Perspective pubs.acs.org/ac
Opportunities for Sensitive Plasma Proteome Analysis Ulf Landegren,* Johan Van̈ elid, Maria Hammond, Rachel Yuan Nong, Di Wu, Erik Ullerås, and Masood Kamali-Moghaddam Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, The Rudbeck Laboratory, 75185 Uppsala, Sweden S Supporting Information *
ABSTRACT: Despite great interest, investments, and efforts, the ongoing search for plasma protein biomarkers for disease so far has come up surprisingly empty-handed. Although discovery programs have revealed large numbers of biomarker candidates, the clinical utility has been validated for only a very small number of these. While this disappointing state of affairs may suggest that plasma protein biomarkers have little more to offer for diagnostics, we take the perspective that experimental conditions might not have been optimal and that analyses will be required that offer far greater sensitivity than currently available, in terms of numbers of molecules needed for unambiguous detection. Accordingly, techniques are needed to search deep and wide for protein biomarker candidates. The requirements and feasibility of such assays will be discussed.
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A more ambitious, if somewhat arbitrary, cutoff for potentially diagnostic plasma protein levels might be placed at one molecule per microliter. This concentration, which could still avoid excessive stochastic noise in realistic sample volumes, is more than 14 orders of magnitude below that of albumin, but it is nonetheless 50-fold higher than the current detection threshold applied in routine screening for HIV particles in blood, as analyzed at the RNA level.4 Kary Mullis, the inventor of PCR, on realizing the possibility to detect even single DNA molecules with very high efficiency, proposed a system for reporting actual numbers of molecules to avoid the abstraction of expressing low numbers of molecules as very small fractions of Avogadro’s number.5 Will it be possible to reach similar levels of sensitivity for protein detection, motivating the use of the same measure? Figure 1 illustrates concentrations for proteins in plasma and in cells, highlighting concentrations of plasma proteins that are currently used for diagnostic purposes.6−14 The figure also illustrates typical reported sensitivities of measurement for some techniques used in the search for plasma protein biomarkers. Protein concentrations are described using three scales for translation purposes: the weight per volume measure popular in clinical contexts, a conversion to molarities, with a reasonable assumption that plasma proteins have molecular weights somewhere around 50 000 Da, and finally, the numbers of molecules per microliter, expressed as proposed by Mullis. See supplementary figure legends (Supporting Information) for some further comments. The implications of the different
n 1937, Arne Tiselius demonstrated, using his protein electrophoresis technique, that blood serum includes four protein components: albumin and alpha, beta, and gamma globulins.1 He cautiously added in a concluding section of the paper: “This does not necessarily mean that our components represent chemical individuals”. Indeed, we now know that blood serum and plasma are rich sources of untold numbers of proteins. The combination of convenient, minimally invasive access via venipuncture and the potential to represent any disease process anywhere in the body as changes in plasma protein composition has rendered plasma proteomics an attractive field of study. In this view, the potential for diagnostics by plasma protein measurements is vast and compelling, with a potential to revolutionize medicine by allowing life-threatening diseases to be identified at early, perhaps still curable stages, while also vastly improving prediction of outcome and selection and monitoring of therapy. Despite this source of bright optimism, new useful protein biomarkers have proven elusive and rather than a floodgate, a trickle of new biomarkers of modest diagnostic value is brought to clinical use, a disappointing situation that has been the subject of considerable soul searching. This conundrum and possible remedies will be the theme for this Perspective, with a focus on affinity-based detection technologies.
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THE COMPLEXITY OF THE PLASMA PROTEOME The plasma proteome confronts the intrepid explorer with many challenges. The oft-quoted number of 10 to the power of 10-fold for the range of plasma protein concentrations refers to the ratio between albumin and interleukin 6, one of the least abundant proteins currently used for routine diagnostic analyses.2,3 However, there are good reasons to expect that useful protein biomarkers may be found at still lower concentrations. © 2012 American Chemical Society
Received: December 5, 2011 Accepted: January 16, 2012 Published: January 16, 2012 1824
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gene may exhibit radically different functions or diagnostic significance.18−21 Proteins are also subject to processing and posttranslational modification steps, and they often occur as covalent or noncovalent complexes or, for example, embedded in the membranes of nanometer-scale particles called exosomes.22 The question about how many plasma protein variants to distinguish for diagnostic purposes is unlikely to receive a clearcut answer anytime soon.
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WHY GO DEEP? It is striking that many current biomarker proteins are present at low concentrations in plasma, indeed far lower than those typically accessed in most screens for new markers (Figure 1). Not only can far more protein species be accessed using assays of greater detection sensitivity, increasing the probability that useful markers will be found, but it is also reasonable to assume that proteins capable of revealing a localized disease process will be present at very low concentrations early in disease, before extensive tissue damage. Accordingly, proteins normally present in plasma at low to undetectable concentrations and showing very restricted tissue expression may represent particularly promising targets for diagnostics, and more sensitive detection of such marker proteins may translate to earlier detection of the disease. Finally, much fishing for biomarkers has been tried in the shallow waters of the more abundant proteins without much to show for, further underlining the need to now probe the depths. A discussion about how sensitive detection can be achieved will require some words about current detection techniques and their limitations.
Figure 1. Concentration ranges of human proteins in plasma and in cells. Proteins concentrations are expressed as weights per volume, or assuming an average protein size of 50 000 Da, as molarities or numbers of protein molecules per microliter. We use the concentration notation suggested by Mullis5 where 1U5 is short for 1 × 105 per microliter. The figure also indicates the concentrations of plasma protein markers in current clinical use,2,3 as well as the reported sensitivities for some techniques currently used for validating new, putative protein biomarkers. See also the supplementary figure legend (Supporting Information).
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HOW TO RECOGNIZE A PROTEIN WHEN YOU SEE ONE Two principally different means can be contemplated for identifying proteins in biological samples: the identity of protein molecules can be read out from their sequence of amino acids. Alternatively, binding by specific affinity reagents can be used to record and report their presence in a sample.23 Despite the ease of constructing affinity reagents for DNA and RNA analyses via the basepairing rules, we are currently witnessing a trend where the use of affinity reagents, hybridization probes, gives way to identification and enumeration of DNA and RNA molecules by recording the sequence of nucleotides in individual molecules through next generation sequencing. For proteins, mass spectrometry (MS) similarly decodes protein identities by recording the information embodied in their amino acid sequence. MS, therefore, has the attraction of offering an overview of proteins present in plasma with no need to bias the investigation by selecting particular targets. Regarding the affinity approach, proteins are at a severe disadvantage compared to nucleic acids as no simple rules exist for the generation of affinity reagents for proteins. The reagents must, therefore, be selected and/or evolved from large pools of binder candidates, either using the immune systems of organisms such as rabbits and mice or through in vitro approaches.24 This situation would seem to argue strongly in favor of MS for biomarker discovery, but while sequences in genomes are characteristically equimolar and transcripts show relatively modest concentration differences, the dramatic concentration differences among proteins in plasma have so far precluded highly sensitive detection by MS, where the more abundant proteins tend to swamp proteins present at far lower concentrations (Figure 2). Selective MS techniques, or MS
detection sensitivities demonstrated in this diagram will be discussed below.
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HOW MANY PLASMA PROTEINS ARE THERE? The discussion of plasma protein concentration ranges also evokes questions about how many different proteins there are that might be targeted for analysis. The number of proteins in plasma is well-defined at the top of the pyramid with albumin accounting for around 60% of the total mass, the five next most abundant proteins representing 25%, and the following 14 proteins comprising 12%.15,16 There is no convincing estimate, however, of how many molecular species can be found at substantially lower concentrations. Among the more abundant proteins at least, numbers of protein species in plasma appear to be distributed according to a power law, such that assays capable of detecting 10-fold lower concentrations of proteins can be expected to access some factor more protein species, some of which may well prove suitable as biomarkers. This also means that, when proteins at lower concentrations are targeted, far more protein species have to be distinguished, placing increasing demands on the specificity of detection. It is well-known that the approximately 20 50017 human protein coding genes give rise to many more distinct protein variants but not how many variants there may be. The vast majority of human genes are subject to variable splicing, and proteins translated from different splice variants of the same 1825
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homologous or nonhomologous proteins or by also binding entirely different epitopes. Polyclonal antibodies may provide a form of majority-vote effect by enhancing detection signals when several epitopes on the target molecules are detected, while these epitopes are unlikely to all be present on irrelevant molecules. This specificity-enhancing effect is offset, however, by the greater opportunities for crossreactivity for irrelevant molecules by the many different antibodies in a polyclonal antibody preparation. A tendency to crossreact with nontarget molecules is commonly seen for all classes of affinity reagents. By way of example, Kramer et al. extensively analyzed the binding promiscuity of a well-characterized monoclonal antibody directed against the p24 protein of HIV-1 by analyzing its ability to bind a synthetic combinatorial library of peptides. The analysis revealed five nonhomologous peptides that were recognized by the same antibody region and were able to compete with recombinant p24 for binding to the antibody. By positional scanning, more than 1000 single amino acid substitution analogs derived from these five different peptides were also shown to bind the antibody. Through bioinformatic analysis, this information was used to identify other proteins that the anti-p24 monoclonal antibody could then be shown to bind.27 The considerable risk of detecting irrelevant target molecules in assays that depend on binding by single affinity reagents is also illustrated in a recent paper.28 The authors used a large number of affinity purified polyclonal antibodies to stain three different cell lines using immunofluorescence. Some 500−700 out of the 3000 antibodies that were scored as positive for the different cell lines had been raised against proteins that would not be expected to be present in those cell lines, since the corresponding transcripts could not be demonstrated by deep sequencing. This suggests that possibly around 20% of the antibodies exhibited false positive signals in this study. The problem of crossreactive binding by single affinity reagents is likely far greater when targeting plasma proteins because of the much wider concentration range, compared to the situation in a cell line. Even minimal tendencies by the affinity reagents to crossreact with nontargets may obliterate any chance of correct detection if a crossreactive target happens to be present in far higher concentrations than the intended one.
Figure 2. Complexity of nucleic acids and proteins in human cells and in plasma. The challenge of specifically detecting DNA, RNA, or protein molecules is illustrated by plotting the proportion out of all nucleotides or amino acids that have to be identified for correct detection of a specific target molecule. For example, a strand-specific detection reaction for a particular single nucleotide polymorphism in a patient sample has to search through all genomic DNA. This represents a task of specifically identifying one nucleotide out of 13 billion. For comparison purposes, the corresponding numbers for the complexity of proteins in cells or plasma are calculated as the proportion out of all amino acids in proteins that must be detected to pinpoint a particular amino acid in a given protein. The numbers for albumin, IL6, and for a hypothetical average-size protein present at one copy per microliter in plasma are indicated in the figure for reference. See also the supplementary figure legend (Supporting Information).
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applied in combination with affinity purification, can extend the dynamic range of protein analysis in plasma, such as in the SISCAPA technique.25 Nonetheless, affinity-based approaches for protein detection still seem to have the upper hand for sensitive detection (Figure 1) and maybe also for convenience. We will, therefore, now focus on opportunities for sensitive and specific protein detection using affinity reagents.
HOW TO BUILD A BETTER PROTEIN ASSAY Highly sensitive protein detection in principle is a simple matter. All one has to do is ensure that efficiency of target recognition is close to 100%, while avoiding all sources of nonspecific detection signals. Obviously, realities present difficulties, and existing assays remain orders of magnitude away from detecting every single protein molecule. Nonetheless, efficiency of target binding by affinity reagents in fact can reach high levels. By analogy, in PCR, it is a matter of routine to have primers bind practically all target DNA sequences in a sample in just a few seconds during each amplification cycle. Similarly, target protein capture efficiency approaching 100% can be routinely accomplished in seconds, given suitable reaction conditions.29,30 Factors such as affinity and concentration of binding reagents, as well as incubation times and average diffusion distances, are relevant. However, conditions that increase target binding may also tend to increase nonspecific signals. A few sources of background dominate affinity-based protein detection reactions, thus limiting assay sensitivity (Figure 3).
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MONOSPECIFIC AFFINITY REAGENTS−DON’T HOLD YOUR BREATH There are many classes of binding reagents for proteins, both antibodies and artificial molecular constructs, some of them reaching dissociation constants in an impressive picomolar range. For example, the selection of high-affinity modified DNA aptamers, characterized by a very slow off-rate, has been shown to provide good sensitivity of detection, and these reagents have been successfully combined in sets of over 1000 aptamers for parallel protein biomarker screens in plasma.8,26 Nonetheless, even affinity reagents with excellent affinities for their targets may exhibit crossreactivity for irrelevant molecules, either because the very same motif is present in 1826
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washing reaction vessels. Another approach to minimize the problem of nonspecific binding by detection reagents is to add these reagents in a form where they cannot give rise to detection signals unless brought together, i.e., by jointly binding their target molecules. This approach can be exemplified by protein complementation assays, where two parts of a protein have to be brought together by jointly binding target molecules in order to be detectable by activating their fluorescence or enzymatic activity.36,37 Crossreactive Binding by Detection Reagents for Nontarget Proteins. Impressive protein detection sensitivity is frequently reported using novel reporting mechanisms in assays that depend on binding by single affinity reagents in buffer solutions.38,39 Unfortunately, the same analyses applied in biological samples tend to exhibit elevated background, reducing sensitivity of detection. As discussed, an important reason for this is the tendency to nonspecific target recognition by affinity reagents, increasing assay background. It is well-known from nature and in molecular technology that specificity of molecular recognition can be improved by applying the principle of proofreading, as illustrated by the process of protein translation from an RNA template.40 PCR derives its specificity from proofing via the required coincidence of two oligonucleotides hybridizing to and priming synthesis of the same double stranded DNA molecule. In the context of protein detection, this same effect is achieved using sandwich assays where specific detection depends on coincident binding of target molecules by pairs of affinity reagents, thus serving as logical AND gates, where both criteria have to be fulfilled for a positive reaction.41 Despite their significantly improved specificity and hence greater sensitivity of detection, sandwich assays have proven difficult to perform in great multiplex, due to the rapidly increasing tendency to binding by noncognate pairs of affinity reagents as more proteins are targeted, gradually eroding the added specificity of pairwise binding (Figure 4).
Figure 3. Ideal and more realistic standard curves for protein detection. Ideally, even single protein molecules should be detected over background, and signals would continue increasing proportionally as the amount of the protein increases. In practice, detection reactions typically exhibit detection thresholds due to nonspecific background, and only a fraction of all available proteins are detected, until at some target concentration a signal plateau is reached or even a so-called hook above which concentration signals begin to decrease again. Some of these effects are illustrated in the figure, along with some terms commonly used to characterize detection reactions. The limits of detection are defined as the numbers of target molecules required to give rise to a detection signal 2 (or 3) standard deviations (signal variation, as represented by the light gray area) above the background or below the maximal signal. See also the supplementary figure legend (Supporting Information).
Nonspecific Signals from Media. Most standard protein assays exhibit some general background in the way of fluorescence or optical absorption by the reaction vessels or solutions. A number of recent papers report improved sensitivity of detection by counting individual detection reagents, rather than averaging the detected quality across whole reaction vessels.31−34 Besides affording a digital measure of bound affinity reagents, these approaches also share the advantage that background from media can be ignored under the conditions used. In a related fashion, only detection reagents are recorded in immuno-PCR,35 where antibody detection is measured by amplification of a DNA molecule attached to the antibody. All these methods thus avoid some sources of background, but inappropriate binding by detection reagents remains an important factor that limits sensitivity of detection. Nonspecific Absorption of Detection Reagents. Detectable affinity reagents have to be added in sufficient concentration for efficient recognition of the intended target molecules. For example, in a standard enzyme linked immunosorbent assay (ELISA), around 10 to the power of 10 and 10 to the power of 11 enzyme-conjugated antibodies are added to each reaction well. In any assay, some small fractions of these reagents are likely to resist washes, for example, due to nonspecific binding to surfaces independently of any target molecules, thus contributing to background. Much ingenuity is spent on optimizing surface chemistry and blocking substances in the reaction media, as well as on more efficient approaches for
Figure 4. Assay formats for affinity-based protein detection. Several different assay architectures are in use for affinity-based protein detection. In single-binder assays, either target molecules (A) or affinity reagents (B) are immobilized on a solid phase, followed by detection of labeled solution-phase molecules that have been captured on the solid phase. (C) In sandwich immunoreactions, target molecules are captured to a solid phase via one affinity reagent and visualized via a second, labeled affinity reagent. (D) Proximity reactions come in many variants either in homogeneous phase or using a solid support. The figure illustrates the process of capturing a target protein via one affinity reagent, followed by binding by two additional affinity reagents with attached DNA strands. Upon proximal binding, these oligonucleotides can be joined to form an amplifiable reporter DNA strand that serves to reveal the identity and the amounts of the detected protein. See also the supplementary figure legend (Supporting Information).
As a consequence, single-binder assays remain the most commonly used approaches in broad searches for protein biomarkers, 1827
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the first time elevated levels of prostasomes in plasma from patients with prostate cancer, potentially furnishing a novel cancer marker of improved performance compared to the wellknown PSA test.51
despite their generally lower sensitivity. However, highthroughput assays of improved performance are becoming available.
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PROXIMITY LIGATION−ENGINEERING ENHANCED PROTEIN DETECTION We will illustrate the possibility to construct high performance protein assays with the proximity ligation reactions developed in our lab, and the technique is compared to earlier assay formats in Figure 4.42,43 The proximity ligation assay (PLA) has the potential to avoid many sources of background in order to reach high levels of detection sensitivity. In this assay, sets of affinity reagents that can jointly bind different epitopes on a target protein molecule are functionalized by attaching DNA strands. Either matched clonal affinity reagents or aliquots of polyclonal antibodies can be used. The latter approach relies on the likelihood that polyclonal antibodies raised against whole proteins will contain antibodies capable of jointly binding different epitopes of the target molecule. Upon proximal binding to the same target molecule by the antibodies, the attached DNA strands are brought close to one another. This allows the strands to participate in ligation reactions or, in the case of proximity extension assays, to undergo a polymerization reaction,44 in both cases giving rise to amplifiable reporter DNA molecules. The assays can be performed with pairs of oligonucleotide-modified affinity reagents in a homogeneous format, i.e., with no washes or separations. Alternatively, target molecules are captured by a first affinity reagent, followed by washes before introducing the pairs of oligonucleotide-modified affinity reagents.45 In these assays, background from media and from individual nonspecifically adsorbed detection reagents do not result in erroneous detecting signals as only amplification products from pairs of reacted reagents are recorded. The requirement for binding by pairs of affinity reagents to generate detectable reaction products provides improved specificity, i.e., lower risk of crossreactive detection, compared to singlerecognition assays.46 Furthermore, the solid-phase variant with its unique requirement for binding by trios of affinity reagents further improves specificity of detection, and it allows for removal of sample components that could compromise detection efficiency. The DNA-based enzymatic ligation and/ or polymerization reactions can be designed to ensure that only cognate sets of affinity reagents give rise to detection signals, thus avoiding the mounting problems of crossreactivity that complicates multiplexing sandwich assays. Proximity ligation or extension assays have been successfully applied for sensitive detection of several tens of proteins in aliquots of 1 or 5 μL of plasma,9,44,47−49 and evidence has been obtained that the assays could be multiplexed much further with modest increase of background.8 In addition, multiple recognition assays allow the combination of protein-specific affinity reagents with reagents recognizing specific posttranslational modifications such as phosphorylations, expanding the range of targets with a modest need for additional affinity reagents. As a final point, the proximity ligation mechanism also serves to illustrate the potential to engineer specialized assays for particular detection purposes. A proximity assay where five antibodies must jointly detect a target structure in order to trigger the formation of an amplifiable reporter molecule was designed to target exosome-like microvesicles called prostasomes. These membrane-coated particles are released in high amounts by prostate epithelial cells into seminal fluid.50 The adapted proximity ligation assay was able to demonstrate for
A CALL TO ARMS In conclusion, development of repertoires of plasma protein biomarkers, potentially capable of revealing disease anywhere in the body, remains an important mission whose feasibility is still essentially unproven. Future progress will depend on concerted efforts combining resources that are only available in part at the present time. These include the following: (i) Suitable patient samples must be secured to screen for potential protein biomarkers, and in particular, consecutive samples from the same individuals are of great value to follow specific protein levels before, at, and after clinical diagnosis and therapy. Extensive biobanks providing such samples are now becoming available and complemented by comprehensive clinical information as needed to identify and validate new biomarkers.52 (ii) Absent convincing means of analyzing or even properly defining whole proteomes, biological and medical insights will be required to guide the selection of promising molecular targets for purposes of distinguishing signs of health versus disease, predicting prognosis, and selecting and monitoring therapy. (iii) In the case of affinity-based detection reactions, replenishable, i.e., clonal resources of suitable detection reagents must be developed that can be shared between laboratories and functionalized for specific assays.53 Two, three, or more reagents capable of binding the same target molecule without mutual interference should be available against any analyte in order to reach the required specificity of detection. (iv) Finally, as discussed herein, high-performance protein detection techniques are needed, and promising approaches exist that could provide the required sensitivity/ specificity, robustness, precision, and capacity for parallel analyses of many markers in numerous samples using limited sample volumes. Ideally, it should be possible to adapt the same molecular assay mechanisms used for discovery and validation also for routine diagnostics, ultimately even at the point of care, although this will place considerable demands on factors such as user-friendliness and speed of the assays.
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ASSOCIATED CONTENT
S Supporting Information *
Supplementary text for Figures 1−4. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +46184714910. Fax: +46184714808. Notes
The authors declare the following competing financial interest(s): UL is the founder of, and a stockholder in, Olink Bioscience, having rights to the proximity ligation technique.
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ACKNOWLEDGMENTS Dr. Spyros Darmanis provided helpful comments on the manuscript. Work in the Landegren laboratory is supported by grants from the Swedish Foundation for Strategic Research (SSF), the Swedish Governmental Agency for Innovation Systems (Vinnova), the Knut and Alice Wallenberg Founda1828
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tion, the Swedish Research Council for medicine, and the European Community’s 7th Framework Program (FP7/20072013) under grant agreement Nos. 259796 (DiaTools), 222635 (AffinityProteome), and 241481 (Affinomics) and by the Innovative Medicines Initiative.
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