Quantification of Cardiovascular Biomarkers in Patient Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution

Verification of candidate biomarkers requires specific assays to selectively detect and quantify target proteins in accessible biofluids. The primary objective of verification is to screen potential biomarkers to ensure that only the highest quality candidates from the discovery phase are taken forward into preclinical validation. Because antibody reagents for a clinical grade immunoassay often exist for a small number of candidates, alternative methodologies are required to credential new and unproven candidates in a statistically viable number of serum or plasma samples. Using multiple reaction monitoring coupled with stable isotope dilution MS, we developed quantitative, multiplexed assays in plasma for six proteins of clinical relevance to cardiac injury. The process described does not require antibodies for immunoaffinity enrichment of either proteins or peptides. Limits of detection and quantitation for each signature peptide used as surrogates for the target proteins were determined by the method of standard addition using synthetic peptides and plasma from a healthy donor. Limits of quantitation ranged from 2 to 15 ng/ml for most of the target proteins. Quantitative measurements were obtained for one to two signature peptides derived from each target protein, including low abundance protein markers of cardiac injury in the nanogram/milliliter range such as the cardiac troponins. Intra- and interassay coefficients of variation were predominantly <10 and 25%, respectively. The configured multiplex assay was then used to measure levels of these proteins across three time points in six patients undergoing alcohol septal ablation for hypertrophic obstructive cardiomyopathy. These results are the first demonstration of a multiplexed, MS-based assay for detection and quantification of changes in concentration of proteins associated with cardiac injury in the low nanogram/milliliter range. Our results also demonstrate that these assays retain the necessary precision, reproducibility, and sensitivity to be applied to novel and uncharacterized candidate biomarkers for verification of proteins in blood.Discovery of disease-specific biomarkers with diagnostic and prognostic utility has become an important challenge in clinical proteomics. In general, unbiased discovery experiments often result in the confident identification of thousands of proteins, hundreds of which may vary significantly between case and control samples in small discovery studies. However, because of the stochastic sampling of proteomes in discovery “omics” experiments, a large fraction of the protein biomarkers “discovered” in these experiments are false positives arising from biological or technical variability. Clearly discovery omics experiments do not lead to biomarkers of immediate clinical utility but rather produce candidates that must be qualified and verified in larger sample sets than were used for discovery (1).Traditional, clinical validation of biomarkers has relied primarily on immunoassays because of their specificity and sensitivity for the target analyte and high throughput capability. However, antibody reagents for a clinical grade immunoassay often only exist for a short list of candidates. The development of a reliable sandwich immunoassay for one target protein is expensive, has a long development time, and is dependent upon the generation of high quality protein antibodies. For the large majority of new, unproven candidate biomarkers, an intermediate verification technology is required that has shorter assay development time lines, lower assay cost, and effective multiplexing of dozens of candidates in low sample volumes. Ideally the approach should be capable of analyzing hundreds of samples of serum or plasma with good precision. The desired outcome of verification is a small number of highly credentialed candidates suitable for traditional preclinical and clinical validation studies.Multiple reaction monitoring (MRM)¹ coupled with stable isotope dilution (SID) MS has recently been shown to be well suited for direct quantification of proteins in plasma (2–4) and has emerged as the core technology for candidate biomarker verification. MRM assays can be highly multiplexed such that a moderate number of candidate proteins (in the range of 10–50) can be simultaneously targeted and measured in the statistically viable number of patient samples required for verification (hundreds of serum samples). However, sensitivity for unambiguous detection and quantification of proteins by MS-based assays is often constrained by sample complexity, particularly when the measurements are being made in complex fluids such as plasma.Many biomarkers of current clinical importance, such as prostate-specific antigen and the cardiac troponins, reside in the low nanogram/milliliter range in plasma and, until recently, have been inaccessible by non-antibody approaches. Our laboratory has recently shown for the first time that a combination of abundant protein depletion with limited fractionation at the peptide level prior to SID-MRM-MS provides robust limits of quantitation (LOQs) in the 1–20 ng/ml range with coefficient of variation (CV) of 10–20% at the LOQ for proteins in plasma (3).Here we demonstrate that this work flow can be extended to configure assays for a number of known markers of cardiovascular disease and, more importantly, can be deployed to measure their concentrations in clinical samples. We modeled a verification study comprising six patients undergoing alcohol septal ablation treatment for hypertrophic obstructive cardiomyopathy, a human model of “planned” myocardial infarction (PMI), and obtained targeted, quantitative measurements for moderate to low concentrations of cardiac biomarkers in plasma. This work provides additional evidence that MS-based assays can be configured and applied to verification of new protein targets for which high quality antibody reagents are not available.     

Accurate inclusion mass screening: a bridge from unbiased discovery to targeted assay development for biomarker verification

Verification of candidate biomarker proteins in blood is typically done using multiple reaction monitoring (MRM) of peptides by LC-MS/MS on triple quadrupole MS systems. MRM assay development for each protein requires significant time and cost, much of which is likely to be of little value if the candidate biomarker is below the detection limit in blood or a false positive in the original discovery data. Here we present a new technology, accurate inclusion mass screening (AIMS), designed to provide a bridge from unbiased discovery to MS-based targeted assay development. Masses on the software inclusion list are monitored in each scan on the Orbitrap MS system, and MS/MS spectra for sequence confirmation are acquired only when a peptide from the list is detected with both the correct accurate mass and charge state. The AIMS experiment confirms that a given peptide (and thus the protein from which it is derived) is present in the plasma. Throughput of the method is sufficient to qualify up to a hundred proteins/week. The sensitivity of AIMS is similar to MRM on a triple quadrupole MS system using optimized sample preparation methods (low tens of ng/ml in plasma), and MS/MS data from the AIMS experiments on the Orbitrap can be directly used to configure MRM assays. The method was shown to be at least 4-fold more efficient at detecting peptides of interest than undirected LC-MS/MS experiments using the same instrumentation, and relative quantitation information can be obtained by AIMS in case versus control experiments. Detection by AIMS ensures that a quantitative MRM-based assay can be configured for that protein. The method has the potential to qualify large number of biomarker candidates based on their detection in plasma prior to committing to the time- and resource-intensive steps of establishing a quantitative assay.     

Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

Selected reaction monitoring (SRM) - also known as multiple reaction monitoring (MRM) - has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for, e.g. detecting low-abundance biomarkers likely present at the low ng/mL to pg/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in cells or tissues. Herein, we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides including posttranslational modifications, as well as advances in MS instrumentation which have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low- to sub-ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed.     

Candidate verification of iron-regulated Neisseria meningitidis proteins using isotopic versions of tandem mass tags (TMT) and single reaction monitoring

Tandem Mass Tags (TMT) are suited to both global and targeted quantitation approaches of proteins and peptides. Different versions of these tags allow for the generation of both isobaric and isotopic sets of reagents sharing the same common structure. This feature allows for a straightforward transfer of data obtained during discovery studies into targeted investigations. In prior discovery studies, an isobaric set of these reagents was used to identify Neisseria meningitidis proteins expressed under iron-limitation. Here, we apply isotopic versions of those reagents in combination with single reaction monitoring to verify selected candidates found to be differentially regulated in these discovery studies, representing both well-known and novel iron-regulated proteins, such as the MtrCDE drug efflux pump. In this targeted approach (TMT–SRM), the selectivity of SRM is maintained while allowing the incorporation of an internal reference standard into the experiment. By monitoring 184 transitions, TMT–SRM resulted in the quantitation of 33 peptides representing 12 proteins. The acquired data corroborated the results obtained during the discovery phase. Furthermore, these data obtained by MS-based quantitation of peptides were independently confirmed by western blotting results, an orthogonal approach based on quantitation at the protein level.     

Current progress in protein profiling of complex samples

Accurate cancer biomarkers are needed for early detection, disease classification, prediction of therapeutic response and monitoring treatment. While there appears to be no shortage of candidate biomarker proteins, a major bottleneck in the biomarker pipeline continues to be their verification by enzyme linked immunosorbent assays. Multiple reaction monitoring (MRM), also known as selected reaction monitoring, is a targeted mass spectrometry approach to protein quantitation and is emerging to bridge the gap between biomarker discovery and clinical validation. Highly multiplexed MRM assays are readily configured and enable simultaneous verification of large numbers of candidates facilitating the development of biomarker panels which can increase specificity. This review focuses on recent applications of MRM to the analysis of plasma and serum from cancer patients for biomarker verification. The current status of this approach is discussed along with future directions for targeted mass spectrometry in clinical biomarker validation.     

P23-M Limit of Quantitation for Low-Abundance Proteins in Plasma by Targeted MS

Biomarker discovery results in the creation of candidate lists of potential markers that must be subsequently verified in plasma.¹ The most mature methods at present require abundant protein depletion and fractionation at the protein/peptide levels in order to detect and quantitate low ng/mL concentrations of plasma proteins by stable isotope dilution mass spectrometry. Sample-processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate bio-marker proteins were evaluated by adding five non-native proteins to immunoaffinity-depleted female plasma at varying concentrations (1000, 100, 50, 25, and 10 ng/mL). Each protein was monitored by one or more representative synthetic tryptic peptides labeled with [¹³C₆]leucine or [¹³C₅] valine. Following reduction, carbamidomethylation, and enzymatic digestion, two separate processing paths were compared. In path 1, digested plasma was diluted 1:10 and [¹³C] internal standards were added just prior to direct analysis by multiple reaction monitoring with LC-MS/MS (MRM LC-MS/MS). In path 2, peptides were separated by strong cation exchange, and [¹³C] internal standards were added to corresponding SCX fractions prior to analysis by MRM LC-MS/MS. Detection and quantitation by MRM used the response of at least two product ions from each of the signature peptides. Using processing path 1, we achieved detection and quantitation down to 50 ng/mL in depleted plasma. However, using processing path 2, we achieved detection and quantitation of all spiked proteins, including the non-native protein at 10 ng/mL. While analysis of non-fractionated plasma achieved higher recovery of those proteins detected in both processes, SCX fractionation at the peptide level clearly increases detection and LOQs for potential biomarker proteins in plasma.     

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, ''hook-effect'').¹ An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).² In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules ^{3, 4} and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.^5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.^8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.     

Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)

A method (denoted SISCAPA) for quantitation of peptides in complex digests is described. In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled). In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin, alpha1 antichymotrypsin, interleukin-6, and tumor necrosis factor-alpha) from a pool of 10,203 in silico tryptic peptide candidates representing 237 known plasma components. Rabbit polyclonal antibodies raised against the chosen peptide sequences were affinity purified and covalently immobilized on POROS supports. Binding and elution from these supports was shown to provide an average 120-fold enrichment of the antigen peptide relative to others, as measured by selected ion monitoring (SIM) or selected reaction monitoring (SRM) electrospray mass spectrometry. The columns could be recycled with little loss in binding capacity, and generated peptide ion current measurements with cycle-to-cycle coefficients of variation near 5%. Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays. The method appears suitable for rapid generation of assays for defined proteins, and should find application in the validation of diagnostic protein panels in large sample sets.     

Clinical Quantitation of Prostate-specific Antigen Biomarker in the Low Nanogram/Milliliter Range by Conventional Bore Liquid Chromatography-Tandem Mass Spectrometry (Multiple Reaction Monitoring) Coupling and Correlation with ELISA Tests

Proteomics discovery leads to a list of potential protein biomarkers that have to be subsequently verified and validated with a statistically viable number of patients. Although the most sensitive, the development of an ELISA test is time-consuming when antibodies are not available and need to be conceived. Mass spectrometry analysis driven in quantitative multiple reaction monitoring mode is now appearing as a promising alternative to quantify proteins in biological fluids. However, all the studies published to date describe limits of quantitation in the low μg/ml range when no immunoenrichment of the target protein is applied, whereas the concentration of known clinical biomarkers is usually in the ng/ml range. Using prostate-specific antigen as a model biomarker, we now provide proof of principle that mass spectrometry enables protein quantitation in a concentration range of clinical interest without immunoenrichment. We have developed and optimized a robust sample processing method combining albumin depletion, trypsin digestion, and solid phase extraction of the proteotypic peptides starting from only 100 μl of serum. For analysis, mass spectrometry was coupled to a conventional liquid chromatography system using a 2-mm-internal diameter reverse phase column. This mass spectrometry-based strategy was applied to the quantitation of prostate-specific antigen in sera of patients with either benign prostate hyperplasia or prostate cancer. The quantitation was performed against an external calibration curve by interpolation, and results showed good correlation with existing ELISA tests applied to the same samples. This strategy might now be implemented in any clinical laboratory or certified company for further evaluation of any putative biomarker in the low ng/ml range of serum or plasma.Used for years across a wide range of pathologies, proteomics studies based on semiquantitative mass spectrometry of proteins have already led to the discovery of numerous protein biomarker candidates. Often tens of putative biomarkers have been described for a single disease, but the subsequent phase of clinical evaluation on large cohorts for each candidate is clearly the bottleneck as revealed by the meager number of newly approved biomarkers for clinical use. One of the critical limitations of discovery work flows arises when no antibody is available to initiate an immunoassay because 1–3 years are required to conceive de novo a reliable immunoassay. Such a delay is a serious drawback when tens of putative markers are concerned. Quantitation of small organic molecules by mass spectrometry has been used extensively for years in the field of environmental contaminant analysis or pharmacokinetic profiling of drug candidates during clinical studies. More recently, absolute quantitation of proteins using mass spectrometry by single (SRM)¹ or multiple reaction monitoring (MRM) and stable isotope dilution has thus naturally emerged as an alternative to immunoassays. Basically the absolute quantitation of a protein is provided by the integration of the specific MRM signals corresponding, respectively, to a proteotypic peptide (1) obtained from enzymatic hydrolysis of the target protein (usually by trypsin) and to its synthetic stable isotope-labeled isotopomer (2). The validation criteria of an MS-based method in terms of accuracy and precision are relatively easy to fulfill when addressing small molecules or proteins below 10 kDa in plasma or serum. Indeed they may be easily extracted from the bulk of high molecular mass proteins simply by selective precipitation. The quantitation of higher molecular mass proteins has proven to be more challenging because of the complexity and large dynamic range of proteins in e.g. plasma. In a pioneering study Anderson and Hunter (3) successfully demonstrated generation of a multiplexed assay for proteins covering high (tens to hundreds of micrograms/milliliter) to medium (hundreds of nanograms/milliliter to a few micrograms/milliliter) abundance ranges in plasma when combined with immunodepletion. However, these ranges remain problematic because clinically relevant biomarkers are usually present in plasma or serum in the low nanogram/milliliter range or below. To significantly improve the limit of quantitation (LOQ) of LC-MRM mass spectrometry, Keshishian et al. (4) evaluated a combination of immunodepletion of the most abundant plasma or serum proteins with strong cation exchange (SCX) chromatography for sample preparation prior to LC-MRM analysis. LOQs in the 1–10 ng/ml range were obtained with a coefficient of variation from 3 to 15% for five exogenous non-human proteins and the human prostate-specific antigen (PSA) protein spiked together into immunodepleted plasma from a healthy female donor. Very recently, a new approach using product ion scanning on a linear ion trap was proposed by Diamandis and co-workers (5) that allowed reaching a limit of quantitation of 1 ng/ml for PSA spiked into control plasma. This study marked a gain in sensitivity compared with previous attempts by others on similar instrumentation (6–9) but applied immunopurification of the target protein.Interestingly all the strategies published to date have been dealing with analytical development of work flows for the validation of biomarker candidates using microbore, nanoflow chromatography. Nanoflow is without any doubt appealing over conventional microflow during the proteomics discovery phase when the amount of biological material, for instance from a tumor biopsy, is often limited. Nonetheless this technique inherently still suffers from a lack of robustness and requires skilled personnel to be operational on a daily basis. As a consequence, nanoflow chromatography is not easily adaptable for the high throughput analysis environment encountered in clinical laboratories or good laboratory practice-certified or contract research organization companies where hundreds of samples are handled per day. In such organizations only microflow separations using 1- or 2-mm-internal diameter HPLC columns are compatible with the requirements of robustness and sample throughput.Therefore, the present work was centered on how a simple work flow could, in the near future, enable the large scale verification phase of putative biomarkers in the ng/ml of plasma range by the use of conventional LC equipment, i.e. using a 2-mm-internal diameter HPLC column. To address this question, we have considered that the absolute quantitation of PSA in true clinical samples could represent a challenging model. Combining immunodepletion of serum albumin and peptide fractionation simply by solid phase extraction (SPE), we were able to demonstrate for the first time the absolute quantitation of PSA by LC-MRM mass spectrometry in clinical serum samples of patients with benign prostate hyperplasia (BPH) or prostate cancer (PCa) within concentrations ranging from 4 to 30 ng/ml. Furthermore a good correlation was observed between the clinical ELISA tests and the mass spectrometry-based assays. We believe that these results are an unprecedented demonstration that the clinical relevance of putative biomarkers issued from proteomics investigation may now be confidently evaluated in the ng/ml range by robust coupling between conventional bore LC and mass spectrometry.     

High dynamic range characterization of the trauma patient plasma proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this "divide-and-conquer" strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3,654 different proteins with 1,494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 "classic" cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2,910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1,553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.     

P202-S Expanding the Capabilities of Peptide MRM-Based Assays in Plasma Using a Hybrid Triple-Quadrupole Linear Ion-Trap Mass Spectrometer

As the study of protein biomarkers increases in importance, technical limitations to the detection of low-abundance proteins and high-throughput, high-precision quantitation remain to be overcome. The complexity and dynamic range of the plasma proteome makes the task of specific, quantitative detection even more challenging. Multiple reaction monitoring (MRM) capabilities of triple quadrupole MS systems have been explored as solutions to this challenge due to their well-known sensitivity and selectivity for components in complex matrices such as plasma. Recently, a suite of >100 MRMs representing ~50 plasma protein markers were monitored quantitatively in a single assay using the MRM-based technique showing detection of proteins down to the level of L-selectin (~1μg/mL) with minimal sample preparation and no peptide or protein standards for most of the plasma protein markers.¹As more extensive candidate biomarker panels are being identified, MRM assays will need to be more rapidly developed to verify the expression changes of these proteins across larger clinical sample sets. To do this, the unique combination of triple-quadrupole and ion-trapping capabilities of the hybrid triple quadrupole–linear ion trap mass spectrometer have been utilized. A strategy for rapid MRM assay development for larger-scale profiling and qualification of biomarker candidates without having to first prepare synthetic peptide standards is currently being investigated and involves a chemical labeling strategy to create global reference standards to enable quantitative comparisons between clinical samples. Single assays consisting of ~500s of MRM transitions have been developed for this rapid qualification phase, facilitated by intelligent use of retention time windows during an LC analysis, while maintaining an optimum number of data points for improved precision of peak area and quantitative profiling. This presentation will demonstrate the details of this workflow with human plasma examples.     

Combining ultracentrifugation and peptide termini group-specific immunoprecipitation for multiplex plasma protein analysis

Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques.     

The Plasma Proteome: High Abundance versus Low Abundance

The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. To bridge this gap, targeted liquid chromatography (LC) tandem MS (MS/MS) analysis of tryptic peptide surrogates using selected reaction monitoring detection has emerged as a viable option for rapid quantification of target proteins. The precision of this approach has been enhanced by the use of stable isotope-labeled peptide internal standards to compensate for variation in recovery and the influence of differential matrix effects. Unfortunately, the complexity of proteinaceous matrices, such as plasma, limits the usefulness of this approach to quantification in the mid-nanogram per milliliter range (medium-abundance proteins). This article reviews the current status of LC/MS/MS using selected reaction monitoring for protein quantification, and specifically considers the use of a single antibody to achieve superior enrichment of either the protein target or the released tryptic peptide. Examples of immunoaffinity-assisted LC/MS/MS are reviewed that demonstrate quantitative analysis of low-abundance proteins (subnanogram per milliliter range). A strategy based on this technology is proposed for the expedited evaluation of novel protein biomarkers, which relies on the synergy created from the complementary nature of MS and ELISA.     

Coupling immunoaffinity techniques with MS for quantitative analysis of low-abundance protein biomarkers

The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. To bridge this gap, targeted liquid chromatography (LC) tandem MS (MS/MS) analysis of tryptic peptide surrogates using selected reaction monitoring detection has emerged as a viable option for rapid quantification of target proteins. The precision of this approach has been enhanced by the use of stable isotope-labeled peptide internal standards to compensate for variation in recovery and the influence of differential matrix effects. Unfortunately, the complexity of proteinaceous matrices, such as plasma, limits the usefulness of this approach to quantification in the mid-nanogram per milliliter range (medium-abundance proteins). This article reviews the current status of LC/MS/MS using selected reaction monitoring for protein quantification, and specifically considers the use of a single antibody to achieve superior enrichment of either the protein target or the released tryptic peptide. Examples of immunoaffinity-assisted LC/MS/MS are reviewed that demonstrate quantitative analysis of low-abundance proteins (subnanogram per milliliter range). A strategy based on this technology is proposed for the expedited evaluation of novel protein biomarkers, which relies on the synergy created from the complementary nature of MS and ELISA.     

Enhanced detection of low abundance human plasma proteins using a tandem IgY12-SuperMix immunoaffinity separation strategy

The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MS-based proteomics technologies as it makes it especially difficult to detect low abundance proteins in human biofluids such as blood plasma, which is an essential aspect for successful biomarker discovery efforts. Here we present a novel tandem IgY12-SuperMix immunoaffinity separation system for enhanced detection of low abundance proteins in human plasma. The tandem IgY12-SuperMix system separates approximately 60 abundant proteins from the low abundance proteins in plasma, allowing for significant enrichment of low abundance plasma proteins in the SuperMix flow-through fraction. High reproducibility of the tandem separations was observed in terms of both sample processing recovery and LC-MS/MS identification results based on spectral count data. The ability to quantitatively measure differential protein abundances following application of the tandem separations was demonstrated by spiking six non-human standard proteins at three different levels into plasma. A side-by-side comparison between the SuperMix flow-through and IgY12 flow-through samples analyzed by both one- and two-dimensional LC-MS/MS revealed a 60-80% increase in proteome coverage as a result of the SuperMix separations, suggesting significantly enhanced detection of low abundance proteins. A total of 695 plasma proteins were confidently identified in a single analysis (with a minimum of two peptides per protein) by coupling the tandem separation strategy with two-dimensional LC-MS/MS, including 42 proteins with reported normal concentrations of approximately 100 pg/ml to 100 ng/ml. The concentrations of two selected proteins, macrophage colony-stimulating factor 1 and matrix metalloproteinase-8, were independently validated by ELISA as 202 pg/ml and 12.4 ng/ml, respectively. Evaluation of binding efficiency revealed that 45 medium abundance proteins were efficiently captured by the SuperMix column with >90% retention. Taken together, these results illustrate the potential broad utilities of this tandem IgY12-SuperMix strategy for proteomics applications involving human biofluids where effectively addressing the dynamic range challenge of the specimen is imperative.     

The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins

Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.     

A quantitative proteomic approach to prion disease biomarker research: delving into the glycoproteome

Mass spectrometry (MS) -- based proteomic approaches have evolved as powerful tools for the discovery of biomarkers. However, the identification of potential protein biomarkers from biofluid samples is challenging because of the limited dynamic range of detection. Currently there is a lack of sensitive and reliable premortem diagnostic test for prion diseases. Here, we describe the use of a combined MS-based approach for biomarker discovery in prion diseases from mouse plasma samples. To overcome the limited dynamic range of detection and sample complexity of plasma samples, we used lectin affinity chromatography and multidimensional separations to enrich and isolate glycoproteins at low abundance. Relative quantitation of a panel of proteins was obtained by a combination of isotopic labeling and validated by spectral counting. Overall 708 proteins were identified, 53 of which showed more than 2-fold increase in concentration whereas 58 exhibited more than 2-fold decrease. A few of the potential candidate markers were previously associated with prion or other neurodegenerative diseases.     

Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma

Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ([¹³C₆]Arg or [¹³C₆]Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.MS is capable of sensitive and accurate protein quantitation based on the quantitation of proteolytic peptides as surrogates for the corresponding intact proteins. Over the past 10 years, MS-based protein quantitation based on the analysis of peptides (in other words, based on “bottom-up” proteomics) has had a profound impact on how biological problems can be addressed (1, 2). Although advances in MS instrumentation have contributed to the improvement of MS-based protein quantitation, the use of stable isotopes in quantitative work flows has arguably had the greatest impact in improving the quality and reproducibility of MS-based protein quantitation (3–5).The ongoing development of untargeted MS-based quantitation work flows has focused on increasingly exhaustive sample prefractionation methods, at both the protein and peptide levels, with the goal of detecting and quantifying entire proteomes (6). Although untargeted MS-based quantitation work flows have their utility, they are costly in terms of lengthy MS data acquisition and analysis times, and as a result, they are often limited to quantifying differences between small sample sets (n < 10). To facilitate rapid quantitation of larger, clinically relevant sample sets (n > 100) there is a need to both simplify sample preparation and reduce MS analysis time.Multiple reaction monitoring (MRM)¹ is a tandem MS (MS/MS) scan mode unique to triple quadrupole MS instrumentation that is capable of rapid, sensitive, and specific quantitation of analytes in highly complex sample matrices (7). MRM is a targeted approach that requires knowledge of the molecular weight of an analyte and its fragmentation behavior under CID. MRM is capable of highly reproducible concentration determination when stable isotope-labeled internal standards are included in work flows and has been used for decades for the quantitation of low molecular mass analytes (<1000 Da) in pharmaceutical, clinical, and environmental applications (7, 8).The combination of triple quadrupole MS instrumentation with nanoliter flow rate high performance LC and nanoelectrospray ionization provides the necessary sensitivity for detection and quantitation of biological molecules such as peptides in complex samples such as plasma by MRM. When combined with the use of isotopically labeled synthetic peptide standards, MRM analysis is capable of sensitive (attomole level) and absolute determination of peptide concentrations across a wide concentration scale spanning a dynamic range of 10³–10⁴ (1, 9–13).Several recent studies involving MRM-based analysis of plasma proteins have focused on increasing MRM detection sensitivity by fractionating plasma using either multidimensional liquid chromatography, affinity depletion of high abundance proteins (11, 14, 15), or affinity enrichment of low abundance peptides (16, 17). Anderson and Hunter (14) have shown that LC-MRM/MS analysis is capable of detecting 47 moderate to high abundance proteins in plasma without depletion even though ∼90% of the total protein by weight in trypsin-digested plasma can be attributed to 10 high abundance proteins (18).Relative abundance of a protein does not preclude its involvement in disease. In fact, 32 of the 47 plasma proteins detected by Anderson and Hunter (14) have been implicated as putative markers for cardiovascular disease. The ability to rapidly quantify proteins in a highly multiplexed manner using MRM and internal standard peptides expands the potential application of MRM quantitation beyond biomarker validation and into the field of biomarker discovery. Targeted, simultaneous quantitation of hundreds of proteins in a single analysis will enable rapid protein expression profiling of large (n > 100) clinically relevant sample sets in a manner similar to DNA microarray expression profiling. By allowing researchers to look at patterns of expression levels of a large number of proteins in a large number of samples (as opposed to looking at the expression levels of only a single protein), multiplexed MRM-based quantitation will allow the correlation of expression patterns with particular diseases. Once these characteristic patterns have been established, physicians will be able to use these protein expression patterns to diagnose diseases in the same way they currently use blood chemistry panels or comprehensive metabolic panels.When considering the clinical utility of MS-based assays, direct comparisons are often made to ELISA, which is considered the “gold standard” for protein quantitation in clinical samples. Attributes of ELISAs, such as “time to first result” (1–2 h (19)) and the ability to quantify 96 or 384 samples in parallel because of their microtiter plate-based format, are currently difficult to match with MS-based protein assays. However, MRM protein assays may surpass ELISA in the rapid development of clinically useful, multiplexed protein assays. The impact of multiplexed assays in the field of genomics has increased interest in multiplexed quantitation of many proteins in individual clinical samples (19). Development and characterization of MRM-based protein assays using isotopically labeled peptides is rapid and inexpensive compared with the time and cost associated with the generation and characterization of antibodies for ELISA development.In this study, we describe the creation of a customizable mixture of concentration-balanced stable isotope-labeled standard (SIS) peptides representing an initial panel of 45 human plasma proteins. We used this mixture of SIS peptides to develop a suite of multiplexed, rapid, and reproducible MRM-based assays for expression profiling of these 45 proteins in simple tryptic digests of whole plasma. Additionally we characterized the analytical performance of these MRM peptide assays with respect to their reproducibility, and we demonstrated their utility for absolute protein concentration determination.Multiplexed MRM quantitation of peptides for protein quantitation has the potential to replace iTRAQ or other isotope label and label-free quantitative proteomics approaches because the approach is much faster than these other methods (30–60 min per analysis compared with 4 days for LC-MALDI-based iTRAQ), has greater reproducibility (CV <5% versus iTRAQ CV >20%), and enables absolute quantitation (concentration and copy number versus only x-fold up- or down-regulated). Additionally MRM-based quantitation with SIS peptides does not “miss” peptides because the SIS peptide must be detected in every sample: this means that if an endogenous peptide is not observed then it is below the limit of detection.     

Multiplexed LC‐MS/MS analysis of horse plasma proteins to study doping in sport

The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC‐MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race‐horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis‐driven discovery of doping biomarker candidates.     

Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome

In order to discover novel protein markers indicative of disease processes or drug effects, the proteomics technology platform most commonly used consists of high resolution protein separation by two-dimensional electrophoresis (2-DE), mass spectrometric identification of proteins from stained gel spots and a bioinformatic data analysis process supported by statistics. This approach has been more successful in profiling proteins and their disease- or treatment-related quantitative changes in tissue homogenates than in plasma samples. Plasma protein display and quantitation suffer from several disadvantages: very high abundance of a few proteins; high heterogeneity of many proteins resulting in long charge trains; crowding of 2-DE separated protein spots in the molecular mass range between 45-80 kD and in the isoelectric point range between 4.5 and 6. Therefore, proteomic technologies are needed that address these problems and particularly allow accurate quantitation of a larger number of less abundant proteins in plasma and other body fluids. The immunoaffinity-based protein subtraction chromatography (IASC) described here removes multiple proteins present in plasma and serum in high concentrations effectively and reproducibly. Applying IASC as an upfront plasma sample preparation process for 2-DE, the protein spot pattern observed in gels changes dramatically and at least 350 additional lower abundance proteins are visualized. Affinity-purified polyclonal antibodies (pAbs) are the immunoaffinity reagents used to specifically remove the abundant proteins such as albumin, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha-1-antitrypsin, hemopexin, transthyretin, alpha-2-HS glycoprotein, alpha-1-acid glycoprotein, alpha-2-macroglobulin and fibrinogen from human plasma samples. To render the immunoaffinity subtraction procedure recyclable, the pAbs are immobilized and cross-linked on chromatographic matrices. Antibody-coupled matrices specific for one protein each can be pooled to form mixed-bed IASC columns. We show that up to ten affinity-bound plasma proteins with similar solubility characteristics are eluted from a mixed-bed column in one step. This facilitates automated chromatographic processing of plasma samples in high throughput, which is desirable in proteomic disease marker discovery projects.