Comparative Urine Protein Phenotyping Using Mass Spectrometric

Immunoassay
Urban A. Kiernan, Kemmons A. Tubbs, Dobrin Nedelkov, Eric E. Niederkofler,
Elizabeth McConnell, and Randall W. Nelson*,
Intrinsic Bioprobes, Inc., 625 South Smith Road, Suite 22, Tempe, Arizona 85281, and Arizona State University,
Tempe, Arizona 85287
Received October 1, 2002

Reported here, human urine samples were analyzed for -2-microglobulin (2m), transthyretin (TTR),
cystatin C, urine protein 1 (UP1), retinol binding protein (RBP), albumin, transferrin, and human
neutrophil defensin peptides (HNP) using mass spectrometric immunoassay (MSIA). MSIA is a unique
analytical technique, which allows for the generation of distinct protein profiles of specific target proteins
from each subject, which may be subsequently used in comparative protein expression profiling
between all subjects. Comparative profiling allows for the rapid identification of variations within
individual protein expression profiles. Although the majority of analyses performed in this study revealed
homology between study participants, roughly one-quarter showed variation in the protein profiles.
Some of these observed variants included a point mutation in TTR, absence of wild-type RBP,
monomeric forms UP1, a novel 2m glycated end product and altered HNP ratios. MSIA has been
previously used in the analysis of blood proteins, but this study shows how MSIA easily transitions to
the analysis of urine samples. This study displays how qualitative urine protein differentiation is readily
achievable with MSIA and is useful in identifying proteomic differences between subjects that might
be otherwise overlooked with other analytical techniques due to complexity of the resulting data or
insufficient sensitivity.
Keywords: proteomics protein variations MALDI-TOF urine biomarker discovery

Introduction
Urine is an easily accessible biological fluid that has lately
become more intensely studied in the quest to identify protein
and peptide biomarkers that may potentially be used to assess
kidney function and identify the presence of disease in the
individual. Many small proteins and peptides freely pass though
the glomerulus, where they are then either catabolized within
the tubular cells of the kidney or are excreted in the urine.1
Abnormalities in kidney function, and the presence of disease,
often result in variations in urine protein excretion rate and
content, both of which have been historically monitored via
enzyme-linked immunosorbent assays (ELISA).2,3 This, along
with the fact that the acquisition of urine is normally a
noninvasive procedure, makes it an ideal biological fluid for
human proteomics studies.
The field of proteomics is developing new technologies and
methodologies toward the analysis of proteins from a variety
of biological fluids, including urine. A common proteomic
approach to analyzing urine proteins involves 2-dimensional
polyacrylamide gel electrophoresis (2D-PAGE) for protein
separation. Even though this method is capable of separating
* To whom correspondence should be addressed. Tel: (480) 804-1778.
Fax: (480) 804-0778. E-mail: rnelson@intrinsicbio.com.
Intrinsic Bioprobes, Inc..

Arizona State University.

10.1021/pr025574c CCC: $25.00

2003 American Chemical Society

hundreds to thousands of proteins in a single analysis, it is not

without weakness. 2D-PAGE has in the past had poor results
in the analysis of peptides due to their high mobilities.4 The
identification of separated proteins, as well as the detection of
low-abundance proteins has also been historically problematic.
A more recent innovation, which incorporates 2D-PAGE with
mass spectrometry (2DE/MS), provides more accurate results,
but requires enzymatic digestion of the isolated proteins.5
Although able to accurately identify genes from which proteolytic fragments originate,6 the 2DE/MS approaches are not
readily able to yield information on the full-length protein and
oftentimes subtle details on the analyte (e.g., the presence of
point mutations and post-translational modifications) are
missed. Because this information can be lost, analysis of intact
proteins is becoming more widely accepted as a mean of
proteomic analysis.7
A proteomics technology that has great potential in the area
of intact urine protein analyses is the mass spectrometric
immunoassay (MSIA). MSIA combines the selectivity of an
immunoassay with the sensitivity, resolution, and mass accuracy of matrix assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS). A major advantage
of mass spectrometric detection over other conventional immunoassay schemes is the ability to discriminate between
protein variants in a single assay.8 Because retrieved species
Journal of Proteome Research 2003, 2, 191-197

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Kiernan et al.

are detected at precise molecular masses, mass-shifted variants

of a protein (i.e., post-translational modifications or point
mutations) are readily detected in a single assay. This approach
is contrary to conventional immunoassays, where each protein
variant would require an individual, monospecific assay, which
obviously requires an a priori knowledge of the variants under
investigation (i.e., a monospecific assay must be constructed
for each variant after the variant is either discovered or
hypothesized). Conversely, the MSIA approach is able to
discover as yet unidentified variants of proteins through the use
of pan antibodies towards the protein of interest. Thus, MSIA,
when applied to the routine screening of known protein
variants (i.e., wild-type), holds much potential in the discovery
and identification of variants resulting from post-translational
modifications, splicing variations or point mutations.
Moreover, MSIA in the form of an affinity pipettor tip is
capable of analyzing very low abundance proteins via a
repetitive pipetting action. The flowing action of the pipettor
tip concentrates and purifies the target protein prior to MALDITOF MS analysis allowing for routine analyses of protein targets
in the picomolar and sub-picomolar range.9 Once in the mass
spectrometer, different forms of the same protein are readily
distinguishable by measurable alterations in molecular mass,
thus allowing for protein isoform identification.
The general utility of MSIA is not that of generating global
gene product expression profiles, as done by many other
proteomics technologies, but is that of targeting a specific
protein and analyzing all endogenous forms of the intact target
protein from within a specific human biological fluid. In this
manner, the exact form(s) of a proteinsrather than those
presumed from a genomic database searchscan be determined
within an individual, and slight changes in structure discerned
for (ultimately) correlation with disease. Previously, MSIA has
been demonstrated in the high throughput quantification and
characterization of various human plasma proteins10-12 but still
has been largely unexploited in the field of urine protein
analyses, with the only reported application being the quantitation of -2-microglobulin (2m)13 and the comparative
profiling of retinol binding protein (RBP).14 Described here is
the development of further urine-based MSIA assays targeting
transthyretin (TTR), cystatin C (CYSC), urine protein 1 (UP1),
albumin (ALB), transferrin (TRFE), and human neutrophil
defensins (HNP), and a brief study illustrating their use in
profiling these proteins between individuals.

Experimental Section
Study Subjects. Urine samples were collected from 5 unrelated male subjects, ages ranging from 26 to 79. Four subjects,
ages 26-68, were healthy study participants, whereas one
individual, age 79, was diagnosed with pancreatic cancer. Urine
samples were obtained via protocols approved through Intrinsic
Bioprobes Inc.s Internal Review Board (IRB). The individuals
had read and signed an Informed Consent form.
Sample Preparation. Urine samples, 25 mL mid-stream
voids, from five individuals were collected. The urine was
collected directly into sterile urine collection cups that were
pretreated with 50 L of the protease inhibitor cocktail consisting of AEBSF (100 mM); aprotin (80 M); bestatin (5 mM); E-64
(1.5 mM); leupeptin (2 mM); pepstatin A (1 mM) to prevent
any enzymatic breakdown or modification. Samples were
collected and stored at -70C until ready for analysis. Samples
were thawed in a warm water bath (37 C) just prior to analysis.
192

Journal of Proteome Research Vol. 2, No. 2, 2003

Figure 1. Mass spectrum of urine diluted 1:20. Small peptides

were present, whereas target proteins were not observed.

Sample Analysis. Each sample was combined 1:1 (v/v) with

2M ammonium acetate (to adjust the pH to 6.8-7.2) and
poured into an individual poly(vinyl chloride) solution basin
prior to analysis. Each sample was individually addressed with
individual MSIA-Tips (Intrinsic Bioprobes, Inc.) derivatized with
anti-2m, anti-TTR, anti-CYSC, anti-UP1, anti-RBP, anti-ALB,
anti-TRFE, or carboxylic acid surface (cation exchange for HNP
analysis). All eight analyses were run in parallel through each
sample with MSIA-Tips loaded onto an octapette.
The manual incubation of each urine sample consisted of
300 cycles (150 L of sample) through each MSIA-Tip. After
incubation, tips were thoroughly rinsed using HBS buffer (10
cycles, 150 L), doubly distilled water (5 cycles, 150 L), 20%
acetoniltrile/1M ammonium acetate wash (10 cycles, 150 L)
and finally with doubly distilled water (15 cycles, 150 L).
Retained species were eluted by drawing 4 L of MALDI matrix
solution (saturated aqueous solution of sinapic acid (SA), in
33% (v/v) acetonitrile, 0.4% (v/v) trifluoroacetic acid) into each
tip and depositing directly onto a 96-well formatted hydrophobic/hydrophilic contrasting MALDI-TOF target.10 Because
of the larger sample volumes of urine and the number of
iterations used, the time spent to run the assays were 20minutes/sample. MALDI-TOF mass spectrometry and data
analysis were performed on all samples as described previously.10 Acquired mass spectra all had mass accuracy within
0.01%, which was sufficient to correctly identify target proteins.

Results and Discussion

The analysis of urine directly with MALDI-TOF MS is shown
in Figure 1. The spectrum was produced by diluting human
urine (Individual 1) by a factor of 20 in double distilled water
and serves as a control (point of reference) for the MSIA
process. A dilution of urine was required in order to reduce
the high salt content of the biological fluid, which would
otherwise disrupt the MALDI process. Only small peptides are
observed.
-2-Microglobulin (2m). 2m is a low molecular weight
protein (11 729 Da), which was identified as a light chain of
the class I major histocompatability antigens.15 Found in most
biological fluids, elevated levels of 2m in blood and urine can
result from a number of ailments (i.e., AIDS,16 rheumatoid
arthritis,17 leukemia18). Glycation of 2m, the covalent attachment of a reducing sugar, is commonly observed in individuals

Comparative Urine Protein Phenotyping

Figure 2. Mass spectrometric results of urinary 2m MSIA. Signal

from wild-type 2m is consistent in all five traces, however Trace
E also contains a high proportion of glycated 2m. Glycation
resulted in a + 146 Da mass shift due to the covalent modification
by a deoxyhexose.

with one of a number of metabolic disorders (i.e., diabetes

mellitus, uremia, hypoglycemia, etc.) resulting in advanced
glycation end products (AGEs).19,20 Moreover, 2m has also
been pathogenically associated in many amyloid disorders
including dialysis related amyloidosis (DRA).21
Analyses of the human urine samples with anti-2m MSIA
are shown in Figure 2 with wild-type (wt-)2m seen in the mass
spectra of all study participants. Inter-individual results are all
very similar, as shown in Figure 2 inlet, except for Trace E in
which a glycated 2m variant is present in relatively high
abundance. Glycated 2m is the result of excess dietary
reducing sugars present in the blood nonenzymatically attaching to free amine groups of local proteins through the Maillard
reaction that can result in a number of possible Amidori
products.22,23 The observed glycated 2m in Figure 2-Trace E
has a measured mass increase of 146 Da, which corresponds
to an imidazolone formation, an Amidori product consisting
of a cyclic ring formed between a reduced sugar and an arginine
residue. This 2m variant was originally observed by Niwa et
al. in amyloid plaque deposits,22 but was then postulated to
be artifacts of the overexposure of the insoluble 2m plaques
to normal levels of blood reducing sugars. The data presented
here is the first report of imidazolone-modified 2m from a
human biological fluid and demonstrates that this unique form
of glycation can be found on native unplaqued 2m. The
individual whose results were shown in Trace E was diagnosed
with pancreatic cancer, and clinical studies have shown a
strong correlation between the development of the pancreatic
cancer and diabetes mellitus.24,25 Hence, the presence of AGEs
in this patient might be suggestive of the presence of a
metabolic disease, such as diabetes mellitus, but due to a lack
of symptoms, the patient was not tested at the time of sample
collection.
Transthyretin (TTR). TTR, the second target, is a thyroid
hormone carrier found in high levels in both serum and
cerebral spinal fluid. Produced mainly in the liver, TTR forms
a homotetramer26,27 and is often complexed with other proteins
in the transport of various biologically active compounds.
Structurally, wt-TTR comprises 127 amino acids and has a MW
of 13 762. Over 80 point mutations have been cataloged for
TTR, with all but 10 potentially leading to severe complica-

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Figure 3. Comparison of urinary TTR MSIA. Both the wild-type

and a post-translationally modified (cysteinylation; m ) +128
Da) forms are seen in all five traces. While, varied amounts of
cysteinylated TTR are seen present in each sample, Trace B also
shows the presence of a variant form of the TTR protein. Peak
splitting, m ) +30, is observed in the wild-type and the
cysteinylated forms of TTR because of two forms of the protein
being expressed, a wild-type and mutant, that are eventually
excreted into urine.

tions.28 The majority of these mutation-related disorders are

caused by amyloid plaques depositing on various tissues,
eventually leading to complex dysfunctions; including carpal
tunnel syndrome, drussen, and familial amyloid polyneuropathy.29-32 Multiple TTR post-translational modifications
have been previously detected in the plasma of healthy
individuals,33 due mostly to TTR having a free cysteine residue,
which commonly react with other sulfhydrils, cysteines, glutathions, etc.
Figure 3 shows the result of the urinary MSIA analyses of
TTR. Both wt- and post-translationally modified (PTM) forms
of intact TTR are readily apparent in all five traces. This in itself
is novel, due to conflicting reports regarding proximal tubular
reabsorption and the presence of TTR in urine.34,33 The most
abundant PTM observed is the cysteinylated (cys) form, m )
+119 Da. Expanded views of the singly charged TTR signals,
shown in each corresponding Figure 3 inlet, clearly show that
ratios between the PTM and the wild-type forms of TTR vary
between all individuals analyzed. Moreover, the TTR signal
shown in Figure 3-Trace B exhibits peak splitting, which is
indicative of the presence of a heterozygous point mutation.
The resolution of the linear TOF MS (m/m ) 1000) is
sufficient to determine the mass shift of the variant to be +
30 Da. This approximate mass shift is accurate enough to
decrease number of possible variants from 80 to 7. The results
shown in Figure 3-Trace B would normally warrant further
protein characterization, i.e., enzymatic digestion; however,
previous MSIA plasma-based studies involving the same individual have already determined the point mutation to be a
Thr119Met substitution.11
Cystatin C (CYSC). Cystatin C (CYSC) is an extracellular
cysteine protease inhibitor found in most biological fluids. Even
though CYSC is freely filtered by the glomerulus, urinary levels
of CYSC are a poor marker for glomerular filtration due to
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Figure 4. Results of urinary CYSC MSIA analysis. Both the wildtype and hydroxylated (m ) +16 Da) forms of CYSC are present
in all five traces. Varied amounts of hydroxylated CYSC are seen
in each individual as well as multiple truncated forms of the
protein. These truncations include the systematic N-terminal
cleavage of S-, SSP-, and SSPG-. Extensive cleavage of CYSC,
with the loss of SSPGKPPR-, SSPGKPPRL-, and SSPGKPPRLV-,
are only seen in Traces D and E.

tubular reabsorption,35 but have been used as a reliable

measure of proximal tubular reabsorption, which has been
linked to renal failure.36 Moreover, hereditary cerebral hemorrhage with amyloidosis (HCHWA), an autosomal dominant
disorder prevalent in Icelandic, Dutch, and Finish populations,
is the result of a CYSC Leu68fGln variant.37 This variant of
CYSC results in amyloid deposits of the walls of cerebral
arteries. A number of carcinoma cell lines have also been
reported to secrete CYSC, leading to investigations of its role
as a possible tumor marker. CYSC also has several PTM
associated with it, most notable is the hydroxylation of a Pro
residue at position 336 which results in a mass shift of the wtCYSC protein by +16 Da.
The results of the anti-CYSC MSIA analyses are shown in
Figure 4 in which very similar protein profiles are observed
between all subjects. The mass spectrometric analysis was able
to sufficiently resolve the wt- (13 344 Da) and the hydroxylated
form (13 360 Da) of CYSC, m/m ) 1000. Varied amounts of
hydroxylation are seen between each individual. Multiple
N-terminally truncated forms of CYSC are also present, most
notable are the S- (13 256 Da) and SSP- (13 072 Da). Hydroxylation still occurs in the S- variant (13 272 Da), but is
lost with the cleavage of the P- at position 3. Further truncated
forms of CYSC are observed in Traces D and E in which
SSPGKPPR- (12 536 Da), SSPGKPPRL- (12 423 Da), and SSPGKPPRLV- (12 324 Da) are also present. The degradation of CYSC
has been reported from a significant portion of native urine
samples to date,36 but this CYSC profiling clearly shows that
this catabolic process is conserved a N-terminal proteolytic
process.
Urine Protein 1. UP1, also known as Clara cell protein, CC10,
or uteroglobin, is a biomarker for a variety of pulmonary
ailments and urinary tract dysfunctions. UP1 is a small protein
MW ) 7909 that is primarily secreted by Clara cells in the
bronchi alveolar lining in mammalian lung tissue is an anti194

Journal of Proteome Research Vol. 2, No. 2, 2003

Kiernan et al.

Figure 5. Mass spectrometric results of urinary UP1 MSIA.

Dimerized UP1 (MW ) 15 819) is seen in all five traces, but Traces
A and B both contain large proportions of UP1 monomer
conjugated to glutathion (m ) +305 Da).

inflammatory agent,38 but to date its physiological role is still

largely unclear. The native state of UP1 is a covalently associated homodimer, which results from the disulfide cross bridging between two UP1 monomers.39 When damage occurs to
the respiratory tract, plasma and urine UP1 levels increase due
to increased bronchioalveolar permeability and the overloading
of the tubular reabsorption process, respectively.40,41 Moreover,
increased UP1 concentration in urine alone, basal levels 5-10
g/L,41 is often an indication of proximal tubular dysfunction,42
whereas decreased UP1 plasma levels have been found in
smokers,43 asthmatics,44 and schizophrenics.45
Figure 5 shows the results of the qualitative urinary UP1
MSIA analysis. Dimerized UP1 with MW ) 15 819 is present in
all five traces. Although multiple charging of UP1 during the
MALDI process is unable to directly differentiate between
potential UP1 monomers and the +2 state of the UP1 dimer,
conjugated monomers are readily identifiable. Because wt-UP1
monomer would have exposed free cysteine groups some sort
of chemical modification through these reactive sulfhydryls
would be expected, as seen in TTR. Closer examination in the
Figure 5 inlets shows that both Traces A and B contain UP1
monomer with varied amounts of glutathion conjugate (m )
+305 Da) associated. Although being virtually undetectable in
Traces C-E, this is the first reported incidence of UP1
monomer being detected. These results suggest that the
individuals results shown in Traces A and B have more
glutathion and/or UP1 monomer present in their systems.
Whether this observation is associated with health state or
significant to disease has yet to be determined.
Retinol Binding Protein (RBP). RBP was the fifth urine
protein target. A member of the lipocalin family, RBP has a
plasma concentration level of 50 mg/L and serves the role of
the major carrier of retinol (vitamin A) from the liver to
peripheral tissues.46 With a molecular weight of 21 065, RBP is
believed to escape glomerular filtration by associating with the
homotetramer of transthyretin in its holo- (retinol bound)
form.47 RBP has been previously reported to exist in two posttranslationally truncated versions; one missing the C-terminal
Leucine (RBP-(Leu)) and the second missing two C-terminal
leucines (RBP-(Leu-Leu)), which are believed to be nonfunc-

Comparative Urine Protein Phenotyping

Figure 6. MSIA results of RBP analysis. Conserved C-terminal

cleavage pattern is seen in Traces A-D, with the loss of -L, -LL,
-RNLL, and -RSERNLL. Trace E displays an abnormal RBP
profile due to the noticeable decrease in the amount of wt-RBP
present compared to the truncated forms.

tional variants of RBP due to their lower binding affinities to

the transthyretin complex.48
The results of the urinary RBP analysis are shown in Figure
6. Conserved protein profiles are seen in Traces A-D, with wtRBP along with -L, -LL, -RNLL, and -RSERNLL C-terminally
truncated variants. The source and function of these variants
are still unknown, but have been determined to be the result
of some unreported enzymatic process that occurs after the
RBP is filtered from the blood.14 Interestingly, the individual
with pancreatic cancer in Figure 6-Trace E displays an altered
RBP profile. Most notable is the marked relative decrease in
the amount of wt-RBP present. Similar results were reported
with the analysis of urinary RBP of a 94-year old woman with
renal failure stemming from chronic diabetes mellitus, in which
wt-RBP was completely absent from the RBP protein expression
profile.14
Albumin (ALB). ALB, the sixth urine protein target, is the
best studied of all plasma proteins.49 At 66.3 kDa, ALB is
considerably larger than any of the previously discussed protein
targets. As a multipurpose house-keeping protein, ALB serves
a multitude of functions including; the binding and transport
of many metallic, organic, and biochemical compounds, antioxidant effects as well as plasma buffering.50,51
Figure 7 shows the results of the urinary ALB MSIA with the
+1 and the +2 states of ALB are present in all traces. Interindividual results are all very similar, as shown in Figure 7 inlet,
except for Trace E in which extensive peak broadening is
observed. Since albumin participates in the transport of so
many biological, inorganic, and pharmacological compounds,
adduct formation with one or many of these compounds is
possible. Albumin is also known to undergo glycation, like 2m,
hence the observed peak broadening may be the result of the
formation of an ALB-AGEs.
Transferrin (TRFE). TRFE, the seventh protein target, is a
large globular glycoprotein (MW ) 79.6 kDa) used in the
transport of dietary iron in human plasma.52 TRFE readily
crosses the glomerular membrane, despite its large size, due
to its strong cationic nature53 resulting in urine levels <0.19
mg/L.54 With two N-linked glycosylation sites, the heterogeneity
within the 4.4 kDa of associated glycan can greatly vary.
Figure 8 displays the results of the anti-TRFE MSIA analysis.

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Figure 7. Comparison of ALB MSIA analysis. ALB +1 and +2

states were detected in all five samples. Even though all traces
were acquired using the same instrument settings, Trace E
exhibits significant peak broadening of the ALB signal, shown
in trace insets, as compared to the other ALB signals in the other
traces.

Figure 8. Results of TRFE MSIA analysis. TRFE (MW ) 79.6 kDa)

was detected in all five samples. Only very minor differences
were observed.

Strong homology is seen in the results in all five Traces. This

homology in TRFE shows that chronic alcoholizm and carbohydrate deficient glycoprotein syndromes (CDGSI or II) were
not present in any of the participants of this study. Only Traces
B and E have detectable amounts of TRFE in the +2 state,
whereas Trace A does exhibit some tailing in the TRFE signal.
Whether these minute differences, seen in the TRFE profiles,
are related to any disease state or from genetic modification,
has yet to be determined.
Human Neutrophil Defensin Peptides (HNP). HNP, also
known as the R-defensins, was the final urine target in this
study. HNP are a family of cysteine-rich, cationic peptides, 2942 amino acids in length, secreted from neutrophils. The three
most common human neutrophil defensin peptides are HNP-1
(MW ) 3443), HNP-2 (MW ) 3372), and HNP-3 (MW ) 3487),
which are found in plasma, urine, saliva and sputum.55,56 These
peptides have demonstrated remarkable antibacterial, antifungal and antiviral activities, thus suggesting that HNP plays as
strong role innate immunity.55
Journal of Proteome Research Vol. 2, No. 2, 2003 195

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Kiernan et al.

Table 1. Summary of Protein Profile of All Eight Assays Run on All Five-Study Subjectsa
TTR

2m

CYTC

sample A

sample B

point
mutation

sample C

sample D

sample E

glycation

extended
truncations
extended
truncations

UP1

RBP

glutathion
conjugated
monomer
glutathion
conjugated
monomer
o

ALB

TRFE

HNP

tailing

altered
ratios
o

decreased
wt-RBP

peak
broadening

altered
ratios

mia, but neither of these conditions had been previously

diagnosed in this individual. Moreover, broadening of this
individuals ALB signal was also observed and may be associated to albumins function in bio- and organic-molecule
transport, whereas other differences included the increase
truncation of CYSC and altered ratios of HNP-1 and -2 were
seen, but not isolated to this individual. The analysis of TTR
showed that one individual had a heterozygous point mutation,
demonstrating the utility of MSIA in the identification of genetic
variation at the protein level. The application of MSIA was able
to also identify monomeric forms of UP1, modified with
glutathion, in two individuals. In total, approximately onequarter of the analyses provided variant results from the wildtype.

Conclusion
Figure 9. Mass spectrometric results of HNP analysis. Signals
from HNP-1 (MW ) 3443), -2 (MW ) 3372), and -3 (MW ) 3487)
were detected in all samples. Differences in relative amounts of
HNP-1 to HNP-2 are observed between each sample.

Previous studies into the roles of HNP in various systemic

responses have shown that they partake in large number of
responses including pro-inflammation with histamine release57
as well as cell proliferation and mitogenic effects.58 Because of
this last effect, more recent studies have suggested a correlation
between HNP and certain cancers.59 Other studies have shown
that HNP may also serve as a marker for certain inflammatory
disease states and sepsis.60,61
Because these peptides are highly cationic, immuno-affinity
retrieval is not required. Cation exchange MSIA ionically retains
sufficient amounts of HNP to allow for mass spectrometric
detection. The results of this analysis are shown in Figure 9, in
which all three major HNP species were detected in all five
samples, as shown by the triple peaks. Intra-sample concentration of each HNP species varies from each sample. Traces C
and E show that these individuals had more HNP-2 relative to
the other two HNP forms than the other individuals involved
in this study. Whether the HNP species ratios are linked to
disease states or are subject to diurnal variation has yet to be
determined.
The results of all 40 analyses were summarized for comparison in Table 1, which describes structural variations observed
in the target proteins. Interestingly, the individual diagnosed
with pancreatic cancer showed the most protein variation
compared to the other samples. The presence of glycated 2m
and the decrease in wt-RBP were most prominent of all the
differences. Similar types of variations have been previously
described from individuals with diabetes mellitus and/or ure196

Journal of Proteome Research Vol. 2, No. 2, 2003

This study demonstrates that mass spectrometric immunoassay is a powerful analytical technique in the study of intact
urinary proteins. MSIA allows for the rapid retrieval of specific
protein targets, which permits concise identification of each
target species to be achieved. Unlike indirect detection, as used
in ELISAs, mass spectrometry is able to discriminate between
variant forms of a protein target that are present, making
identification of all species possible. Moreover, MSIA is an
analytical technique that allows for comparative protein profiling to look for differences between individuals in specific
protein targets. Many of these differences are subtle and could
not be readily distinguished without mass spectrometric detection. As shown here, a small study with 40 data points produced
almost a dozen observable differences between 5 individuals.
The analysis of 8 protein targets detected 29 different observed
forms of these proteins, making mass spectrometry integral for
protein phenotyping. The observation of such variation within
such a small study population necessitates the need for larger
urine protein population studies in order to correlate such
findings to possible disease states.

Acknowledgment. This publication was supported in

part by Grant No. R44 GM56603-01 and Contract No. N43-DK1-2470 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.
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