Characterization of the prion protein in human urine.

Abstract

The presence of the prion protein (PrP) in normal human urine is controversial and currently inconclusive. This issue has taken a special relevance because prion infectivity has been demonstrated in urine of animals carrying experimental or naturally occurring prion diseases, but the actual presence and tissue origin of the infectious prion have not been determined. We used immunoprecipitation, one- and two-dimensional electrophoresis, and mass spectrometry to prove definitely the presence of PrP in human urine and its post-translational modifications. We show that urinary PrP (uPrP) is truncated mainly at residue 112 but also at other residues up to 122. This truncation makes uPrP undetectable with some commonly used antibodies to PrP. uPrP is glycosylated and carries an anchor which, at variance with that of cellular PrP, lacks the inositol-associated phospholipid moiety, indicating that uPrP is probably shed from the cell surface. The detailed characterization of uPrP reported here definitely proves the presence of PrP in human urine and will help determine the origin of prion infectivity in urine.

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J Biol Chem. 2010 October 1; 285(40): 30489–30495.
Published online 2010 July 29. doi: 10.1074/jbc.M110.161794
PMCID: PMC2945542
Characterization of the Prion Protein in Human Urine
Ayuna Dagdanova,
McAnulty, Lequn Huang, Wenquan Zou, Qingzhong Kong, Pierluigi Gambetti,
Serguei Ilchenko,
!
Silvio Notari,
‡
Qiwei Yang, Mark E. Obrenovich, Kristen Hatcher, Peter
and Shu G. Chen
From the Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106,
§
the Center for Proteomics and Mass Spectrometry, Case Western Reserve University, Cleveland, Ohio 44106,
¶
the Non-Clinical Consultancy, DK-4000 Roskilde, Denmark, and
!
the Nanjing University Medical School, Nanjing 210093, China
2
To whom correspondence may be addressed: Dept. of Pathology, Case Western Reserve University, 2085 Adelbert Rd., Cleveland, OH
44106., Tel.: Phone: 216-368-0586; Fax: 216-368-4090; E-mail: pierluigi.gambetti@case.edu.
3
To whom correspondence may be addressed: Dept. of Pathology, Case Western Reserve University, 2103 Cornell Rd., WRB 5533,
Cleveland, OH 44106., Tel.: Phone: 216-368-8925; Fax: 216-368-0494; E-mail: shu.chen@case.edu.
1
These authors contributed equally to this work.
Received July 6, 2010
Copyright © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Abstract
The presence of the prion protein (PrP) in normal human urine is controversial and currently
inconclusive. This issue has taken a special relevance because prion infectivity has been demonstrated in
urine of animals carrying experimental or naturally occurring prion diseases, but the actual presence
and tissue origin of the infectious prion have not been determined. We used immunoprecipitation, one-
and two-dimensional electrophoresis, and mass spectrometry to prove definitely the presence of PrP in
human urine and its post-translational modifications. We show that urinary PrP (uPrP) is truncated
mainly at residue 112 but also at other residues up to 122. This truncation makes uPrP undetectable with
some commonly used antibodies to PrP. uPrP is glycosylated and carries an anchor which, at variance
with that of cellular PrP, lacks the inositol-associated phospholipid moiety, indicating that uPrP is
probably shed from the cell surface. The detailed characterization of uPrP reported here definitely
proves the presence of PrP in human urine and will help determine the origin of prion infectivity in
urine.
Keywords: Glycosylphosphatidylinositol Anchors, Mass Spectrometry (MS), Post-translational
Modification, Prions, Urine, Prion Protein
Introduction
The normal or cellular prion protein (PrP ) is predominantly a cell surface protein that is sensitive to
proteases and is soluble in non-ionic detergents (1,–4). In humans, the post-translational modified, full-
length PrP is made of 209 amino acids (residues 23–231), which include two sites of N-glycosylation,
and carries a glycosylphosphatidylinositol (GPI) anchor (Fig. 1A) (5, 6).
*
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Because glycosylation is not obligatory, three PrP glycoforms, diglycosylated, monoglycosylated, and
unglycosylated, are commonly observed (6). In addition to the full-length PrP , truncated forms of PrP
have been found in brains and cultured cells (7,–9). A major PrP C-terminal fragment named C1 is
generated during normal PrP processing by a cleavage at residues 111/112 that has been suggested to
take place in the endosomal recycling (10), or in a late compartment of the secretory pathway such as
the Golgi apparatus (11). Like the full-length PrP , the C1 fragment is mostly glycosylated with an
electrophoretic mobility of ~28 kDa which is reduced to ~18 kDa following deglycosylation (8). The GPI
anchor links both full-length PrP and the C1 fragment to the external surface of the plasma membrane
of the cell. The structure of the GPI anchor, which is thought to be identical for the full-length and the C1
fragment, is made of a glycan core attached to the PrP C terminus through a phosphodiester linkage of
phosphoethanolamine, and a hydrophobic phospholipid component made of phosphatidylinositol and
fatty acid that mediates the attachment to the membrane (12, 13). Although the GPI-linked membrane
isoform accounts for most of the PrP, minor trans-membrane and cytosolic forms of PrP have also been
detected (14). Because the GPI anchor is subject to a variety of natural cleavages, secretion or shedding
of various isoforms of PrP in the medium of cultured cells, cerebrospinal fluid, serum, plasma, and
milk has been reported (6, 15,–18).
A rogue post-translational modification of PrP can trigger prion diseases, a group of fatal
encephalopathies that affect both humans and animals (19). The basic pathogenic mechanism
underlying prion diseases is the conversion of the normal PrP into a misfolded, disease-associated
isoform, termed prion or scrapie prion protein (PrP ), which accumulates predominantly in the brain
and to a lesser extent in other organs (19,–21).
Although PrP and PrP share the amino acid sequence and the normal post-translational
modifications, they differ in that PrP is insoluble in nondenaturing detergents, mostly resistant to
proteases. and aggregated (22, 23). It is thought that the conversion of PrP to PrP is associated with a
transition from α-helical to β-sheet-rich conformations. Prion transmissions between animals, from
cattle to humans through food ingestion, and from humans to humans through blood transfusion, solid
tissue grafting, and injection of tissue-extracted hormones has been widely documented (24, 25).
The presence of infectious PrP in human urine would pose serious risks to public health because of the
medicinal use of urine-extracted proteins, hormones, urokinase, and the risk of environmental prion
contamination. The search for PrP in urine has yielded controversial results. Shaked et al. (26)
originally reported the detection of PrP by Western blotting with the monoclonal antibody 3F4 in the
urine of prion-affected Syrian hamsters and human subjects. They also described the presence of
protease-sensitive PrP, apparently full-length PrP , in controls that were free of prion disease, which
suggests that normal urine contains PrP whereas urine from individuals affected with prion disease
also contains PrP . However, three subsequent studies using the same antibody failed to detect PrP in
urine from normal and prion disease-affected individuals and demonstrated that the false positive
results arose from the cross-reaction of anti-mouse IgG with either contaminating bacterial proteins
(27) or urinary IgG fragments (28, 29). Nonetheless, a series of more recent studies have observed prion
infectivity in urine from experimentally and naturally affected animals (30,–33). In an additional report,
prion infectivity was observed in urine from prion-infected mice affected by concomitant nephritis but
not in prion-infected but nephritis-free mice (34). Thus, despite numerous reports of prion infectivity in
urine in the course of prion diseases, the nature of the infectious prion agent in urine as well as the
presence of PrP in urine remain to be determined.
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Antibodies
used (Fig. 1A). They included two rabbit antibodies to the N and C termini (36) and the monoclonal
antibodies 3F4 (37) (Signet Laboratories, Dedham, MA), 6H4 (Prionics, Zurich), and 8H4 (38) to
internal sequences of PrP (Fig. 1A).
Immunoprecipitation
0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM Tris-HCl, pH 7.5) containing protease inhibitors
(Roche Applied Science). Human urine was concentrated 100-fold by ultrafiltration with a 10-kDa cutoff
membrane filter (Centriprep, Millipore). Anti-PrP monoclonal antibodies 3F4, 6H4, 8H4 and were
conjugated to tosyl-activated magnetic beads (M-280 Dynabeads; Dynal), and immunoprecipitation was
performed as described previously (39). The resulting immunoprecipitates were used for
immunoblotting with anti-C antibody. Samples were subjected to one- or two-dimensional SDS-PAGE
followed by immunoblotting using anti-C antibody, as described elsewhere (36, 40).
Purification and Mass Spectrometric Detection of Urinary PrP
concentrated 200-fold by ultrafiltration followed by deglycosylation with N-glycosidase F (PNGase F;
New England Biolabs). Deglycosylated proteins were then separated in 14% Tris-glycine tube gel (Bio-
Rad). PrP-containing fractions were detected with the anti-C antibody and further purified by two-
dimensional chromatofocusing and reverse phase HPLC. After each purification step, the fractions were
collected and analyzed. Urinary PrP (uPrP) was detected by immunoblotting with anti-C, and the purity
of the fractions was assessed by silver staining. The final preparations were subjected to two-
dimensional SDS-PAGE, and Coomassie-stained spots matching those identified on immunoblots were
excised and digested in-gel with trypsin. Tryptic peptides were separated by reverse phase HPLC
followed by nanoelectrospray ionization MS using a hybrid ion trap Fourier-transform ion cyclotron
resonance mass spectrometer (LTQ-FT) equipped with a 7 T superconducting electromagnet and a
packed-Tip nanospray ionization probe (LTQ-FT; Thermo Electron Corp). Chromatographic separation
of the protein digest was performed by an Ultimate 3000 nano-HPLC (Dionex; Germering, Germany)
with a trapping precolumn (C18, PepMap100, 300 µm ×5 mm, 5-µm particle size, 100 Å; Dionex)
followed by a reverse phase column (C18, 75 µm ×150 mm, 3 µm; Dionex), using a mobile phase A (0.1%
formic acid in water) and B (80% acetonitrile, 0.04% formic acid in water) with a linear gradient of 2%
B/min. The peptides were electrosprayed at a flow rate of 300 nl/min via the silica noncoated PicoTip
emitter (FS360-20-10-C12; New Objective Inc., Woburn, MA) at 2.2 kV. The capillary temperature was
kept at 200 °C. Full MS spectra were recorded in the Fourier-transform ion cyclotron resonance cell,
and data-dependent tandem MS spectra of the six highest intensive ions were simultaneously recorded
by the linear ion trap LTQ at collision energy of 35 eV, isolation width of 2.5 Da, and activation Q of
0.250. Peptide assignments were made by searching tandem MS spectra against NCBI protein data base
using the BioWorks software (Thermo Electron).
Because PrP serves as substrate and precursor in the formation of infectious PrP , a critical step in
understanding the mechanism of prionuria is to establish definitively the presence and characteristics of
PrP in urine. We previously reported the detection of a PrP-immunoreactive protein in human urine
(35). Here, we report on the use of advanced mass spectrometry combined with other techniques to
demonstrate definitively the presence, primary structure, and post-translational modifications of PrP in
human urine.
EXPERIMENTAL PROCEDURES
A panel of five antibodies recognizing epitopes spanning the entire length of human PrP was
Brain homogenate was prepared at 4 °C in lysis buffer (100 mM NaCl, 10 mM EDTA,
Urine collected from healthy subjects was
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Characterization of the GPI Anchor
according to the manufacturer's protocol, and urinary proteins were dissolved in Tris-buffered saline
(pH 7.5) containing 2% Triton X-114 at 4 °C. The sample was subjected to phase transition for 15 min at
35 °C followed by centrifugation. The resulting aqueous phase and detergent phase were collected and
prepared for immunoblotting analysis. The lipid component of the GPI anchor was removed from the
urinary proteins by incubating the deglycosylated samples with phosphatidylinositol-specific
phospholipase C (PI-PLC) (Sigma) 1 unit/ml for 1 h at 37 °C.
Antibody Mapping of uPrP
homogenate with a panel of antibodies (Abs) to three regions of full-length PrP revealed that, although
all of the Abs immunoprecipitated PrP-immunoreactive proteins from normal brain homogenate, in
urine preparations the immunoprecipitations were positive only with the two Abs to the more C-
terminal region of PrP (Fig. 1). Immunoblotting with the anti-N Ab also failed to detect the PrP-
immunoreactive protein in urine.
Peptide Sequencing of uPrP by Mass Spectrometry
the human urine indeed was PrP, we subjected purified and deglycosylated uPrP to tryptic digestion and
peptide sequencing using highly accurate Fourier-transform mass spectrometry (MS). The peptides
detected by tandem MS were matched with the sequence of human PrP (see “Experimental
Procedures”). Except for a few small peptides, all other tryptic peptides were identified by MS with high
degree of confidence as belonging to the C-terminal region of PrP. This region comprised residues 112–
228 with the PrP 112–136 sequence being the most N-terminal peptide detected (Table 1 and Fig. 2A).
Additional evidence identifying PrP was the observation that peptides 157–185 and 195–204, which
carries two predicted sites of N-glycosylation, contained aspartic acid rather than asparagine as residues
181 and 197 of the human PrP sequence (Table 1 and Fig. 2, B and C). These results indicate that
deglycosylation of uPrP by PNGase F had resulted in the substitution of asparagine with aspartic acid at
the two glycosylation sites, as expected (42).
N-Linked glycans were removed by PNGase F (New England Biolabs)
To remove the entire GPI anchor from urinary proteins, the deglycosylated samples were treated with
48% hydrofluoric acid (HF) for 48 h at 4 °C. The HF-treated sample was neutralized by the addition of 4
M unbuffered Tris. Samples equivalent of one-tenth of the starting material and its fractions were loaded
onto 16% SDS-PAGE and immunoblotted with anti-C.
RESULTS
Parallel immunoprecipitations of concentrated urine and normal brain
As expected, the electrophoretic mobilities of the PrP immunoprecipitated from the brain spanned from
35 to 18 kDa, and deglycosylation resulted in two PrP-reactive immunoblot bands of about 27 and 18
kDa, corresponding to the full-length and C1 fragment of PrP, respectively (8). In contrast, only one PrP-
immunoreactive band of ~28 kDa was detected in the urine immunoprecipitates, which, following
deglycosylation, shifted to about 18 kDa (Fig. 1C). These findings strongly suggest that normal human
urine contains an isoform of PrP that is truncated at the N terminus at residues that are at, or more C-
terminal than, the 106–110 epitope of 3F4 (Fig. 1A).
On two-dimensional immunoblots, the 28-kDa uPrP was distributed as at least nine spots with acidic pI
values of 3.8–5.3 likely reflecting the major heterogeneity conferred to uPrP by the glycans (Fig. 1D).
After deglycosylation only three less acidic spots of 18 kDa were detected at pI 5.4–6.0. This remaining
heterogeneity might derive from the presence of different GPI anchors (41) and/or the presence in urine
of PrP isoforms with slightly ragged ends which would carry different charges.
To prove that the PrP-immunoreactive band observed in
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Characterization of the GPI Anchor in uPrP
anchor, we first subjected urinary proteins to phase transition following deglycosylation and treatment
with 2% Triton X-114. Following these procedures, uPrP was recovered mostly in the aqueous phase
rather than in the detergent phase (Fig. 3A), suggesting that the anchor associated with uPrP, if present,
is soluble and largely hydrophilic thus likely lacking the phospholipid component. As second step, we
digested uPrP with the PI-PLC that cleaves off the phospholipid moiety of the GPI anchor. In brain
tissue, used here as control, it is well known that the cleavage of the anchor phospholipid moiety
paradoxically causes a slower migration of the anchor-cleaved PrP (15, 40, 43). We reproduced this
phenomenon in both full-length PrP and the C1 PrP C-terminal fragment (Fig. 3B). In contrast, this
effect was not observed when PI-PLC digestion was performed on uPrP (Fig. 3B). Combined phase
transition and PI-PLC cleavage experiments suggested that the phospholipid component of the anchor is
not present in uPrP. To verify whether the glycan core of the anchor was retained in the uPrP, we used
HF, which hydrolyzes the phosphodiester bonds and releases the entire anchor from the PrP (40, 43,
44). HF treatment of uPrP indeed generated a faster migrating fragment with a reduction of molecular
mass of about 3–4 kDa, consistent with the removal of the anchor (Fig. 3C). Again, following HF
digestion the C1 PrP fragment from brain preparations showed precisely the same change in migration (
Fig. 3C). This set of studies clearly indicates that uPrP lacks the phospholipid component but retains the
hydrophilic glycan core of the GPI anchor (Fig. 3D). Primary and secondary structures and post-
translational modifications of uPrP are shown diagrammatically in Fig. 4.
Overall, our MS study definitely proves that PrP is present in normal human urine and shows that uPrP
is truncated at the N terminus (Figs. 1 and 2). Although the residue methionine 112 definitely is one, and
perhaps the most common, N terminus of uPrP, isoforms with N termini at residues 113, 117, 118, 120,
121, or 122 may also be present because peptides with these N termini were also detected. This
possibility is supported by the consideration that none of these N termini, including the 112 N terminus,
involves residues arginine or lysine, which are the obligatory trypsin cleavage sites, indicating that the
uPrP N termini are generated by endogenous proteolytic enzyme(s) and not by our trypsin digestion.
The finding that the C terminus is residue 228, rather than residue 231 as in PrP , is likely due to the
trypsin digestion, which is expected to cleave the C-terminal region of PrP at the arginine 228 residue
releasing the PrP 229–231 (GSS sequence) attached to the GPI anchor. This interpretation is in keeping
with the strong uPrP immunoreactivity with the Ab anti-C to residues 220–231. However, this finding
raises a question concerning the presence of the GPI anchor in uPrP.
To investigate the presence and the characteristics of the GPI
DISCUSSION
The present study demonstrates unequivocally that PrP is present in normal human urine. Our MS
analyses provide the first description of the primary structure of uPrP and show that human uPrP is
truncated at N terminus residue 112. Although other N terminus forms, truncated at residues 113, 117,
118, 120, or 122, might also exist, the major uPrP backbone matches that of the PrP 112–231 fragment
called C1 that is generated by a metabolic pathway of PrP. This fragment as the full-length PrP is
mostly tethered by a complete GPI anchor to the cell plasma membrane (8, 13). In contrast, our analyses
of the uPrP C-terminal region and anchor show the presence of an incomplete anchor. Our inability to
demonstrate the C-terminal sequence 229–231 by MS is very likely due to the trypsin treatment that
cleaved PrP at arginine or lysine residues before MS analysis, thereby releasing the most C-terminal
three residues attached to the anchor. Similar findings were reported in a study on hamster brain PrP in
which trypsin treatment of the 221–231 C-terminal PrP linked to the GPI anchor ESQAYYDGRRS-GPI
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generated the C-terminal truncated sequence 221–229 ESQAYYDGR ending with arginine as expected
(44). This conclusion is strongly supported by the set of studies we carried out which includes Trion X-
114 phase transition, PI-PLC digestion, and HF treatment. The results show that uPrP carries an anchor
which, unlike that of C1 fragment, lacks the phospholipid component. Therefore, uPrP likely results
from the shedding that has been observed in cell culture and body fluids such as cerebrospinal fluid,
serum, sperm, and milk (6, 16, 17, 45,–48). Two mechanisms of PrP shedding have been proposed (15).
The first postulates that PrP is shed by proteolytic cleavage, possibly zinc metalloprotease, resulting in
a PrP isoform truncated at C terminus 228 lacking the GPI anchor (15, 40, 43). According to the second
mechanism, which matches our observations in uPrP, shedding would occur following either disruption
of the lipid rafts or GPI cleavage by a phospholipase, or both. Either of these two latter processes would
generate PrP linked to a soluble anchor lacking the phospholipid component (15, 47). It has been
reported that in cultured cells, the PrP shed by either of these two mechanisms has the full-length N
terminus or is truncated at or around residue 112 as PrP fragment C1 (16). Whether uPrP originates from
the shedding of the C1 fragment, as has been shown in baby hamster kidney cells, or from the shedding
of the full-length PrP that is N-terminal-truncated following the shedding, remains to be determined.
Our findings raise a few considerations related not only to the normal metabolism of PrP in the urinary
system but also to the origin of the PrP isoform(s) that carry the prion infectivity demonstrated in the
urine of animals affected by prion diseases. First, if uPrP results from shedding, it is important to
determine whether it originates from tissues involved in producing and storing urine or derives directly
from PrP shed into the blood. Our finding of a large overrepresentation of C1 over the full-length PrP in
the urinary bladder and, to a minor extent, in the kidney supports the possibility that uPrP derives
from one or both these tissues. On the other hand, if uPrP originates in blood, it likely has the same
characteristics of blood plasma PrP, which has not been fully characterized (49, 50). These
considerations raise the question of whether soluble, hydrophilic, and shed uPrP are capable of
sustaining prion infectivity in urine. This possibility may not be ruled out despite at least three lines of
evidence that suggest that C1 is not a good substrate for PrP replication. First, the C1 fragment is
generated from a PrP N-terminal cleavage that breaks the 100–130-residue domain (the so-called
amyloidogenic region of PrP), considered important for the conversion of PrP into PrP (8, 51,–53).
Second, C1 also lacks two of three regions recently shown to be important for PrP -PrP interaction,
specifically 23/33, 98/110, and 136/158 (54). Finally, the conversion of C1 to PrP has never been
reported.
Unquestionably, to elucidate the origin of prion infectivity in urine it is necessary to characterize the
infectious PrP present in urine and related tissues. The present characterization of uPrP will likely
facilitate future studies toward these goals and help develop a possible premortem diagnostic test of
prion diseases.
Acknowledgments
We thank Drs. Mark Chance and Janna Kiselar (Center for Proteomics and Mass Spectrometry, Case
Western Reserve University) for access to MS instrumentation and helpful discussions and Yvonne
Cohen for contributing to the PrP characterization.
This work was supported, in whole or in part, by National Institutes of Health Grant AG14359. This work was also
supported by Centers for Disease Control and Prevention Grant UR8/CCU515004, Department of Agriculture National
Research Initiative Grant 2002-35201-2608, Department of Defense National Prion Research Program Grant DAMD17-
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PrP
Ab
GPI
HF
PI-PLC
PNGase F
PrP
uPrP
PrP
03-1-0283, and by the Charles S. Britton Fund.
5
S. Notari, S. G. Chen, and P. Gambetti, unpublished data.
The abbreviations used are:
C
cellular PrP
antibody
glycosylphosphatidylinositol
hydrofluoric acid
phosphatidylinositol-specific phospholipase C
N-glycosidase F
scrapie PrP
urinary PrP
prion protein.
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Figures and Tables
FIGURE 1.
Immunoprecipitation and immunoblotting of brain and uPrP. A, Primary and secondary
structures and post-translational modifications of human PrP. β-sheet 1,128–131; β2,161–164; α-helix 1,
144–154; α2, 173–194; α3, 200–220 are indicated. Sugars at Asp
Cys-Cys and GPI anchor are represented. Antibodies used in immunoblot and
immunoprecipitation with relative epitopes are indicated. B, immunoblot (IB) detection of PrP from
brain (B) and urine (U) in crude homogenate and after PrP immunoprecipitation (IP) with the indicated
Abs. Anti-N and anti-C antibodies were used for detection (IB Abs). C, comparative immunoblotting of
uPrP and brain PrP before and after deglycosylation with PNGase F. Although upon PNGase F treatment
brain PrP, which untreated migrates to 27–35 kDa, shifts to two major bands of 27 kDa and 18 kDa,
untreated uPrP shows a mobility of 28 kDa that shifts to 18 kDa upon treatment. D, two-dimensional gel
electrophoresis of uPrP. Upper panel, uPrP not treated with PNGase F. At least nine spots likely
corresponding to PrP glycoforms with acidic isoelectric points of 3.8–5.3 are detected. Lower panel,
uPrP treated with PNGase F. The spots are reduced to 3–4 with less acidic pI values of 5.4, 5.7, and 6.0.
, Asp residues, disulfide bond
TABLE 1
uPrP peptides identified by LTQ-FT-MS
MH
2274.068
zm/z (ion)m/z (ion)Xcor PositionPeptide sequence
112–136 H.M*AGAAAAGAVVGGLGGYM*LGSAM*SR.P
181 197
79214
+Th
a b
Exp
c
Th
defg

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2 1137.5511137.5377.1
3 758.701758.694 7.2
2127.0322 1064.0331064.0206.16113–136 M.AGAAAAGAVVGGLGGYM*LGSAM*SR.P
3 709.687709.682
1856.8992928.961 928.9535.75 117–136 A.AAGAVVGGLGGYM*LGSAM*SR.P
1785.8622893.441893.4354.61118–136 A.AGAVVGGLGGYM*LGSAM*SR.P
1657.8042829.408829.4055.64120–136 G.AVVGGLGGYM*LGSAM*SR.P
1586.7672793.891793.887 5.08121–136 A.VVGGLGGYM*LGSAM*SR.P
1487.698744.358744.353 3.95122–136 V.VGGLGGYM*LGSAM*SR.P
1448.6812724.847724.8444.05137–148 R.PIIHFGSDYEDR.Y
3483.567483.5654.63
988.4011988.409988.4012.45 140–148 H.FGSDYEDR.Y
1125.4592 563.236563.233 3.29141–148 I.HFGSDYEDR.Y
1102.5312 551.772 551.7702.70 157–164 R.YPNQVYYR.P
3638.6313 1213.56 1213.5487.2157–185 R.YPNQVYYRPM*DEYSNQNNFVHDC#VDITIK.Q
4910.419910.413 4.6
1140.5051 1140.5111140.5052.64195–204 K.GEDFTETDVK.M
2570.758570.7563.52
1571.7192 786.367786.3634.02209–220 R.VVEQM*C#ITQYER.E
1555.7042778.372778.3664.38209–220 R.VVEQMC#ITQYER.E
1044.4751 1044.4791044.4752.19221–228 R.ESQAYYQR.G
2522.743522.7382.53
MH, theoretical mass of the protonated molecule.
z, number of charges.
m/z (ion), experimentally determined mass to charge ratio of the ionized molecule.
Exp
m/z (ion) , theoretical mass to charge ratio of ionized molecule.
Th
Xcorr, cross-correlation score, a parameter for the quality of peptide matches in the Sequest software.
The higher the score, the better the confidence of peptide.
Position, location of the peptide in the PrP sequence.
Symbols * and # in the peptide sequences indicate, oxidation of methionine and alkylation of cysteine,
respectively. Dots identify residues immediately preceding or following the indicated sequence.
FIGURE 2.
a+Th
b
c
d
e
f
g

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Mass spectra of tryptic peptides of uPrP. A, mass spectrum of the N-terminal peptide 112–136.
The 112–136 peptide is identified based on the parent ions with isotopic resolution as they are detected

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in the full scan MS spectrum (inset). The monoisotopic peak of the 112–136 peptide with m/z value of
1137.551 is selected for fragmentation via collision-induced dissociation. This dissociation produces
daughter b and y ions which are then detected in the tandem MS spectrum. The b and y ions result from
the sequential cleavage of peptide bonds corresponding to N-terminal and C-terminal fragments of the
112–136 peptide, respectively. The detected b and y ions are consistent with the 112–136 peptide
sequence shown on the top part of the panel. The b and y ions are indicated by their positions in the
peptide sequence (subscript) and charges they carry (superscript). * indicates oxidized methionine. B
and C, mass spectra of the peptides 157–185 and 195–204 containing the 181 and 197 N-glycosylation
sites, respectively. The peptides are identified as described in A based on isotopic parent ions detected in
the full scan MS spectra (insets). The monoisotopic peaks with m/z values of 910.418 (B) and 570.758
(C) are selected for fragmentation via collision-induced dissociation, producing daughter ions detected
in the tandem MS spectrum (see A for more details). The b and y ions are consistent with the peptide
sequences shown on the top of B and C. * and # indicate oxidized methionine and
carboxyamidomethylated cysteine, respectively. Note that residues 181 and 197 are detected as aspartic
acid and not asparagine, consistent with the PNGase F deglycosylation, carried out before MS analyses,
which converts asparagine into aspartic acid by deamidation. In all of the sequences, dots indicate the
residues immediately preceding and following the sequence examined.
FIGURE 3.
Characterization of the GPI anchor of uPrP. A, phase transition. The immunoblots of
deglycosylated Triton X-114 (Tx-114)-treated urine fractions show that uPrP is recovered mostly in the
aqueous phase (Aq) instead of the detergent phase (Det). The Triton X-114-untreated urine used as
control is indicated (lane −). B, PI-PLC cleavage. In the brain preparation, full-length PrP and, to a
lesser extent, the C1 fragment, show the paradoxical slower electrophoretic migration effect caused by
the cleavage of the GPI phospholipid component by PI-PLC. This effect is not detectable in uPrP. C, HF
treatment of uPrP results in a 3–4-kDa electrophoretic downshift, which matches that of the PrP C1
fragment from the brain. D, diagrammatic representation of GPI anchor. The GPI anchor is represented

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with its glycan core and lipid component. Hydrolysis sites of phosphodiester bonds by HF and of
phospholipid portion by PI-PLC are indicated.
FIGURE 4.
Linear sequences and locations of the amino acids definitely identified by FT-MS with
diagram of the GPI anchor in uPrP. A, sequences of uPrP definitely identified by MS are
underlined. Letters in bold indicate the N termini. The two aspartate residues generated by PNGase F
deglycosylation at N-glycosylation sites 181 and 197 (see “Results”) are shown in bold italic. B, primary
and secondary structures of uPrP with post-translational modifications including N-linked glycans (○),
disulfide bonds, and hydrophilic GPI anchor are shown. The linear sequences definitely identified by MS
are aligned on the top. Secondary structures are hypothetical based on the assumption that they are the
same as those of PrP .
Articles from The Journal of Biological Chemistry are provided here courtesy of American Society for Biochemistry
and Molecular Biology
C