Development of protein microarray technology to monitor biomarkers of rheumatoid arthritis disease

Most biological processes are mediated by complex networks of molecular interactions involving proteins. The analysis of protein expression in biological samples is especially important in the identification and monitoring of biomarkers for disease progression and therapeutic endpoints. In this paper, the development of a protein microarray format for multiplexed quantitative analysis of several potential markers for rheumatoid arthritis (RA) is described. Development of a high-performance protein microarray system depends on several key parameters such as surface chemistry, capture agents, immobilization technology, and methods used for signal detection and quantification. Several technical possibilities were investigated and compared: poly-L-lysine versus self-assembled monolayer of octadecyl phosphoric acid ester for surface chemistries; noncontact piezoelectric versus contact printing technology for antibody deposition; CCD camera capture versus fluorescent scanning for image detection; and the concentration of coating antibody. On the basis of reproducibility, signal-to-noise ratio, and sensitivity we have selected self-assembled monolayer, noncontact piezoelectric printer, and high-read-out fluorescence scanning for our microarray format. This format was used to perform multiplexed quantitative analysis of several potential markers of disease progression of rheumatoid arthritis: IL-1, IL-6, IL-8, MCP-1, and SAA. Some assays, such as MCP-1, provided a working range that covered physiologically relevant concentrations. Other assays, such as IL-6 and SAA, lacked sensitivity or were too sensitive for measuring biological concentrations, respectively. The results described demonstrate the applicability of protein microarrays to monitor RA markers; however, sandwich assay methodologies need to be further optimized to measure the appropriate biological ranges of these markers on one chip.     

Multiplexed mass spectrometric immunoassay in biomarker research: a novel approach to the determination of a myocardial infarct

Reported here is the development of a multiplexed mass spectrometric immunoassay (MSIA) for the detection of myocardial infarction (MI). The assay is the product of a study that systematically progresses from biomarker discovery--to identification and verification--to assay design, data analysis, and statistical challenge. During targeted population proteomics investigations, two novel biomarkers, serum amyloid A1alpha and S-sulfated transthyretin, were found to be responsive to MI. These putative markers were subsequently screened in larger cohorts of individuals to verify their responsiveness toward MI. Upon verification, a multiplexed assay was designed that was capable of simultaneously monitoring the new markers plus a previously established MI-marker (myoglobin). The multiplexed MSIA was applied to two 96-sample sets comprised of 48-MI/48-healthy and 19-MI/77-healthy, which served as training and case cohorts, respectively. Data evaluation using either preset reference levels or multivariate analysis exhibited sensitivities and specificities of >97%. These findings illustrate the importance of using systematic approaches in clinical proteomics to discover biomarkers and produce high-performance assays relevant to disease.     

Anticoagulants used in plasma collection affect adipokine multiplexed measurements

Obesity is an important health problem worldwide. Adipose tissue acts as an endocrine organ that secretes various bioactive substances, called adipokines, including pro-inflammatory biomarkers such as TNF-α, IL-6, leptin and C-reactive protein (CRP) and anti-inflammatory molecules such as adiponectin. The deregulated production of adipokines in obesity is linked to the pathogenesis of various disease processes and monitoring their variation is critical to understand metabolic diseases.The aim of this study was to determine the plasma concentration of adipokines in healthy subjects by multiplexed measurements and the effect of anticoagulants on their levels.Plasma samples from 10 healthy donors were collected in two different anticoagulants (sodium citrate or heparin).All markers, excluding TNF-α, showed significantly higher concentrations in heparinized compared to citrate plasma. However, levels of adipokines in different plasma samples were highly correlated for most of these markers.We reported that different anticoagulants used in the preparation of the plasma samples affected the measurements of some adipokines. The importance of the present results in epidemiology is relevant when comparing different studies in which blood samples were collected with different anticoagulants.     

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.     

Fluorescent nanoparticles as labels for immunometric assay of C-reactive protein using two-photon excitation assay technology

We describe the use of fluorophore-doped nanoparticles as reporters in a recently developed ArcDia TPX bioaffinity assay technique. The ArcDia TPX technique is based on the use of polymer microspheres as solid-phase reaction carrier, fluorescent bioaffinity reagents, and detection of two-photon excited fluorescence. This new assay technique enables multiplexed, separation-free bioaffinity assays from microvolumes with high sensitivity. As a model analyte we chose C-reactive protein (CRP). The assay of CRP was optimized for assessment of CRP baseline levels using a nanoparticulate fluorescent reporter, 75 nm in diameter, and the assay performance was compared to that of CRP assay based on a molecular reporter of the same fluorophore core. The results show that using fluorescent nanoparticles as the reporter provides two orders of magnitude better sensitivity (87 fM) than using the molecular label, while no difference between precision profiles of the different assay types was found. The new assay method was applied for assessment of baseline levels of CRP in sera of apparently healthy individuals.     

Competitive, non-competitive, and mixed format cleavable tag immunoassays

Immunoassays are one of the most useful diagnostic techniques in disease assessment, drug metabolite analysis, and environmental applications due largely in part to the selectivity and sensitivity provided by antibody-antigen interactions. Here, a multiplexed immunoassay termed cleavable tag immunoassay (CTI) was performed in competitive, non-competitive, and mixed formats for the analysis of proteins and small molecule biomarkers of inflammation and tissue damage. Microchip capillary electrophoresis (MCE) with fluorescence detection was employed for the analysis of fluorescently labeled tags corresponding to the analytes of interest cleaved from the detection antibodies. For this work we have selected 3-nitrotyrosine (3-NT) a molecule indicative of reactive nitrogen species (RNS), thyroxine (T4) a molecule used to monitor thyroid gland function, and C-reactive protein (CRP) a marker of chronic inflammation as model analytes to demonstrate the assay principles. The simultaneous detection of 3-nitrotyrosine (3-NT) and thyroxine (T4) was carried out as a proof-of-principle for the competitive CTI while non-competitive CTI performance was demonstrated via the analysis of C-reactive protein (CRP). Limit of detections (LOD) and dynamic ranges were investigated. LOD for 3-NT, T4, and CRP were 0.5μg/mL, 23nM, and 5μg/mL, respectively thus demonstrating the ability of the CTI to detect proteins and small molecules within clinical reference ranges. Moreover, this is the first report of the use of mixed format CTI chemistry for the simultaneous detection of proteins (CRP) and small molecules (3-NT) in a single assay. The success of this work demonstrates the ability of CTI to analyze intact proteins and small molecule biomarkers simultaneously.     

Development of a bead-based aptamer/antibody detection system for C-reactive protein

A multiplexing bead-based platform provides an approach for the development of assays targeting specific analytes for biomonitoring and biosensing applications. Multi-Analyte Profiling (xMAP) assays typically employ a sandwich-type format using antibodies for the capture and detection of analytes of interest, and the system permits the simultaneous quantitation of multiple targets. In this study, an aptamer/antibody assay for the detection of C-reactive protein (CRP) was developed. CRP is an acute phase marker of inflammation whose elevated basal levels are correlated with an increased risk for a number of pathologies. For this assay, an RNA aptamer that binds CRP was conjugated to beads to act as the capture agent. Biotinylated anti-CRP antibody coupled to fluorescently labeled streptavidin was used for quantification of CRP. The detection limit of the CRP assay was 0.4 mg/L in diluted serum. The assay was then used to detect spiked CRP samples in the range of 0.4 to 10 mg/L in diluted serum with acceptable recoveries (extrapolated values of 70–130%), including that of a certified reference material (129% recovery). The successful incorporation of the CRP aptamer into this platform demonstrates that the exploration of other aptamer–target systems could increase the number of analytes measurable using xMAP-type assays.     

A targeted proteomics-based pipeline for verification of biomarkers in plasma

High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.     

An Automated and Multiplexed Method for High Throughput Peptide Immunoaffinity Enrichment and Multiple Reaction Monitoring Mass Spectrometry-based Quantification of Protein Biomarkers

There is an urgent need for quantitative assays in verifying and validating the large numbers of protein biomarker candidates produced in modern “-omics” experiments. Stable isotope standards with capture by anti-peptide antibodies (SISCAPA) has shown tremendous potential to meet this need by combining peptide immunoaffinity enrichment with quantitative mass spectrometry. In this study, we describe three significant advances to the SISCAPA technique. First, we develop a method for an automated magnetic bead-based platform capable of high throughput processing. Second, we implement the automated method in a multiplexed SISCAPA assay (nine targets in one assay) and assess the performance characteristics of the multiplexed assay. Using the automated, multiplexed platform, we demonstrate detection limits in the physiologically relevant ng/ml range (from 10 μl of plasma) with sufficient precision (median coefficient of variation, 12.6%) for quantifying biomarkers. Third, we demonstrate that enrichment of peptides from larger volumes of plasma (1 ml) can extend the limits of detection to the low pg/ml range of protein concentration. The method is generally applicable to any protein or biological specimen of interest and holds great promise for analyzing large numbers of biomarker candidates.The current gold standard for quantifying protein biomarkers is the ELISA. A well functioning ELISA can be run at high throughput and has excellent sensitivity; however, the cost associated with development is very high, the lead time is very long, and the failure rate can be high. In addition, sandwich immunoassays are subject to potential interference from endogenous antibodies (1). Unfortunately, there are no quantitative assays available for the majority of biomarker candidates, and a considerable investment is required to generate assays de novo, creating a bottleneck in the biomarker pipeline (2, 3).A technique that has shown potential for bridging the gap between discovery and validation of biomarkers is stable isotope standards with capture by anti-peptide antibodies (SISCAPA)¹ (4) coupled to multiple reaction monitoring (MRM) MS. SISCAPA has several advantages over other immunoassays in that the mass spectrometer provides excellent specificity for the analyte of interest; the sample (including endogenous immunoglobulins) is digested to peptides, avoiding potential interference from endogenous antibodies; and precise, relative quantification is possible via the use of an internal standard. Additionally, although it is very difficult to combine multiple analytes into one assay (i.e. multiplex) using ELISAs, SISCAPA assays can in theory be highly multiplexed as many analytes can be measured from a single enrichment step. To date, individual SISCAPA assays have been successfully configured to a number of analytes (4–9), and up to three peptides have been enriched simultaneously (7, 8). In this study, we sought to advance the utility of SISCAPA for testing large numbers of biomarker candidates in large numbers of patient samples by automating the method to improve throughput and performance, testing the performance of multiplexing analytes, and improving sensitivity.     

Multiplexed MRM‐based quantitation of candidate cancer biomarker proteins in undepleted and non‐enriched human plasma

An emerging approach for multiplexed targeted proteomics involves bottom‐up LC‐MRM‐MS, with stable isotope‐labeled internal standard peptides, to accurately quantitate panels of putative disease biomarkers in biofluids. In this paper, we used this approach to quantitate 27 candidate cancer‐biomarker proteins in human plasma that had not been treated by immunoaffinity depletion or enrichment techniques. These proteins have been reported as biomarkers for a variety of human cancers, from laryngeal to ovarian, with breast cancer having the highest correlation. We implemented measures to minimize the analytical variability, improve the quantitative accuracy, and increase the feasibility and applicability of this MRM‐based method. We have demonstrated excellent retention time reproducibility (median interday CV: 0.08%) and signal stability (median interday CV: 4.5% for the analytical platform and 6.1% for the bottom‐up workflow) for the 27 biomarker proteins (represented by 57 interference‐free peptides). The linear dynamic range for the MRM assays spanned four orders‐of‐magnitude, with 25 assays covering a 10³–10⁴ range in protein concentration. The lowest abundance quantifiable protein in our biomarker panel was insulin‐like growth factor 1 (calculated concentration: 127 ng/mL). Overall, the analytical performance of this assay demonstrates high robustness and sensitivity, and provides the necessary throughput and multiplexing capabilities required to verify and validate cancer‐associated protein biomarker panels in human plasma, prior to clinical use.     

Relative quantification of serum proteins from pancreatic ductal adenocarcinoma patients by stable isotope dilution liquid chromatography-mass spectrometry

We report an innovative multiplexed liquidchromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS)-based assay for rapidly measuring a large number of disease specific protein biomarkers in human serum. Furthermore, this approach uses stable isotope dilution methodology to reliably quantify candidate protein biomarkers. Human serum was diluted using a stable isotope labeled proteome (SILAP) standard prepared from the secretome of pancreatic cell lines, subjected to immunoaffinity removal of the most highly abundant proteins, trypsin digested, and analyzed by LC-MRM/MS. The method was found to be precise, linear, and specific for the relative quantification of 72 proteins when analyte response was normalized to the relevant internal standard (IS) from the SILAP. The method made it possible to determine statistically different concentrations for three proteins (cystatin M, IGF binding protein 7, and villin 2) in control and pancreatic cancer patient samples. This method proves the feasibility of using a SILAP standard in combination with stable isotope dilution LC-MRM/MS analysis of tryptic peptides to compare changes in the concentration of candidate protein biomarkers in human serum.     

Rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics

A significant challenge for all biosensor systems is to achieve high assay sensitivity and specificity while minimizing sample preparation requirements, operational complexity, and sample-to-answer time. We have achieved multiplexed, unamplified, femtomolar detection of both DNA and proteins in complex matrices (including whole blood, serum, plasma, and milk) in minutes using as few as two reagents by labeling conventional assay schemes with micrometer-scale magnetic beads, and applying fluidic force discrimination (FFD). In FFD assays, analytes captured onto a microarray surface are labeled with microbeads, and a controlled laminar flow is then used to apply microfluidic forces sufficient to preferentially remove only nonspecifically bound bead labels. The density of beads that remain bound is proportional to the analyte concentration and can be determined with either optical counting or magnetoelectronic detection of the magnetic labels. Combining FFD assays with chip-based magnetoelectronic detection enables a simple, potentially handheld, platform capable of both nucleic acid hybridization assays and immunoassays, including orthogonal detection and identification of bacterial and viral pathogens, and therefore suitable for a wide range of biosensing applications.     

Microfluidic electrochemical immunoarray for ultrasensitive detection of two cancer biomarker proteins in serum

A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL?1 levels. MPs were conjugated with ～90,000 antibodies and ～200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL?1 for PSA and 0.30 pg mL?1 for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers.     

An optimised electrochemical biosensor for the label-free detection of C-reactive protein in blood

C-reactive protein (CRP) is an acute phase protein whose levels are increased in many disorders. There exists, in particular, a great deal of interest in the correlation between blood serum levels and the severity of risk for cardiovascular disease. A sensitive, label-free, non-amplified and reusable electrochemical impedimetric biosensor for the detection of CRP in blood serum was developed herein based on controlled and coverage optimised antibody immobilization on standard polycrystalline gold electrodes. Charge transfer resistance changes were highly target specific, linear with logCRPconcentration across a 0.5-50nM range and associated with a limit of detection of 176pM. Significantly, the detection limits are better than those of current CRP clinical methods and the assays are potentially cheap, relatively automated, reusable, multiplexed and highly portable. The generated interfaces were capable not only of comfortably quantifying CRP across a clinically relevant range of concentrations but also of doing this in whole blood serum with interfaces that were, subsequently, reusable. The importance of optimising receptor layer resistance in maximising assay sensitivity is also detailed.     

A Multiplex Immunoassay Method for Simultaneous Quantification of Iron,Vitamin A and Inflammation Status Markers

Deficiencies of vitamin A and iron affect a significant portion of the world''s population, and efforts to characterize patterns of these deficiencies are hampered by a lack of measurement tools appropriate for large-scale population-based surveys. Vitamin A and iron are not easily measured directly, so reliable proxy markers for deficiency status have been identified and adopted. Measurement of inflammatory markers is necessary to interpret vitamin A and iron status markers, because circulating levels are altered by inflammation. We developed a multiplex immunoassay method for simultaneous measurement of five markers relevant to assessing inflammation, vitamin A and iron status: α-1-acid glycoprotein, C-reactive protein, retinol binding protein 4, ferritin and soluble transferrin receptor. Serum and plasma specimens were used to optimize the assay protocol. To evaluate assay performance, plasma from 72 volunteers was assayed using the multiplex technique and compared to conventional immunoassay methods for each of the five markers. Results of the new and conventional assay methods were highly correlated (Pearson Correlations of 0.606 to 0.991, p<.0001). Inter-assay imprecision for the multiplex panel varied from 1% to 8%, and all samples fell within the limits of quantification for all assays at a single dilution. Absolute values given by the multiplex and conventional assays differed, indicating a need for further work to devise a new standard curve. This multiplexed micronutrient immunoassay technique has excellent potential as a cost effective tool for use in large-scale deficiency assessment efforts.     

Nano-bio-chips for high performance multiplexed protein detection: Determinations of cancer biomarkers in serum and saliva using quantum dot bioconjugate labels

The integration of semiconductor nanoparticle quantum dots (QDs) into a modular, microfluidic biosensor for the multiplexed quantitation of three important cancer markers, carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), and Her-2/Neu (C-erbB-2) was achieved. The functionality of the integrated sample processing, analyte capture and detection modalities was demonstrated using both serum and whole saliva specimens. Here, nano-bio-chips that employed a fluorescence transduction signal with QD-labeled detecting antibody were used in combination with antigen capture by a microporous agarose bead array supported within a microfluidics ensemble so as to complete the sandwich-type immunoassay. The utilization of QD probes in this miniaturized biosensor format resulted in signal amplification 30 times relative to that of standard molecular fluorophores as well as affording a reduction in observed limits of detection by nearly 2 orders of magnitude (0.02 ng/mL CEA; 0.11 pM CEA) relative to enzyme-linked immunosorbent assay (ELISA). Assay validation studies indicate that measurements by the nano-bio-chip system correlate to standard methods at R² = 0.94 and R² = 0.95 for saliva and serum, respectively. This integrated nano-bio-chip assay system, in tandem with next-generation fluorophores, promises to be a sensitive, multiplexed tool for important diagnostic and prognostic applications.     

Multiplexed quantification of estrogen receptor and HER2/Neu in tissue and cell lysates by peptide immunoaffinity enrichment mass spectrometry

Access to a wider range of quantitative protein assays would significantly impact the number and use of tissue markers in guiding disease treatment. Quantitative mass spectrometry-based peptide and protein assays, such as immuno-SRM assays, have seen tremendous growth in recent years in application to protein quantification in biological fluids such as plasma or urine. Here, we extend the capability of the technique by demonstrating the application of a multiplexed immuno-SRM assay for quantification of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) levels in cell line lysates and human surgical specimens. The performance of the assay was characterized using peptide response curves, with linear ranges covering approximately four orders of magnitude and limits of detection in the low fmol/mg lysate range. Reproducibility was acceptable with median coefficients of variation of approximately 10%. We applied the assay to measurements of ER and HER2 in well-characterized cell line lysates with good discernment based on ER/HER2 status. Finally, the proteins were measured in surgically resected breast cancers, and the results showed good correlation with ER/HER2 status determined by clinical assays. This is the first implementation of the peptide-based immuno-SRM assay technology in cell lysates and human surgical specimens.     

Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 μl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.