Proteomics and diagnostics: Let's Get Specific, again

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    of historical interest are antibodies; we shall try in this shortreview to compare binding specificity for both antibodies

    an effecter of much of biology, often because of secretedproteins (such as growth factors) whose job is to moveextreme sensitivity and specificity.and aptamers and the ways in which proteomics might bescaled to deliver on the promise of high analyte density and

    through the blood to a nearby or distant site. In addition, avariety of proteins make their way into blood when somepathology causes localized cell death and the uninten-DOI 10.1016/j.cbpa.2008.01.016

    IntroductionSome years ago we published a short paper called LetsGet Specific [1] in which we tried to understand what wethought to be the extraordinary binding specificity ofaptamers [2,3]. We thought that elements of aptameraffinity and binding specificity were derived in part thelarge libraries used to find aptamers [often as many as 1015

    molecules for a SELEX experiment [4,5], and recentdevelopments [6]], as well as the structural possibilitiesexplored by single-stranded oligonucleotides. Morerecently we have focused our attention on the more generalnature of biochemical specificity, and we have wonderedabout the connection between reagent specificity andproteomics. When one considers proteomics, the reagentsProteomics and diagnostics: LetDom Zichi1, Bruce Eaton1,2, Britta S

    DNA array technology has changed all discussions about

    proteomics. Whole genome arrays allow unbiased

    experimentation, and the surprises that flow from those

    approaches. Whole proteome proteomics is not possible

    today, and might never be possible unless experiments are

    guided by careful evaluation of reagent specificity. In this paper

    we explore some possible ways to increase the content of

    proteomic analysis.

    Addresses1 SomaLogic, 1775 38th Street, Boulder, CO 80301, USA2Department of Chemistry and Biochemistry, University of Colorado,

    Boulder, CO 80309, USA3Department of Molecular, Cellular and Developmental Biology,

    University of Colorado, Boulder, CO 80309, USA

    Corresponding author: Zichi, Dom (dzichi@somalogic.com), Eaton,

    Bruce (beaton@somalogic.com), Singer, Britta

    (bsinger@somalogic.com), and Gold, Larry (lgold@somalogic.com),

    Current Opinion in Chemical Biology 2008, 12:7885

    This review comes from a themed issue on

    Proteomics and Genomics

    Edited by Natalie Ahn and Andrew H.-J. Wang

    Available online 7th March 2008

    1367-5931/$ see front matter

    # 2008 Elsevier Ltd. All rights reserved.A beautifully written (and referenced) recent review byBorrebaeck and Wingren [7] is aimed at a substantially

    Current Opinion in Chemical Biology 2008, 12:7885Get Specific, againger1 and Larry Gold1,3

    different question than we address in this article. Borre-baeck and Wingren have focused attention on theimpressive (and growing) list of improvements to variouscomponents of an antibody-based proteomic array. Ourfocus is on what specificity is possible with variousreagents, and to raise the possibility that array formatsmust solve any intrinsic limitations of those reagents.

    High analyte density is often abbreviated as content inthe case of nucleic acid arrays, content eventuallyincluded probes for entire genomes of viruses, bacteria,yeast, model organisms (flies, worms, and the mouse), andhumans. Using large arrays, scientists have utilizedmRNA and SNP profiling, along with epigenetic DNAmethylation, as genome-wide biomarker-discovery tech-nologies. All three platforms are possible because gen-ome-wide specific hybridization is possible that is, byjudicious use of proper probe lengths and appropriatebase composition along with the right temperature, bufferconditions, and hybridization time, specific sequencescan be recognized in the face of an entire genome.DNA chip technologies are remarkable as engineeringmarvels, but their discovery power flows from the highspecificity of nucleic acid hybridization. In fact, notsurprisingly, this same high specificity is the hallmarkof how nucleic acids perform their functions in biochem-istry. Remarkably, however, the array manufacturers havereached elegant solutions even as they built their contenton to compromised platforms. Probes bound to surfaces,be they beads (Luminex), slides (Agilent, Affymetrix,NimbleGen), or things in the middle (Illumina) presentless than perfect kinetics and slow approaches to equi-librium. These approaches are quite unlike hybridizationin solution as it was originally developed [8,9]. Only rarelydid platform builders include approaches that overcamethe slow kinetic approach to equilibrium (NanoGen,MetriGenix, PamGene, etc.), and those sensible plat-forms seem to have lost in the market place.

    Human diagnostics is better served by protein measure-ments than by nucleic acid measurements, and servedbest by protein measurements in blood samples (a matrixwith a vast number of proteins see below). Human bloodis an integrator of much of what happens in the body, andtional release of proteins. Since blood equilibrates quicklywith all human tissues, including brain, panels of proteinbiomarkers should become the earliest warnings one has

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  • Proteomics and diagnostics: Lets Get Specific, again Zichi et al. 79for the early stages of disease, even when a person isasymptomatic. Of course these same biomarkers can bepresent at vanishingly low concentrations and measuringthese low abundant proteins is the major hurdle proteo-mics must overcome.

    Of the approximately 23,000 human genes and their>100,000 encoded proteins (comprising splice variants,post-translationally modified proteins, and even more rareevents [10], we do not know how many proteins are foundin blood. Probably every human protein is present in bloodat a very low level (if only from cell death), and perhapsseveral thousand are present between the concentrations ofthe most abundant blood proteins (albumin at just under1 mM) down to protein concentrations at about 1 fM adynamic range of 12 logs or more! The problems in humanproteomics and diagnostics are to scale proteomics to highcontent to discover useful biomarkers and to make avail-able diagnostic products that utilize (smaller) panels ofproteins for specific medical purposes. That is, in the samespirit as made possible by nucleic acid array technologies,one must survey large fractions of the proteome content inan unbiased manner for novel biomarker discovery.

    One might think this is a simple task, given the stunningsuccess with nucleic acid arrays. The problem of course isthat typing nucleic acid complement sequences (probes) isa lot easier than understanding the biophysics of proteinrecognition biochemistry. Indeed, the allure of typingenthralled the antisense, ribozymes, and siRNA thera-peutic researchers with the hope that typing would be agreat way to identify new drugs. For proteomics the ideahas been to replace typing of nucleic acid complementswith typing orders to the antibody suppliers. However,when commercial antibodies are printed (as though theywere analogues of nucleic acid probes) and then testedwith various protein mixtures, performance (meaningspecificity) probably is not adequate. The purpose of thisreview is to discuss these attempts and the aptamer-basedalternatives, and to note the intrinsic kinetic problemsthat must be solved by proteomics.

    Reagent-free proteomicsWe mention briefly that reagent-free proteomics wouldbe a wonderful development, although it appears diffi-cult. Since 1975, with the publication of Pat OFarrellsextraordinary work on 2D gels [11], such reagent-freeproteomics has been possible. The quality of the first 2Dgels was amazingly high (something like 1100 proteinswere visible), major and minor proteins seemed to differquantitatively by three logs or so, and differently chargedspecies of the same molecular weight were not a majorsource of additional spots that cluttered the patterns. InPats thesis seminar he showed wild-type Escherichia coli

    gels versus lactose-operon deletion gels, and the missingspots were a powerful demonstration of what might bedone, at least qualitatively.

    www.sciencedirect.comBlasting through many samples (tissue extracts or plasmaor serum, or urine, or whatever) with 2D gels had amoment (back in the day as our children say . . .).However, the problems with reproducibility and quanti-fication were serious, and the cost per analysis was high eventually people added MS to the methodology, and it isnow common for an entire 2D gel to be extracted (featureby feature) for MS analysis. Furthermore, the limitationsin protein number (content again) have never been solved Pat OFarrells number from E. coli really has remained,after a lot of work, about the number of protein spots onecan visualize, and the proteins observed are thus inevi-tably the most abundant proteins in the sample.

    Mass spectrometry (MS) has had a similar fate, so far.Even though the sensitivity of a great mass spectrometermight enable analysis of samples quite a bit lower thannmolar in a few microliters, when complex matrices areexplored the noise obscures all but the most abundantproteins. In fact, it appears that 2D gels and MS querymore or less the same most abundant proteins from withina complex sample. Clearly, the resolving power of 2D gelscoupled to MS in proteomics has yet to match what iscapable in DNA hybridization micro arrays.

    Reagent-dependent proteomics: antibodiesNo one really knows what fraction of the human pro-teome has been used to generate high quality antibodies[see the Human Protein Atlas (http://www.proteinatla-s.org/), [12]] or protein reagents with alternative frame-works [13]. The assumption has been that antibodyproduction could be s