Quantitative Multiplexed C-Reactive Protein Mass Spectrometric

Immunoassay
Urban A. Kiernan,* Riccardo Addobbati, Dobrin Nedelkov, and Randall W. Nelson
Intrinsic Bioprobes, Inc., 625 S. Smith Rd. Ste. 22, Tempe, Arizona 85281
Received March 23, 2006

Reported in this work is the development and application of a high sensitivity mass spectrometric
immunoassay for the quantitative analysis of C-reactive protein from human plasma. Multiplexed affinity
retrieval devices and methodology were developed to simultaneously target retinol binding protein,
C-reactive protein, serum amyloid P component, as well as an added exogenous internal reference
standard (staphylococcal enterotoxin B) for subsequent MALDI-TOF MS analysis. This approach allows
for semiquantitative analysis of both retinol binding protein and serum amyloid P component while
performing absolute quantitative measurements of C-reactive protein. The ability to qualitatively
differentiate between all three human proteins and their associated variants is also maintained. Standard
curve, QC, and human plasma samples were analyzed in a high throughput manner, which performed
with a CV < 15%. The resultant human plasma sample C-reactive protein quantitative measurements
were then compared to those achieved with a high sensitivity latex immunoturbidimetric assay.
Keywords: mass spectrometry Immunoassay C-reactive protein quantitation multiplexing human plasma

Introduction
The field of proteomics has extended its scope beyond basic
research and is now attempting to enter clinical application
and diagnostics. Historically, clinical biomarker screening has
been reserved for classical immunoassay methodologies, but
recent findings have shown that pertinent clinical data may
lie beyond the fidelity of such approaches 1. The use of protein
mass spectrometry in proteomics holds the key to this issue,
with the intrinsic ability to qualitatively discriminate between
multiple forms of the same target protein. However, a major
hurdle for proteomics, in entering the clinical and diagnostic
arena, is the inability to readily perform absolute protein
quantification. There are many quantitative proteomic approaches currently available, however, methodologies such as
Isotope-Coded Affinity Tags (ICAT)2 are only semiquantitative
in nature and have restricted data content due to being peptide
based. Such peptide based approaches may not allow for
accurate qualitative differentiation between the clinically relevant forms of the same protein.3-5
A potential solution to this problem is the mass spectrometric immunoassay (MSIA), a high content proteomics methodology based on immuno-affinity protein isolation combined with
mass spectrometric detection. This approach has a longstanding track record of performing both relative6-8 and
absolute quantitative analyses,9-12 which is augmented by its
unprecedented ability to qualitatively differentiate between
multiple forms of the same protein target1,13-19 in a single
* To whom correspondence should be addressed. Tel: (480) 804-1778.
Fax: (480) 804-0778. Email: ukiernan@intrinsicbio.com.

Currently at IRCCS Burlo Garofolo, Via dellIstria 65/1, 34127 Trieste,

Italy.

1682

Journal of Proteome Research 2006, 5, 1682-1687

Published on Web 05/27/2006

analysis. This ability to discriminate between multiple proteins

makes MSIA ideal for the development of multiplexed assays,
for combined qualitative and quantitative assessment.
Presented in this work is the development and application
of a novel quantitative multiplexed C-reactive protein (CRP)
MSIA. CRP, a renowned clinical biomarker of inflammation,20
was selected as an ideal protein target because it exhibits a
large dynamic concentration range in human plasma,21 has
little known phenotypic variation, and possesses strong clinical
value.22 This is the first demonstration of a quadraplexed MSIA
approach, which is based upon a previously developed semiquantitative methodology.6 However, this approach has been
enhanced for the absolute quantitative measurement of CRP
through the selective affinity retrieval of an exogenous internal
reference standard, while maintaining its semiquantitative
capacity for two other targeted human plasma proteins (retinol
binding protein and serum amyloid P component) and its
qualitative differential capacity for all protein targets. CRP
measurements made using this assay were then compared to
those generated via a classical immuno-metric method.

Experimental Procedure
Affinity Pipets. MSIA protein purification was achieved using
antibody derivatized affinity pipets (Intrinsic Bioprobes, Inc.,
Tempe, AZ). These pipets (commonly referred to as MSIA-Tips)
were produced using the same protocol as described previously.23 The affinity ligand used in this study was a mixture of
polyclonal antibodies targeting retinol binding protein (RBP:
8.0 mg/mL, Cat No. A0040, DakoCytomation, Carpinteria, CA),
C-reactive protein (CRP: 8.3 mg/mL, Cat No. A0073, DakoCytomation, Carpinteria, CA), serum amyloid P component
10.1021/pr0601133 CCC: $33.50

2006 American Chemical Society

research articles

Quantitative C-Reactive Protein Mass Spectrometric Assay

(SAP: 8.1 mg/mL, Cat No. A0302, DakoCytomation, Carpinteria,

CA) and staphylococcal enterotoxin B (SEB: 1 mg/mL, Cat No.
LBI 202, Toxin Technology, Sarasota, FL). The optimum
antibody ratio was empirically determined to be 3:3:1:5 (v/v),
respectively.
Patient Sample Collection. The samples used in this study
were provided by the National Institute of Diabetes & Digestive
& Kidney Diseases (NIDDK) repository as a part of their preand type 2 diabetes biomarker discovery program. Citrate
plasmas from 35 individuals, which included healthy, prediabetic, and untreated type 2 diabetic patients, were obtained.
These classifications were retrospectively determined after the
administration of an oral glucose tolerance test. Moreover,
samples were provided with pre-screen C-reactive protein
(CRP) values that were determined using the Roche Tina-quant
high sensitivity latex immunoturbidimetric CRP assay (measurements ranging from 0.1 to 20.0 mg/L) performed on a
Hitachi spectrophotometer.
Standard Curve and QC Sample Preparation. This study
utilized an eight point standard curve (run in duplicate) and
low, med and high QC samples (each run in quadruplicate).
The standard curves and QC samples were prepared by doping
highly purified protein targets into horse serum (Cat No. P5552,
Sigma Chemical, St. Louis, MO). Horse serum was selected as
the sample matrix of choice due to a lack of cross reactivity
between the human protein antibodies used and their equine
antigen counterparts, while still being a highly complex biological fluid that is similar in many respects to human. These
samples were initially produced in bulk, by spiking neat horse
serum (1.38 mL) with purified protein antigens. Spiking included 22.2 L of 2.9 mg/mL human RBP (Cat No. 30-AR20,
Fitzgerald, Concord, MA), 75 L of 1.0 mg/mL human SAP (Cat
No. S-5269, Sigma-Aldrich Co., St. Louis, MO) and finally 22.5
L of 1.0 mg/mL solution of SEB (Cat No. BT202, Toxin
Technology, Sarasota, FL) used as the internal reference
standard (IRS). (SEB is a category B toxin, as defined by the
U.S. Department of Defense, which requires specific precautions in handling and storage due to its known toxicity to
humans and its potential as a biological warfare agent.) The
human RBP antigen used in this study was affinity purified from
human urine and was truncated (loss of 4 C-terminal amino
acids) as compared to the endogenous wild-type plasma
protein. Even though the RBP was modified, it was determined
to be suitable as a QC standard since its affinity retrieval and
MS characteristics did not significantly differ from the endogenous plasma forms. The concentrations of human protein
antigens used were selected in order to mimic the physiological
concentrations of these targets within human plasma. At this
point, the prepared standard samples were split and subsequently spiked with their corresponding amount of 1.0 mg/
mL human CRP antigen (Cat No. CP1000U, Cortex Biochem,
San Leandro, CA). The final CRP concentrations of the standard
curve samples ranged from 0.025 to 3.0 g/mL and the QC
concentrations used were 0.075, 0.75 and 2.5 g/mL, corresponding to low, med and high QC, respectively. Fifty microliter
aliquots of each were transferred into individual wells of a 96deep well micro-titer plate (Greiner Bio-One, Longwood, FL)
and diluted to 1 mL with 0.1 M HEPES buffered saline (pH 7.4)
with 0.15 M NaCl, 3 mM EDTA and 0.005% polyoxyethylenesorbitan monolaurate (Tween 20).
Patient Sample Preparation. Patient samples were thawed
at room temperature and 50 L aliquots were pipetted into
individual wells of a 96-deep well micro-titer plate. Each aliquot

was spiked with 7.5 L of 0.1 mg/mL SEB internal reference

standard and was then diluted to 1 mL with HBS-EP.
Sample Analysis. Samples were screened in parallel using a
multichannel robotic pipetting workstation (Beckman Coulter,
Fullerton, CA) outfitted with the prepared multiplexed affinity
pipets. In this study, a total of 63 parallel analyses were
performed in a high-throughput manner. The analytical procedure used involved the repetitive flowing of target samples
through the affinity pipet tips to simultaneously retrieve and
enrich target proteins from the biological fluid. Samples (100
L) were flown 150 times (repetitive aspirations and dispenses)
through the tips, followed by a HBS-EP rinse (100 L, 10 times)
and four serial double distilled water rinses (100 L, 10 times).
At this point, the enriched and purified proteins retained within
the affinity pipet tips were eluted directly onto a contrasted
96-spot MALDI-target.11 The elution process involved aspirating
6.0 L of MALDI matrix solution (saturated sinapinic acid in
acetonitrile/water (1:2 v/v) with 0.4% trifluoroacetic acid) into
the tips followed by eluant deposition. The samples were
allowed to air-dry prior to mass spectrometric analysis.
Mass Spectrometric Analyses. Parent protein mass spectrometry was performed on a linear Autoflex MALDI-TOF mass
spectrometer (Bruker, Billerica, MA). A linear delayed extraction
mode was employed using a 1.45 kV draw out pulse, 670 ns
delay and a full accelerating potential of 20.00 kV. Human CRP
was used as a calibration standard. Each mass spectrum was
of the sum of five 100-laser shot acquisitions. All spectra were
then viewed using Proteome Analyzer software (Intrinsic Bioprobes Inc., Tempe, AZ) in which all spectra were aligned and
normalized to the integral of the SEB parent signal for intersample comparison.

Results and Discussion

Assay Development. This quantitative multiplexed mass
spectrometric immunoassay is based upon a previously developed semiquantitative approach that targets the same three
human proteins.6 These three protein targets were previously
selected for multiplexing based on their size (similar in molecular mass yet different enough to be sufficiently resolved),
amicability to MALDI-TOF MS analysis, tolerance to the same
MALDI-matrix, and the quality of commercially available
antibodies. However, this work is an improvement of the
previous approach because of its new assay design, which has
the additional ability to target for an exogenous protein for use
as an internal reference standard, thus allowing for absolute
protein quantification through the generation of a standard
curve. SEB was selected as a suitable IRS based on the same
criteria listed above as well as it not being an endogenous
human blood protein. Other exogenous proteins were tested
during the assay development phase (data not shown), however, SEB was found to be the best choice for use with these
human protein targets. The appropriate quantity of antigen
used as the IRS and the ratio of SEB antibody were determined
empirically, with a final working amount of 7.5 L of 0.1 mg/
mL SEB per 50 L of human plasma and an antibody ratio of
3 RBP:3 CRP:1 SAP:5 SEB (v/v).
Application. The resultant assays were then applied to the
standard and human samples. Figure 1A is a representative
mass spectrum of the application of the multiplexed MSIA
devices in the analysis of human plasma (sample no. 35). MS
signals from all four-protein targets, and their associated
variants, are clearly observed. A complete list of all identified
signals, along with their theoretical and observed m/z values,
Journal of Proteome Research Vol. 5, No. 7, 2006 1683

research articles

Kiernan et al.

Figure 1. Application of the multiplexed MSIA devices and

methodology. (A) Representative mass spectrum of the MSIA
analysis of human plasma (sample no. 35). M/z signals from RBP,
CRP, SAP, and SEB are present. (B) Mass spectra from the MSIA
analysis of some standard samples used to generate standard
curve 1. Samples displayed were with CRP concentrations of 0.05,
0.25, 1.00, and 3.00 g/mL.
Table 1. Identified Parent Protein Signals from Human Plasma
protein
identified

m/z theoretical
(MH+)

m/z observed
(MH+)

RBP
RBP -Leu
CRP
SAP
SAP-Val
SAP-Sial
SEB

21 066.5
20 953.4
23 029.1
25 463.5
25 364.4
25 172.2
28 367.0

21 065.8 ( 3.7
20 952.9 ( 4.3
23 029.2 ( 3.5
25 466.0 ( 6.6
25 360.9 ( 3.0
25 174.5 ( 3.9
28 368.7 ( 4.4

is presented in Table 1. The analysis of the standard curve and

QC samples result in very similar protein profiles as those
generated from human plasma. The only difference observed
was because the purified RBP antigen used in the standard
samples was a truncated form compared to the human wildtype found in plasma, with a molecular weight of 20 568.9 Da.
Examples of such profiles, normalized to the integral of the
internal reference standard, are shown in Figure 1B, which are
a portion of the data set used to generate one of the two
standard curves. These data clearly illustrate the consistency
of the analysis while demonstrating the ability of this MS-based
immunoassay to monitor change as the concentration of the
CRP antigen is increased.
The normalized CRP peak integrals from both the standard
curve and QC samples were plotted, shown in Figure 2. The
plot in Figure 2A shows how the two standard curves are
identical and correlate well with a power series, with R2 values
of 0.9893 and 0.9935, respectively. Also included in this plot
are the normalized results for the QC samples. For improved
clarity at the lower concentration range of the standard curve
and QC values, the plots were converted to a Log/Log scale,
shown in Figure 2B. The data generated from the two standard
curves and the QC samples were also used to determine
1684

Journal of Proteome Research Vol. 5, No. 7, 2006

Figure 2. Plots of the CRP standard curves and QC samples. (A)

Overlay plot of curves 1 and 2, with points that range from 0.025
to 3.000 g/mL, along with the low (0.075 g/mL), med (0.750
g/mL), and high (2.500 g/mL) QC samples. Plots correlate with
a power series, with R2 values of 0.9893 and 0.9935, respectively.
(B) Overlay log/log plot of curves 1 and 2 along with the QC
samples.
Table 2. Coefficients of Variation Determined from the
Standard Curve and QC Samples
source data

RBP

curve 1
curve 2
low QC (0.075 g/mL)
med QC (0.750 g/mL)
high QC (2.500 g/mL)

13.634
6.242
4.192
13.752
9.676

CRP

SAP

6.373
7.115
10.134

6.252
11.640
2.354
11.590
11.932

reproducibility, with the coefficient of variation (CV) of the

assay. These values are listed in Table 2, demonstrated a mean
CV and standard error of the estimate (SEE) of 8.837 and 13.4,
respectively.
Semiquantitative Determination. The semiquantitative measurements of both RBP and SAP were extracted from the
normalized data sets. Plots showing the normalized integral
of each protein and their associated variants are presented in
Figure 3A and B, respectively. Even though the measurements
are only semiquantitative in nature, the intact protein MS
analysis allows for differentiation between the multiple variants
of each protein that were affinity retrieved. This is a novel
feature of this approach since conventional immunoassays are
normally blind to such subtle variation. These plots clearly
show the varying normalized abundances of three forms of RBP
and three forms of SAP consistently detected in each patient

Quantitative C-Reactive Protein Mass Spectrometric Assay

research articles

Figure 3. Semiquantitative protein determination. (A) Normalized abundances of retinol binding protein and detected variants in each
of the 35 patient plasma samples. (B) Normalized abundances of serum amyloid P component and detected variants in each of the
same patient plasma samples.

sample. Such protein variation may hold significant information regarding the presence of disease.
Absolute CRP Quantitation. Regarding the quantitative CRP
analysis, the two generated standard curves of the normalized
CRP peak integrals verses CRP antigen concentration were
averaged, which resulted in the following equation (CRP/SEB )
1.3092[CRP mg/mL]0.6742). This average equation was then used
to translate the observed normalized CRP/SEB peak integral ratios
determined from each of the patient samples into CRP concentrations (mg/mL). Only two plasma samples (nos. 17 and
26) of the 35 analyzed using this approach resulted in concen-

tration values outside the range of the standard curve. During

the development phase of this assay, we identified that points
outside of the established curve range no longer displayed
characteristics consistent with the power series (data not
shown). Therefore, repeat analyses using sample dilutions
would be necessary for accurate measurements of samples with
high concentration of CRP. These samples were reanalyzed
using a decreased amount of sample plasma (25 L and 16.5
L, respectively), so that the results fell within the range of the
standard curve, but was then factored for when the final
concentration was calculated.
Journal of Proteome Research Vol. 5, No. 7, 2006 1685

research articles

Kiernan et al.

multiplexed proteomic analysis. Moreover, the QC and standard curve analyses demonstrated coefficients of variation that
were significantly better than other potential clinical proteomic
approaches28 again adding to the potential of the mass spectrometric immunoassay for routine and repetitive target protein(s) analysis, as a needed in a clinical and diagnostic application.
Finally, this demonstration of an improved multiplexed MSIA
approach sets the foundation for future developmental work,
which would include increased multiplexing for more protein
targets, as well as the simultaneous generation of multiple
standard curves for absolute multi-protein quantitative measurements in a single analysis.
Figure 4. Human plasma CRP concentrations. Comparison of the
CRP concentrations determined in each of the patient samples
(g/mL) via mass spectrometric immunoassay and immunoturbidimetric assay.

The results of the MSIA CRP-quantitative measurements

performed on the patient samples were then plotted with the
CRP concentrations determined in the same samples via an
immunoturbidimetric assay. This plot is shown in Figure 4,
which clearly illustrates that the same trend in CRP concentrations is observed regardless of the immunoassay methodology
applied. Discrepancies between the two assay methods used
are observed in the two data sets, mostly with the MSIA values
being lower than those determined with the immunoturbidimetric approach. The differences observed range from 88 to
627% lower in calculated concentration. However, such differences may be explained by a number of factors, which include
the following: (1) the MSIA hs-CRP assay and standard curve
were engineered to detect and quantify CRP levels in a
concentration range that differed from the turbidity assay
(MSIA ranged from 0.025 to 3.000 g/mL and the turbidity assay
ranged from 0.1 to 300.0 g/mL, (2) the samples were older
when the MSIA analysis was performed than when the turbidity
assay was performed, and finally (3) the antibodies and
standards used to perform the two immunoassays and generate
the standard curve samples were different. Such variations in
assay development and methodology have shown to differ
results between other assays of the same protein target by as
much as 10 000%.24-27

Conclusion
It has become readily apparent that proteomics has the
potential to become integral in clinical application. However,
the question still remains as to which form of the multiple
proteomics approaches available will translate into the mainstream of clinical and diagnostic use. Presented here was the
development and application of a novel multiplexed high
sensitivity-CRP mass spectrometric immunoassay. This approach allowed for simultaneous semiquantitative analysis of
human retinol binding protein and serum amyloid P component while performing rigorous quantitative measurements of
C-reactive protein. Qualitative protein characterization, of all
targets, is maintained in this high throughput approach. This
methodology described is a novel multiplexed approach that
is an improvement of a previously existing assay. The enhancement was achieved through the specific affinity targeting and
analysis of an exogenous protein (staphylococcal enterotoxin
B) that was added to all samples and co-analyzed for use as
an internal reference standard in peak integral normalization,
thus making the semi- and absolute quantitative measurements
possible. This is the first ever demonstration of such a
1686

Journal of Proteome Research Vol. 5, No. 7, 2006

Acknowledgment. We would like to thank Dr. Allan L.

Bieber for his assistance in preparing and the critical reading
of this manuscript. We would also like to thank Dr. Lawrence
S. Philips from Emory University for kindly providing the
samples and the CRP immunoturbidometric assay data used
in this study through the NIDDK repository as a part of Grant
No. R18-DK066204 and M01-RR000039. This work was funded
by the National Institute of Diabetes & Digestive & Kidney
Diseases under Grant No. 1 R42DK071290-01.
References
(1) Nedelkov, D.; Kiernan, U. A.; Niederkofler, E. E.; Tubbs, K. A.;
Nelson, R. W. PNAS 2005, 102, 10852-10857.
(2) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.;
Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999.
(3) Mahley, R. W.; Huang, Y. Curr. Opin. Lipidol. 1999, 10, 207-217.
(4) Yamauchi, K.; Tozuka, M.; Nakabayashi, T.; Sugano, M.; Hidaka,
H.; Kondo, Y.; Katsuyama, T. J. Neurosci. Res. 1999, 58, 301-307.
(5) Schreiber, G.; Richardson, S. J. Comp. Biochem. Physiol. B
Biochem. Mol. Biol. 1997, 116, 137-160.
(6) Kiernan, U. A.; Nedelkov, D.; Tubbs, K. A.; Niederkofler, E. E.;
Nelson, R. W. Clin. Proteomics J. 2004, 1, 7-16.
(7) Niederkofler, E. E.; Nedelkov, D.; Tubbs, K. A.; Kiernan, U. A.;
Nelson, R. W. Proceedings of the 51st ASMS Conference on Mass
Spectrometry and Allied Topics, 2003.
(8) Kiernan, U. A.; Nedelkov, D.; Niederkofler, E. E.; Tubbs, K. A.;
Bieber, A. L.; Nelson, R. W. Proceedings of the 53st ASMS
Conference on Mass Spectrometry and Allied Topics, 2005.
(9) Nelson, R. W.; Krone, J. R.; Bieber, A. L.; Williams, P. Anal. Chem.
1995, 67, 1153-1158.
(10) Tubbs, K. A.; Nedelkov, D.; Nelson, R. W. Anal. Biochem. 2001,
289, 26-35.
(11) Niederkofler, E. E.; Tubbs, K. A.; Gruber, K.; Nedelkov, D.; Kiernan,
U. A.; Williams, P.; Nelson, R. W. Anal. Chem. 2001, 73, 32943299.
(12) Nelson, R. W.; Nedelkov, D.; Tubbs, K. A.; Kiernan, U. A. J.
Proteome Res. 2004, 3, 851-855.
(13) Kiernan, U. A.; Nedelkov, D.; Tubbs, K. A.; Niederkofler, E. E.;
Nelson, R. W. Am. Biotech. Lab. 2002, 20, 26-28.
(14) Kiernan, U. A.; Nedelkov, D.; Tubbs, K. A.; Niederkofler, E. E.;
Nelson, R. W. Proteomics 2004, 4, 1825-1829.
(15) Kiernan, U. A.; Tubbs, K. A.; Gruber, K.; Nedelkov, D.; Niederkofler, E. E.; Williams, P.; Nelson, R. W. Anal. Biochem. 2002, 301,
49-56.
(16) Kiernan, U. A.; Tubbs, K. A.; Nedelkov, D.; Niederkofler, E. E.;
McConnell, E.; Nelson, R. W. J. Proteome Res. 2003, 2, 191-197.
(17) Kiernan, U. A.; Tubbs, K. A.; Nedelkov, D.; Niederkofler, E. E.;
Nelson, R. W. Biochem. Biophys. Res. Commun. 2002, 297, 401405.
(18) Kiernan, U. A.; Tubbs, K. A.; Nedelkov, D.; Niederkofler, E. E.;
Nelson, R. W. FEBS Lett. 2003, 537, 166-170.
(19) Tubbs, K. A.; Kiernan, U. A.; Niederkofler, E. E.; Nedelkov, D.;
Bieber, A. L.; Nelson, R. W. Proteomics 2005, 5, 5002-5007.
(20) Zimmerman, M. A.; Selzman, C. H.; Cothren, C.; Sorensen, A. C.;
Raeburn, C. D.; Harken, A. H. Arch. Surg. 2003, 138, 220-224.
(21) Kushner, I. Ann. N.Y. Acad. Sci. 1982, 389, 39-48.
(22) Kushner, I. Science 2002, 297, 520-521.
(23) Niederkofler, E. E.; Tubbs, K. A.; Kiernan, U. A.; Nedelkov, D.;
Nelson, R. W. J. Lipid Res. 2003, 44, 630-639.

research articles

Quantitative C-Reactive Protein Mass Spectrometric Assay

(24) Hafner, G.; Peetz, D.; Erbes, H.; Post, F.; Dahm, M.; Peivandi, A.
A.; Sucke, B.; Blankenberg, S.; Rupprecht, H. J.; Prellwitz, W.
Scand. J. Clin. Lab. Invest. 2001, 61, 227-235.
(25) Maggiore, U.; Cristol, J. P.; Canaud, B.; Dupuy, A. M.; Formica,
M.; Pozzato, M.; Panichi, V.; Consani, C.; Metelli, M. R.; Sereni,
L.; De Nitti, C.; David, S.; Tetta, C. J. Lab. Clin. Med. 2005, 145,
305-308.
(26) Christenson, R. H.; Apple, F. S.; Morgan, D. L.; Alonsozana, G.
L.; Mascotti, K.; Olson, M.; McCormack, R. T.; Wians, F. H., Jr.;
Keffer, J. H.; Duh, S. H. Clin. Chem. 1998, 44, 52-60.

(27) Apple, F. S.; Maturen, A. J.; Mullins, R. E.; Painter, P. C.; PessinMinsley, M. S.; Webster, R. A.; Spray Flores, J.; DeCresce, R.; Fink,
D. J.; Buckley, P. M.; Marsh, J.; Ricchiuti, V.; Christenson, R. H.
Clin. Chem. 1999, 45, 206-212.
(28) Aivado, M.; Spentzos, D.; Alterovitz, G.; Otu, H. H.; Grall, F.;
Giagounidis, A. A.; Wells, M.; Cho, J. Y.; Germing, U.; Czibere, A.;
Prall, W. C.; Porter, C.; Ramoni, M. F.; Libermann, T. A. Clin.
Chem. Lab. Med. 2005, 43, 133-140.

PR0601133

Journal of Proteome Research Vol. 5, No. 7, 2006 1687