).

Moreover, further examinations of the signal from the lin-

ear MS clearly establish the presence of residues 14–38

with monosialo- and asialo-variations of the glycopep-

tides. 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-ter-

minal digest fragment (residues 194–204; MW 1285.78)

and a truncated variant (residues 194–203; MW 1186.71)were identified in the mass spectrum shown in Fig. 6, con-

firming the presence of this novel SAP truncated variant

initially observed in the parent protein mass spectrum.

Figure 6. Region of the SAP tryptic digest MS containing

the C-terminal cleavage peptide (residues 194–204;

MW 1285.78). The peptide that corresponds to the SAP-

Val variant (residues 194–203; MW 1186.71) was also pre-

sent, confirming the presence of this novel form of SAP.

4 Concluding remarks

The analysis of SAP via affinity-based proteomic technol-

ogy 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-

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 iden-

tified, the superlative nature of this biofluid for protein

analyses will become more evident. Even though the

analysis of proteins from urine has historically been diffi-

cult, this next generation of protein profiling and charac-

terization 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 tech-

nologies. This clearly illustrates the necessity for popula-

tion 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

screening.

We would like to thank Dr. Allan Bieber for the critical reading of this manuscript. This publication was sup-

ported in part by contract No. N43-DK-1-2470 and N44-

ES-35511 from the National Institutes of Health. Its con-

tents are solely the responsibility of the authors and do

not necessarily represent the official views of the National

Institute of Health.

5 References

[1] Pepys, M. B., Booth, D. R., Hutchinson, W. L., Gallimore, J.R. et al., Amyloid – Int. J. Exp. Clin. Invest. 1997, 4, 274–295.

[2] Pepys, M. B., Herbert, J., Hutchinson, W. L., Tennent, G. A.et al., Nature 2002, 417 , 254–259.

[3] Prelli, F., Pras, M., Frangione, B., J. Biol. Chem. 1985, 260,12895–12898.

[4] Tennent, G. A., Pepys, M. B., Biochem. Soc. Trans. 1994, 22,74–79.

[5] Pepys, M. B., Rademacher, T. W., Amatayakul-Chantler, S.,Williams, P. et al., Proc. Natl. Acad. Sci. USA 1994, 91, 5602–5606.

[6] Hutchinson, W. L., Noble, G. E., Hawkins, P. N., Pepys, M.B., J. Clin. Invest. 1994, 94, 1390–1396.

[7] Maury, C. P. J., Teppo, A.-M., J. Lab. Clin. Med. 1985, 106,619–623.

[8] Niederkofler, E. E., Tubbs, K. A., Gruber, K., Nedelkov, D. et al., Anal. Chem. 2001, 73, 3294–3299.

[9] Kiernan, U. A., Black, J. A., Williams, P., Nelson, R. W., Clin.Chem. 2002, 48, 947–949.

[10] Morell, A. G., Irvine, R. A., Sternlieb, I., Scheinberg, I. H., Ashwell, G., J. Biol. Chem. 1968, 243, 155–159.

[11] Ashwell, G., Morell, A. G., Adv. Enzymol. Relat. Areas Mol.Biol. 1974, 41, 99–128.

[12] Nelson, R. W., Dogruel, D., Krone, J. R., Will iams, P., Rapid Commun. Mass Spectrom. 1995, 9, 1380–1385.

[13] Kiernan, U. A., Tubbs, K. A., Nedelkov, D., Niederkofler, E.E., Nelson, R. W., Biochem. Biophys. Res. Commun. 2002, 297 , 401–405.

[14] Kiernan, U. A., Tubbs, K. A., Nedelkov, D., Niederkofler, E. E.et al., J. Proteome Res. 2003, 2, 191–197.


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