Beruflich Dokumente
Kultur Dokumente
DOI 10.1002/pmic.200300690
1825
Received
Revised
Accepted
16/10/03
17/11/03
18/11/03
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
E-mail: ukiernan@intrinsicbio.com
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
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
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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.
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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,
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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.
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5 References
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-
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