Abstract
Mass spectrometric immunoassay (MSIA) is a proteomics technology that combines the selectivity of anity capture with the
sensitivity and resolution of mass spectrometric detection. This unique approach allows for intact protein identication therefore is
readily capable of discriminating between protein variants, i.e., mutations, posttranslational modications, and truncations. In this
work, MSIA is used in the comparative analyses of retinol binding protein (RBP) from the plasma and urine of a small study
population. Detailed RBP proles were obtained from both biological uids, resulting in the identication of several catabolic RBP
products (present in urine) that have not been previously reported. In addition, comparative analysis of urine samples taken from
healthy and renally impaired individuals revealed dierent breakdown proles. These results illustrate the use of MSIA for the rapid,
sensitive, and accurate proling of RBP both within and between individuals. 2002 Elsevier Science (USA). All rights reserved.
Keywords: MALDI-TOF MS; Humans; Posttranslational modications; Retinol binding protein; Urine protein
0006-291X/02/$ - see front matter 2002 Elsevier Science (USA). All rights reserved.
PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 2 1 2 - X
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als. During these studies, point mutations and alterations in glycosylation were observed between individuals
and subsequently characterized using mass spectrometry
[9,10]. Moreover, MSIA has been applied to the quantitative measurement of urine- and plasma-borne b-2microglobulin (b2m) [11]. Again, variants of the target
protein, in particular glycated-b2m, were observed
during the routine course of the analyses. Thus, it is
apparent the MSIA can be employed in the analysis/
discovery of protein variants present in the urine or
plasma of individuals.
Here, we present the development of urinary and
plasma retinol binding protein (RBP) mass spectrometric immunoassays for the rapid qualitative proling
of RBP isolated from multiple individuals. In previous
work, we have used a complementary technique, biomolecular interaction mass spectrometry (BIA/MS), in
the study of urine-borne RBP. These studies, although
promising, were unable to fully resolve urine-borne RBP
variants with the condence needed for unambiguous
identication [12]. In these studies presented here, which
achieve a higher level of performance, MSIA was used
to qualitatively compare RBP isolated from both the
plasma and urine of individuals within a small study
population. Samples from four healthy controls and one
individual suering from chronic pyelonephritis (stemming from diabetes mellitus) and renal failure were
donated for use in this study. The rst objective of the
study was to verify that no genetic or transcriptional
variants, which could potentially inuence urinary RBP
proles, existed between individuals. This object was
accomplished by analyzing RBP from the plasma of the
individuals. The subsequent objective of the study was
to use MSIA to prole urinary RBP for possible differences related to the (renal) health state of the individual.
U.A. Kiernan et al. / Biochemical and Biophysical Research Communications 297 (2002) 401405
(diluted plasma) was decanted from each sample and interrogated
using MSIA.
The ve plasma samples were addressed in parallel using an eightbarrel repeating pipettor outtted with anti-RBP MSIA-Tips (Intrinsic
Bioprobes). Sample incubation consisted of repeatedly cycling (aspiration and dispense; 50 cycles; 3 s/cycle; 150 lL/cycle) the samples
through individual anity tips. After incubation, tips were rinsed using
HBS (10 cycles, 150 lL), doubly distilled water (5 cycles, 150 lL), and
20% acetonitrile/1 M ammonium acetate wash (10 cycles, 150 lL), and
nally with doubly distilled water (15 cycles, 150 lL). Retained species
were eluted by drawing 4 lL MALDI matrix solution (saturated
aqueous solution of sinapic acid (SA) in 33% (v/v) acetonitrile and
0.4% (v/v) triuoroacetic acid) into each tip and depositing directly
onto a 96-well formatted hydrophobic/hydrophilic contrasting MALDI-TOF target [8]. Samples were allowed to air dry, prior to insertion
into the mass spectrometer. The total time required for the preparation
of the ve plasma samples was approximately 10 min.
Urine samples, 25 mL mid-stream voids, from the same ve individuals were also collected. The urine samples were immediately
combined 1:1 (v/v) with 2 M ammonium acetate (to adjust the pH to
6.87.2) and 50 lL protease inhibitor cocktail described above. Due
to their larger volume, the urine samples were interrogated individually
with anti-RBP MSIA-Tips. Incubation of each urine sample consisted
of 300 cycles (150 lL of sample) through a MSIA-Tip. After incubation, tips were treated using the same rinse/elution protocols described
for the plasma analyses. Because of the larger sample volumes of urine,
time spent in sample preparation was 20 min/sample. MALDI-TOF
MS and data analysis were performed on all samples as described
previously [9].
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U.A. Kiernan et al. / Biochemical and Biophysical Research Communications 297 (2002) 401405
Acknowledgments
Fig. 3. Results of the anti-RBP MSIA analysis of urine. Traces AD
are from healthy study subjects and display a generally similar phenotypic prole. Wild-type RBP, RBP (-L), RBP (-LL), and RBP
(-RNLL) (MW 20,534) are present in all four mass spectra. Observable signal from RBP (-RSERNLL) (MW 20,162) is also present
in three of the four spectra. However, trace E is from the individual
with kidney impairment and mass spectrum is dominated by RBP-L
signal and devoid of wt-RBP.
Conclusion
This preliminary study demonstrates that mass
spectrometric immunoassay is a relatively easy and rapid method for isolating and detecting human retinol
binding protein from both plasma and urine. The use of
mass spectrometry for detection allows for the identication of variant forms of RBP that would possibly have
been overlooked by other methods. Even though only a
small study population was used in this study, dierences in RBP phenotypic proles from both plasma and
urine were readily observed. The sensitivity and resolution of MALDI-TOF MS detection were able to identify
RBP-RNLL and RBP-RSERNLL, two C-terminal
truncated urinary variants that have not been reported
previously. Overt dierences in the RBP phenotypic
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