kiernan et al _2002_comparative phenotypic analyses of human plasma and urinary retinol binding...

8/9/2019 Kiernan et al _2002_Comparative phenotypic analyses of human plasma and urinary retinol binding protein using mass spectrometric immunoassay..pdf

http://slidepdf.com/reader/full/kiernan-et-al-2002comparative-phenotypic-analyses-of-human-plasma-and-urinary 1/5



desorption/ionization time-of-ight mass spectrometry(MALDI-TOF MS) detection (see Fig. 1). This combi-nation allows for the rapid purication/concentrationand mass spectrometric analysis of specic proteinspresent in biological uids. Moreover, the approachyields a detailed structural analysis of full-length pro-

teins with sensitivities of the order of those found inimmunoassay approaches. This ability allows for thediscrimination of mass-shifted structural variants (re-trieved by a pan-antibody) that would otherwise beviewed as a single protein using conventional immuno-assay, or possibly missed using conventional proteomicsapproaches.

More recently, we have used MSIA to differentiatebetween variants of transthyretin and serum amyloid Pcomponent present in the plasma of different individu-

als. During these studies, point mutations and altera-tions in glycosylation were observed between individualsand subsequently characterized using mass spectrometry[9,10]. Moreover, MSIA has been applied to the quan-titative measurement of urine- and plasma-borne b-2-microglobulin ( b2m) [11]. Again, variants of the target

protein, in particular glycated- b2m, were observedduring the routine course of the analyses. Thus, it isapparent the MSIA can be employed in the analysis/discovery of protein variants present in the urine orplasma of individuals.

Here, we present the development of urinary andplasma retinol binding protein (RBP) mass spectro-metric immunoassays for the rapid qualitative prolingof RBP isolated from multiple individuals. In previouswork, we have used a complementary technique, bio-molecular interaction mass spectrometry (BIA/MS), inthe study of urine-borne RBP. These studies, although

promising, were unable to fully resolve urine-borne RBPvariants with the condence needed for unambiguousidentication [12]. In these studies presented here, whichachieve a higher level of performance, MSIA was usedto qualitatively compare RBP isolated from both theplasma and urine of individuals within a small studypopulation. Samples from four healthy controls and oneindividual suffering from chronic pyelonephritis (stem-ming from diabetes mellitus) and renal failure weredonated for use in this study. The rst objective of thestudy was to verify that no genetic or transcriptionalvariants, which could potentially inuence urinary RBPproles, existed between individuals. This object wasaccomplished by analyzing RBP from the plasma of theindividuals. The subsequent objective of the study wasto use MSIA to prole urinary RBP for possible dif-ferences related to the (renal) health state of the indi-vidual.

Materials and methods

Study subjects . Plasma and urine samples were donated by fourhealthy male subjects, ages ranging from 26–49, and from one elderlyfemale suffering from chronic pyelonephritis and diabetes mellitus,

aged 94. All subjects participated in the study under an informedconsent form approved under IRB00001399, which is in compliancewith FWA00000559. Appropriate safeguards were used in collectingsamples using universal precautions.

Sample preparation . Plasma samples were collected from ve indi-viduals and processed for immediate analysis as follows. Whole blood(50 l L per individual) was acquired under sterile conditions through alancet punctured nger using a 50 l L microcolumn (Drummond Sci-entic, Broomall, PA).

Each whole blood sample was immediately combined with 200 l LHepes buffered saline (10 mM Hepes, 150mM NaCl, 3 mM EDTA,and 0.005% polysorbate 20 (v/v), pH 7.4 (HBS)) containing 2 l Lprotease inhibitor cocktail [AEBSF (100 mM); aprotin (80 l M); best-atin (5mM); E-64 (1.5mM); leupeptin (2mM); and pepstatin A(1mM) — added to prevent any enzymatic breakdown] and centrifugedfor 2 min (at 7000 rpm) to pellet red blood cells. The supernatant

Fig. 1. Schematic of MSIA process. Biological uid (i.e., urine, plasma)is repetitively pipetted through the MSIA-Tip derivatized with an af-nity ligand for a specic protein target. The target protein is retrievedand concentrated within the MSIA-Tip, which is then puried with theapplication of multiple rinse steps to remove non-specically boundmaterial. Retained protein is eluted from the MSIA-Tip with MALDImatrix and applied directly onto a MALDI hydrophilic/hydrophobiccontrasted target. Air-dried samples are then interrogated with MA-DLI-TOF MS.

402 U.A. Kiernan et al. / Biochemical and Biophysical Research Communications 297 (2002) 401–405



Interestingly, repeating the analyses on urine sampleleft at native conditions (no addition of pH buffers orprotease inhibitors) and incubated at 37 C for 24h didnot show any increased amount of RBP truncation (datanot shown). These observations indicate that the pro-duction of these C-terminally truncated forms of RBP isnot the result of proteolysis occurring in the urine. Thus,the constant observation of C-terminally truncatedvariants in urine suggests a catabolism mechanism in-volving exopeptidase truncation, prior to clearance intothe bladder (independent of health state). However, theobservation of two different truncation patterns furthersuggests the involvement of multiple exopeptidase,which may be differentially regulated, dependent on thehealth state of the individual.

Conclusion

This preliminary study demonstrates that massspectrometric immunoassay is a relatively easy and ra-pid method for isolating and detecting human retinolbinding protein from both plasma and urine. The use of mass spectrometry for detection allows for the identi-cation of variant forms of RBP that would possibly havebeen overlooked by other methods. Even though only asmall study population was used in this study, differ-ences in RBP phenotypic proles from both plasma andurine were readily observed. The sensitivity and resolu-tion of MALDI-TOF MS detection were able to identifyRBP-RNLL and RBP-RSERNLL, two C-terminaltruncated urinary variants that have not been reportedpreviously. Overt differences in the RBP phenotypic

proles of individuals with healthy and diseased kidneyswere also observed. Therefore, a more concerted studymay be able to provide further insight into the catabo-lism of RBP, or at the very least verify proles capableof serving as diagnostic indicators of renal function.Thus, this preliminary demonstration of MSIA, as ap-

plied to RBP present in plasma and urine, serves as astepping-stone for the study of larger populations toclinically dene ‘‘normal’’ and ‘‘abnormal’’ phenotypicRBP proles and assist in determining the mechanisticcause of differences.

Acknowledgments

This publication was supported in part by Grant No. R44GM56603-01 and Contract No. N43-DK-1-2470 from the NationalInstitutes of Health. Its contents are solely the responsibility of theauthors and do not necessarily represent the official views of the Na-tional Institute of Health.

References

[1] D. Wild, The Immunoassay Handbook, second ed., Nature Pub.Group, New York, 2001.

[2] C.S. Spahr, M.T. Davis, M.D. McGinley, J.H. Robinson, E.J.Bures, J. Beierle, J. Mort, P.L. Courchesne, K. Chen, R.C. Wahl,W. Yu, R. Luethy, S.D. Patterson, Towards dening the urinaryproteome using liquid chromatography–tandem mass spectrome-try. I. Proling an unfractionated tryptic digest, Proteomics 1(2001) 93–107.

[3] M.T. Davis, C.S. Spahr, M.D. McGinley, J.H. Robinson, E.J.Bures, J. Beierle, J. Mort, W. Yu, R. Luethy, S.D. Patterson,Towards dening the urinary proteome using liquid chromato-graphy–tandem mass spectrometry. II. Limitations of complexmixture analyses, Proteomics 1 (2001) 108–117.

[4] D.J. Hampel, C. Sansome, M. Sha, S. Brodsky, W.E. Lawson,M.S. Goligorsky, Toward proteomics in uroscopy: urinary proteinproles after radiocontrast medium administration, J. Am. Soc.Nephrol. 12 (2001) 1026–1035.

[5] E.F. Petricoin, A.M. Ardekani, B.A. Hitt, P.J. Levine, V.A.Fusaro, S.M. Steinberg, G.B. Mills, C. Simone, D.A. Fishman,E.C. Kohn, L.A. Liotta, Use of proteomic patterns in serum toidentify ovarian cancer, Lancet 359 (2002) 572–577.

[6] S.P. Gygi, B. Rist, S.A. Gerber, F. Turecek, M.H. Gelb, R.Aebersold, Quantitative analysis of complex protein mixtures

using isotope-coded affinity tags, Nat. Biotechnol. 17 (1999) 994– 999.

[7] R.W. Nelson, J.R. Krone, A.L. Bieber, P. Williams, Mass-spectrometric immunoassay, Anal. Chem. 67 (1995) 1153–1158.

[8] E.E. Niederkoer, K.A. Tubbs, K. Gruber, D. Nedelkov, U.A.Kiernan, P. Williams, R.W. Nelson, Determination of b-2microglobulin levels in plasma using a high-throughput massspectrometric immunoassay system, Anal. Chem. 73 (2001) 3294– 3299.

[9] U.A. Kiernan, D. Nedelkov, K.A. Tubbs, E.E. Niederkoer,R.W. Nelson, High-throughput analysis of human plasma pro-teins, Am. Biotech. Lab. 20 (2002) 26–28.

[10] U.A. Kiernan, K.A. Tubbs, K. Gruber, D. Nedelkov, E.E.Niederkoer, P. Williams, R.W. Nelson, High-throughput proteincharacterization using mass spectrometric immunoassay, Anal.Biochem. 301 (2002) 49–56.

Fig. 3. Results of the anti-RBP MSIA analysis of urine. Traces A–Dare from healthy study subjects and display a generally similar phe-notypic prole. Wild-type RBP, RBP (-L), RBP (-LL), and RBP(-RNLL) (MW ¼ 20,534) are present in all four mass spectra. Ob-servable signal from RBP (-RSERNLL) (MW ¼ 20,162) is also presentin three of the four spectra. However, trace E is from the individualwith kidney impairment and mass spectrum is dominated by RBP-Lsignal and devoid of wt-RBP.

404 U.A. Kiernan et al. / Biochemical and Biophysical Research Communications 297 (2002) 401–405



[11] K.A. Tubbs, D. Nedelkov, R.W. Nelson, Detection and quanti-cation of b-2-microglobulin using mass spectrometric immuno-assay (MSIA), Anal. Biochem. 289 (2000) 26–35.

[12] D. Nedelkov, R.W. Nelson, Analysis of human urine proteinbiomarkers via biomolecular interaction analysis mass spectrom-etry, Am. J. Kidney Dis. 38 (2001) 481–487.

[13] M. Kanai, A. Raz, D.S. Goodman, Retinol-binding protein: the

transport protein for vitamin A in human plasma, J. Clin. Invest.47 (1968) 2025–2044.[14] H.M. Naylor, M.E. Newcomer, The structure of human retinol-

binding protein (RBP) with its carrier protein transthyretin reveals

an interaction with the carboxy terminus of RBP, Biochemistry 38(1999) 2647–2653.

[15] D.S. Goodman, Plasma retinol-binding protein, Ann. NY Acad.Sci. 348 (1980) 378–390.

[16] R. Beetham, A. Dawnay, J. Landon, W.R. Cattell, A radioim-munoassay for retinol-binding protein in serum and urine, Clin.Chem. 31 (1985) 1364–1367.

[17] S. Jaconi, K. Rose, G.J. Hughes, J.H. Saurat, G. Siegenthaler,Characterization of two posttranslationally processed forms of human serum retinol-binding protein: altered ratios in chronicrenal failure, J. Lipid Res. 36 (1995) 1247–1253.

U.A. Kiernan et al. / Biochemical and Biophysical Research Communications 297 (2002) 401–405 405

kiernan et al _2002_comparative phenotypic analyses of human plasma and urinary retinol binding...

Documents