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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..
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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.
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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.
Kiernan et al.
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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
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.
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.
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PR025574C