- Open Access
Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics
Journal of Translational Medicine volume 13, Article number: 182 (2015)
There is an emerging demand for the use of molecular profiling to facilitate biomarker identification and development, and to stratify patients for more efficient treatment decisions with reduced adverse effects. In the past decade, great strides have been made to advance genomic, transcriptomic and proteomic approaches to address these demands. While there has been much progress with these large scale approaches, profiling at the protein level still faces challenges due to limitations in clinical sample size, poor reproducibility, unreliable quantitation, and lack of assay robustness. A novel automated capillary nano-immunoassay (CNIA) technology has been developed. This technology offers precise and accurate measurement of proteins and their post-translational modifications using either charge-based or size-based separation formats. The system not only uses ultralow nanogram levels of protein but also allows multi-analyte analysis using a parallel single-analyte format for increased sensitivity and specificity. The high sensitivity and excellent reproducibility of this technology make it particularly powerful for analysis of clinical samples. Furthermore, the system can distinguish and detect specific protein post-translational modifications that conventional Western blot and other immunoassays cannot easily capture. This review will summarize and evaluate the latest progress to optimize the CNIA system for comprehensive, quantitative protein and signaling event characterization. It will also discuss how the technology has been successfully applied in both discovery research and clinical studies, for signaling pathway dissection, proteomic biomarker assessment, targeted treatment evaluation and quantitative proteomic analysis. Lastly, a comparison of this novel system with other conventional immuno-assay platforms is performed.
Advances in the molecular analysis of genes, proteins and metabolites have greatly improved our understanding of biological processes and disease, and have increased our ability to monitor treatment response and stratify patients to improve treatment efficacy. Precision medicine facilitated by companion diagnostics is one of the driving forces accelerating the drug development process and improving therapeutic management . For example, targeting HER2 over-expression for breast cancer treatment led to the development of Herceptin (trastuzumab, Genentech), the first approved monoclonal antibody drug . Another well known example is the use of a K-ras mutation test as a predictor of poor response to EGFR inhibitor treatment .
To enable this type of molecular profiling, concerted effort has been put forth in both academic and industry sectors to develop and validate new technologies. In the past decade, many genome and transcriptome profiling technologies, including next-generation sequencing, single-nucleotide polymorphism (SNP), fluorescent in situ hybridization (FISH), and mRNA quantitation technologies, have been developed and successfully applied in areas from discovery and translational research to clinical practice [4–8]. However, DNA sequence information is not sufficient to predict the biological function of a protein, and gene expression does not always correlate with pathway activation level [9, 10]. Based on UniProt, there is no experimental evidence at the protein level for about 38% out of 20,000 protein-coding human genes . In a study of correlation between protein and mRNA abundance in yeast , Gygi et al. found that for some genes, while the mRNA levels were about the same, the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Post-translation modifications (PTM) of proteins, such as phosphorylation, acetylation and glycosylation, can alter a protein’s activity and affect disease development and treatment response [13–15]. Thus it is critical to directly measure proteins and their activation status to confirm genomic predictions and monitor treatment responses.
In contrast to major advances in DNA and RNA profiling, the investigation of signaling molecules at the protein level still faces many challenges, such as limited clinical sample size, poor reproducibility, unreliable quantitation, and difficulties in protocol standardization across different laboratories [16, 17]. There is a strong need to develop automated, high-throughput proteomic technologies for precise and accurate determination of protein levels and analysis of protein activation status, to facilitate both discovery research and clinical practice.
In this manuscript, we review the performance and applications of a novel capillary nano-immunoassay (CNIA) system (The Simple Western System™, marketed by ProteinSimple, CA, USA), a fully automated capillary electrophoresis system for characterization of proteins and their post-translational modifications. This new system overcomes many of the limitations of conventional proteomic approaches: it offers straightforward target-specific detection, easy operation, high quality data quantitation and excellent assay reproducibility using nanogram levels of sample. As discussed below, this technology has been successfully applied in the dissection of signaling pathways, assessment of proteomic biomarkers, and evaluation of targeted therapies.
The CNIA technology
Capillary nano-immunoassay (also named the Simple Western™ system by the manufacturer, Protein Simple) refers to a capillary-electrophoresis immunoassay system, which offers both size-based and charge-based separation formats. Depending on the process, samples are prepared with SDS-containing buffers (for size-based assay) or solution-phase carrier ampholytes (for charge-based assay) and automatically loaded into small (5 cm, 100 µm internal diameter) capillaries for electrophoretic separation. The CNIA system offer both size-based and charge-based separation formats. Figure 1 shows the assay scheme.
The size-based assay (size-CNIA) separates proteins based on molecular weight. The assay is performed in a similar manner to an SDS-PAGE gel. The protein sample is denatured by heat in the presence of a reducing agent (DTT) and is coated with SDS. The SDS coating gives the proteins in the sample a uniform charge-to-mass ratio. Because the proteins have identical charge-to-mass ratio, each protein’s velocity through the polymer matrix will depend solely on the pores in the polymer matrix and the protein’s radius of gyration, which is proportional to molecular weight.
The charge-based assay (charge-CNIA) differs from a traditional iso-electric focusing (IEF) gel in that the pH gradient is in the solution phase. An “ampholyte gradient” consisting of hundreds of zwitterionic species whose isoelectric points cover a range of pH is added to the sample mixture. When the electric field is applied, the ampholytes arrange themselves according to isoelectric-point (pI) and form a pH gradient throughout the length of the capillary. The proteins and standards migrate through the gradient until they reach their pI, at which point they carry no net charge and cease to migrate. The charge-based assay is also historically referred to as NanoPro™ referring to the company-specific platform or generically as NIA (nanofluidic proteomic immunoassay) assays in the literature.
Electrophoresis in either format is followed by UV light cross-linking of the separated proteins to the capillary wall through proprietary chemistry . The capillaries are then automatically shuttled to an adjacent chamber within the instrument and the capillary lumen is loaded and incubated successively with primary and HRP-tagged secondary antibodies which are flowed through the capillary. The resulting chemiluminescence signal is then detected with a CCD camera, and the digital image is analyzed with Compass software (ProteinSimple, CA, USA). Signal strength is presented as peak area and quantitated. Each sample is prepared with fluorescent pI (for charge-CNIA) or molecular weight (for size-CNIA) markers to calibrate the signal peak profile. Once calibrated, peaks can then be compared between capillaries.
CNIA assay performance
This novel analysis system offers a number of significant advantages over conventional immunoblot techniques as discussed below.
Automation and robust performance
By performing all the steps of a charge- or size-based Western blot in a single vessel, the system allows for a level of automation and robust performance that is not possible with conventional immunoblot techniques. The capillaries are ferried between different stations in the instrument for separation/immobilization, antibody incubation, detection, washing and reagent loading. This eliminates all handling during the run and the inconsistencies that can arise from manual intervention. Good intra-, inter-assay reproducibility has been reported with the system . Figure 2a shows an analysis of ERK1 performed by different analysts on different days. An average %CV (coefficient of variation) of less than 10% was observed in this example.
Sensitivity, dynamic range, turnaround time, and data quantitation
By taking advantage of the economies of scale in microfluidics, detection sensitivity is much greater than that of other techniques, with results reported on samples corresponding to fewer than 50 cells .Typical CNIA runs load nanogram rather than microgram amount of total protein, and have been reported able to precisely measure sub-femtomole amount of target protein . The CCD camera provides 10 times higher dynamic range measurement than film . Analysis linearity over several orders of magnitude has been reported for the system [18, 21–23]. In the 96-capillary throughput analysis platforms (Peggy™, Sally™), one cycle of 12-capillary analysis can be completed in about 2–5 h. A measurement run of up to eight cycles, for a total of 96 samples/analytes can be done in fewer than 24 h, demonstrating a fast assay turn-around time. Recently released instrumentation (Wes™) is capable of running 24 samples simultaneously in under 3 h (see manufacture’s website for more detail on different analysis platforms).
The automated operating system and digital data quantitation allows the CNIA system to provide quantitative and precise measurement of signaling molecules and their activation status [24–30]. The system has demonstrated more accurate and reproducible assessment of protein levels when compared with conventional Western blot analysis and has enabled better correspondence of protein level and function [31–35. In a study of TGF-β-mediated epithelial carcinogenesis , Kohn et al. found that Smad3 gene dosage regulates the biological responses to TGF-β. In this study, a mere two-fold reduction in Smad3 was confirmed by precise protein measurements using size-CNIA. This small reduction in protein levels was shown to be sufficient to promote metastasis. Using size-CNIA, Chen et al. developed a method for measuring the absolute amount of endogenous protein at the picogram or sub-picogram level per nanogram of cell lysate . The method provides an approach for precise and accurate assessment of protein levels to correlate with their functions in complex biological settings. The system was also demonstrated to provide quantifiable, consistent and reproducible data that are favorable for clinical translation .
With a through-put of 96 sample/analyte combinations in the Peggy™, Sally™ and NanoPro™ platforms of the CNIA system, multiple targets can be profiled simultaneously in one analysis run with low sample consumption. Multiple-analyte analysis in other assays, such as multiplex bead assays and antibody arrays, is constrained by analyte complexity and the potential impact of cross-reactivity. The CNIA system, however, bypasses these obstacles by performing successive cycles of single-analyte analysis each within their separate individual capillaries. This parallel approach thus offers an efficient method of ‘multiplex’ analysis with more accurate and reliable analysis data.
Assay development and the complementary nature of size-based and charge-based formats
The Size-CNIA assays can be used to detect any protein for which a good antibody is available. Detection of protein modifications is dependent on availability of an antibody specific for that modification. Assay transfer from conventional size-based Western blot to size-CNIA is relatively straight forward for most targets. Based on our experience with developing size-CNIA assays for about two hundred signaling molecule targets, more than 80% of the antibodies that work in conventional size-based Western blot also work in size-CNIA. We observed a good correspondence between conventional Western blot and size-CNIA for the quantitation of PKC isoform down-regulation in U937 cells treated with phorbol esters and bryostatins (Figure 2b) . In the charge-based configuration, using IEF as the separation mode allows for additional levels of information to be obtained. Phosphorylation and other post-translational modification differences in a protein population lead to “charge variants” which can be spatially resolved in IEF. This allows for the simultaneous detection and accurate relative quantitation of these protein variants using a single pan-specific antibody [39, 40]. The identity of the peaks/protein variants has been established experimentally using modification-specific antibodies, site-directed mutagenesis, mass spectrometry and other techniques. See later sections for detailed examples.
The charge-CNIA has been particularly successful in the quantitative analysis of ERK signaling. Using a single detection antibody, ERK1, ERK2 and their phospho-forms can be simultaneously identified, quantitated and compared. Figure 2c shows the IEF profile of ERK isoforms treated with different concentrations of phorbol-12-myristate-13-acetate (PMA). The changes in the dose response curves of individual ERK isoforms (i.e. the dual-phospho-ERK1/2, mono-phospho-ERK1/2, and non-phospho-ERK/1/2) in response to PMA treatment are presented as well. Visualization of the relative abundance of these forms at different concentrations of the drug provides more detail and specific information about signaling molecule activation than is captured by conventional immunoassays.
Using the IEF system to study protein kinase G-I isoform expression in human ovarian cells , Fiscus et al. demonstrated that the technology is more than 100 times more sensitive than the conventional Western blot in detecting low abundance PKG-I expressing cancer cells. The technology is also able to resolve and clearly identify the PKG-Iα and PKG-Iβ and the phosphorylation isoforms, which are not distinguishable in Western blot analysis.
The development of charge-CNIA assays may not be as straightforward as the size-CNIA assay and is highly dependent on the specific antibody. In charge-CNIA assay, proteins are analyzed in their native forms without being denatured and reduced as in size-based assays. However, the majority of commercially available antibodies are developed and characterized by recognizing epitopes on denatured proteins. In addition, the data profiles in the charge-CNIA assays, are generally highly complex, requiring multiple control samples and multiple antibodies to be screened before an assay can be confirmed to be specific to the target. Thus, while hundreds of size-CNIA assays have been developed in the past couple of years, the availability of validated charge-CNIA assays is still limited. Nevertheless, when well-validated antibodies are available, charge-CNIA assays are a uniquely powerful tool for generating information that is cumbersome or impossible to generate using other approaches. Antibodies developed for Human Protein Atlas project (http://www.proteinatlas.org/) has been reported may serve as a good source for charge-CNIA assay development .
The CNIA technology has been demonstrated to be ideal for comprehensive and quantitative protein characterization and profiling of signaling events. It holds great promise as a molecular diagnostic tool to facilitate precision medicine.
Charge-CNIA, a novel platform for detailed analysis of signaling pathways
As noted above, when there are charge variations among post-translationally-modified isoforms of the target protein, the IEF separation is able to separate and discriminate the different forms, and detect them without using antibodies specific to the modifications . This useful feature of the charge-CNIA has been exploited by investigators to discover novel post-translational modifications (PTM) in a number of signaling pathways and their association with important biological processes.
While there were no phospho-specific Pdx1 antibodies available at the time, Frogne et al. used charge CNIA with an anti-pan-Pdx1 antibody to show that Pdx1, a transcription factor for proper regulation of blood glucose homeostasis and pancreatic beta cell function, underwent post-translational modification in mammalian cells but not in bacterial cells . They further identified that one of the post-translationally-modified variants is unique to mature beta-cells only. Together with alanine scanning and mass spectrometric (MS) analysis, the group determined that serine 61 is the site of the most abundant phosphorylation on Pdx1. In another example, Huang et al. identified a new phosphorylation site on TRAF-interacting protein with an FHA domain (TIFA) that was stimulated by tumor necrosis factor alpha (TNF-α) . In conjunction with MS and in vitro kinase assay analysis, the phosphorylation site was confirmed to be threonine 9. The identification of this novel threonine phosphorylation site provided new insight into the TNF-α mediated signaling pathway, as well as a new functional mechanism for the FHA domain.
Others have used the charge-CNIA to provide novel insights about VEGF induced c-Src phosphorylation changes at pY418 and pY527 . Besides the overall VEGF induced signal change of pY418 and pY527 that is observed in Western blot, the assay also revealed multiple additional phosphorylation isoforms that respond differently to VEGF stimulation, and found that the c-Src pY418 might be a minor fraction of the total phosphorylated c-Src comparing with pY527. The charge-CNIA has also proved to be powerful in the analysis of isoform-specific phosphorylation of AKT, an important target in cancer therapy. Two studies demonstrated that the IEF separation allows simultaneously detection and measurement of all three AKT isoforms with a single pan-reactive antibody, which enabled a more sensitive and accurate comparison of differential regulation roles played by the different isoforms [23, 47].
The high resolution of the charge-CNIA technique can even allow species-specific protein isoforms to be resolved in some cases. In a study of the effect of erlotinib on non-small cell lung cancer (NSCLC) xenograft samples, conventional Western blot analysis indicated that EGFR Y1068 was completely dephosphorylated upon treatment of erlotinib, a tyrosine kinase inhibitor, whereas ERK1/2 appeared to be only partly dephosphorylated (Figure 3). However, using charge-CNIA, human ERK1 isoforms can be differentiated from the mouse isoforms based on their pI differences. Erlotinib was shown to almost completely inhibit ERK phosphorylation in the targeted human xenograft cancer cells but had no effect on ERK phosphorylation in the surrounding mouse stromal cells .
Besides phosphorylation, the charge-CNIA assay is also able to detect other PTMs that change the charge of the protein and have functional consequences. Icardi et al. detected a pI shift of STAT3 induced by SIN3 transcription regulator homolog A (Sin3a) and identified K87 acetylation as a negative regulator of STAT3 activity . In a similar approach, Tikhanovich and coworkers identified specific and interactive PTM patterns of phosphorylation, acetylation, methylation and ubiquitination of FOXO3 that were induced by hepatitis C virus, alcohol or the combination of the two, and which alter the activity of this transcription factor .
Together, these studies demonstrate that the charge-CNIA system is able to distinguish and detect specific PTM isoforms that conventional Western blot and other immunoassays fail to identify. The technology thus provides a novel platform for in-depth study of signaling pathway mechanisms.
Quantitative proteomic analysis in limited patient specimens and real-time molecular diagnostics
A key issue in the development of molecular-targeted therapy is the ability to assess whether the drug is hitting the target. Nanogram level sample consumption, digital data processing and quantitation, and proven assay reproducibility make the CNIA system appealing for analysis of clinical specimens to facilitate assessment of pharmacodynamic biomarkers and treatment evaluation. The technology was demonstrated to allow the measurement of stem cell signals from a total of <10,000 cells, which contains a stem cell population of <0.2% .
The first CNIA application in analysis of clinical specimens was reported by Fan and coworkers from Stanford in 2009 . In this study, multiple oncoproteins in human tumor samples were precisely quantified using charge-CNIA (NIA). Differential Myc and Bcl2 expression was identified in different types of non-Hodgkin’s lymphoma subtypes. Furthermore, a specific mono-phosphorylated ERK2 signal, which correlated with patient response to tyrosine kinase inhibitor treatment, was identified in chronic myelogenous leukemia (CML) samples.
In a Phase I/II clinical trial, patients with higher risk red blood cell-dependent myelodysplastic syndrome (MDS) were treated with the multi-kinase inhibitor rigosertib . Seetharam and coworkers used charge CNIA to show a direct correlation between changes in the levels of phospho-AKT2 with patient response to drug treatment. CNIA assays were also used to measure rigosertib treatment response in human clinical specimens that are too small to be analyzed by conventional means, such as CD34+ cells from bone marrows and core biopsies from solid tumor tissues . Using a mouse model of head and neck squamous cell carcinoma, Hubbard and coworkers also demonstrated that the system allows molecular diagnostics to be performed longitudinally before, during and after treatment using a minimally invasive technique for specimen collection . These studies indicated that the CNIA system offers a real-time approach to measure target pathway activities and response to treatment in specimen samples that are limited.
The high sensitivity of the system has also enabled the profiling of signaling molecules in other samples of limited availability [48, 54]. ERK activation in aberrant crypt foci (ACF) was measured and compared to the adjacent normal colonic muscosa in micro-dissected human clinical samples containing as little as 16 ng protein from fewer than 200 cells . Other examples that exploit the high sensitivity of this technique include the analysis of target molecule response in FACS-sorted blast crisis leukemia stem cells derived from bone marrow of tyrosine kinase inhibitor-treated mice , label-retaining cancer stem cells derived from human hepatocellular carcinoma , and assessing the pS962 PTRC/CD45 level in CD34+ cells from CML patients .
Development of pharmacodynamic and predictive biomarkers
Although this application has not been as fully exploited, the CNIA technology shows promise in the development of biomarkers that predict response to therapy [22, 51, 59, 60]. In a study of the dynamic phosphorylation status of signaling molecules in NSCLC cells treated with EGFR tyrosine kinase and MEK inhibitors, a specific on-target MEK response pattern to a MEK inhibitor was identified, which was not detectable by conventional Western blot . The authors also identified a MEK2 signal that may be associated with NSCLC cell sensitivity to the EGFR inhibitor erlotinib (Figure 4). Using a charge-CNIA, Beurlet showed that treatment with the BCL-2 inhibitor ABT-7373 restored wild-type MEK phosphorylation patterns in mice with acute myelogenous leukemia, associated with extended lifespan and increased survival. .
Quality assessment in development and manufacture of biological drugs
The CNIA technology has important industrial applications as well. The CNIA platforms have been reported to be the choice for product quality control analysis based on it being a high-throughput and automated analysis system, and its proven assay precision, accuracy and sensitivity. Michels et al. demonstrated that the charge-CNIA offers specificity, speed and sensitivity advantages over an imaged capillary isoelectric focusing platform when used to analyze charge heterogeneity of monoclonal antibody products in early stage process development . Rustandi and colleagues applied size-CNIA to protein expression and identity in vaccine development. They reported that the technology increased the efficiency of analytical testing and could more effectively guide process development . The group also developed a new improved method for detection of residual BSA to meet quality control requirements in vaccine production .
A comparison of CNIA to other immunoassay technologies
Immunoassay-based analytical technologies have a long history and have been widely used for target-specific protein detection and quantification from discovery research to clinical studies. The following narrative offers a comparison of the CNIA system with other major immunoassay platforms. The advantages and challenges for each technology are highlighted in Table 1.
ELISA (Enzyme-linked immunosorbent assay) and multiplexed bead assays
ELISA is the most widely used and highly validated method for protein concentration measurement. It remains a gold standard immunoassay for drug development programs. A digital ELISA approach has been reported that detects serum proteins at sub-femtomolar concentrations . Multiplex ELISA assays have been developed in recent years to quantify multiple proteins in complex biological samples [66, 67]. Applications of multiplexed assays have been mainly in biological fluid samples, e.g. serum and plasma. Applications in complex bio-specimens, such as tissue or whole cell lysates, are limited due to confounding matrix effects. There are also challenges when optimizing analysis conditions simultaneously for multiple proteins with different affinities for the detecting antibodies. Due to antibody cross-reactivity issues, most commercial kits are limited to fewer than 30 targets in a single measurement [68, 69]. Traditional ELISA assays usually require large amount of samples, and the lead time for development is long (usually several months to more than 1 year) . In addition, though evidence has suggested the importance of protein isoforms in disease progress, ELISA and multiplex-bead assays are limited in accessing this information due to the lack of isoform specific antibodies to differentiate different modifications and/or protein variants [16, 70].
Conventional Western blot
Combining protein separations with target-specific antibody probing, Western blot has been the top choice for studying protein levels and post-translational modifications, due to the low cost and simplicity of the technology. However, the conventional Western blot technology suffers from large sample and reagent usage, time and labor commitment, semi-quantitative data, poor reproducibility, and low throughput. Currently, Western blot is mainly used in discovery research and encounters difficulties in further development for pre-clinical and clinical applications [69, 71].
Protein arrays are somewhat analogous to chip-based transcriptional profiling for analysis of proteins and their functioning status. There are two types of protein arrays, antibody arrays and reverse-phase protein arrays (RPPA). The antibody arrays involve immobilizing well-characterized antibodies on a solid support which are then used for detecting multiple target proteins or PTMs in single sample. Labeled protein mixtures are applied to the array followed by immuno-analysis. In contract, multiple tissue or cell lysates, instead of antibodies, are used to construct RPPAs., The arrays are then probed by target-specific antibodies, thus allowing multiple samples to be queried simultaneously for a single target. Like CNIA protein arrays offer multiplexed protein profiling in low sample volumes. Protein arrays have been successfully applied in clinical proteomics, biomarker identification, therapeutic monitoring, biomarker discovery, and evaluation of pharmaceutical targets. [72–74]. Quantitative proteomic profiling of signaling pathways from surgical and micro-dissected tumor specimens and fine-needle aspirates were shown to be promising for guiding potential therapeutic intervention strategies [73, 75, 76].
The antibody array and RPPA face several technical challenges compared with CNIA. Since the platforms cannot identify nonspecific binding of the antibodies to other proteins, so rigorous antibody validation procedures are required to assure absolute specificity [10, 15, 77], Moreover, additional specific antibodies are needed to detect protein isoform variants . As in multiplexed bead assays, antibody array-based protein measurements are usually limited to 30–50 targets due to antibody cross-reactivity . RPPA has the advantage over CNIA that hundreds of samples can be analyzed in a single slide for a single target, with the detection sensitivities of 1000-5000 molecules/spot . However, the technology can be expensive when only a small number of samples are evaluated across many targets.
Immunohistochemistry (IHC) detects endogenous proteins in situ and thus provides spatial information about heterogeneous expression of proteins in their natural environment within a tissue that is not provided by bulk biochemical analysis techniques such as CNIA . Traditional IHC analysis is the most widely used immunoassay for clinical tissue sample analysis and has demonstrated the power of biomarker analysis to drive clinical decisions. However, IHC data are usually semi-quantitative and lack of reproducibility making the approach difficult to use when quantitative information is important, for example, when assessing pharmacodynamic biomarker responses to targeted therapies. Since IHC data assessment primarily relies on the individual pathologist’s experience, it is difficult to standardize procedures when data interpretation is subjective .
Further improvements of the CNIA technology
As discussed in this manuscript and elsewhere, the CNIA technology has shown considerable promise for quantitative sensitive proteomic analysis. For the future development and translational application of the technology, some improvements are needed.
Expanding the range of target detection with good resolution
To accommodate the analysis of a broad range of protein size and charge, new reagents and assay conditions will need to be developed. In the size-based analysis platform, current reagents and assay conditions are optimized for the separation of proteins with MW between 12 and 230 kDa. Based on our experience, when proteins with molecular weights lower than 20 kD, variation in estimated molecular weight or low signals were observed for some proteins. In addition, although a new kit for 66–440 kD protein analysis has been released (see the ProteinSimple website for product detail), more extensive validation of the kit performance is desired. In the IEF system, good assay resolution and reproducibility are observed between pI 4–8. While this covers most proteins, reduced resolution and lower assay reproducibility can be observed with especially low or high pI targets.
Improving signal-to-noise ratio in size-CNIA
In general, straight-forward assay transfer and good data consistency with conventional Western blot assays has been observed with the size-CNIA assays. Occasionally, high background has been observed in the size-CNIA assays. This phenomenon is mostly primary antibody-dependent. It may be solved by additional blocking of the capillaries, increasing the antibody dilution factor, or switching to an alternative primary antibody. We have seen a higher baseline noise in the higher-sensitivity size-CNIA analysis mode that uses the newer Peggy Sue™ technology. Though this analysis mode offers more than five-fold increase in signal, in our experience, the higher noise level seen with some targets can jeopardize data quantification and assay reproducibility. Also, the Peggy Sue analysis mode has been observed to be sensitive to climate factors, such as low humidity in the winter months. This analysis mode needs further development to restore the robustness that is an important positive attribute of the CNIA assays.
Peak identification for charge-CNIA
The comprehensive in-depth information obtained for signaling molecule activation by the IEF system is exciting. However, it also raises the challenge of determining the identities of the peak components revealed by the system. For a complete understanding of the complicated peak profiles and their correlations to the function of signaling molecules, other technologies will need to be employed. In cases where isoform- and PTM-directed antibodies are available along with knockout cells and/or mutant constructs, peak identity has been determined [23, 46, 47, 50]. Mass spectrometry (MS) peak identification protocols have also been developed and used for identification of previously unknown phosphorylation sites on their targets in the CNIA system [44, 45, 81]. For broader application, peak ID protocols will need to be developed for more complex PTM patterns.
Integrating the CNIA technology with complementary proteomics approaches
It is likely that the specific advantages of many of the different technologies can be usefully combined in ways that exploit the specific strengths of each. There are reports of using RPPA and antibody arrays as an initial discovery screening to identify predictive assays that are later transferred to CNIA analysis with reduced complexity for potential clinical applications [82, 83]. With the further development and improvement of the CNIA system, it is expected that complementary and supplementary applications of this new technology with other immunoassay platforms will emerge.
Like other immunoassays, antibody performance and availability in many circumstance may limited the applications of CNIA assays. In the past decade, the field of MS-based proteomics has grown tremendously. MS-based multiple reaction monitoring (MRM) assays provide targeted, quantitative and multiplexed analysis of proteins and respective PTM isoforms without the requirement for an antibody [84, 85]. The MS technology has demonstrated to be a powerful discovery platform. Though more complicated sample processing and data analysis are involved with MS assays, and more sophisticated expertise is required to operate the system, MRM–MS assays can be rapidly configured for verification of protein biomarker candidates before further moving to clinical-grade immunoassays. An in-depth discussion of the MS technology is beyond the scope of this review. Interested readers may refer to Libeler’s recent review  about MRM for more information. As mentioned in the previous session that MS protocols have been used as a strategy to detect the detail identity of PTM isoforms revealed by the charge-CNIA assays [44, 45, 81]. Integrating the different analysis methods will allow a more comprehensive study of proteins and their functioning mechanisms and will help translate the discovery research data into clinical practice.
As reviewed here, the CNIA system provides comprehensive and quantitative analysis for protein characterization and profiling of signaling events. The automated system offers easy operation, precise and accurate measurement of proteins and their post-translational modifications and a fast turn-around time. The capillary platform allows functional proteomic analysis to be performed with limited sample, such as from stem cells, primary cells, fine needle aspirates, micro-dissection samples and other patient specimens. Good intra-assay, inter-run and inter-person data reproducibility have been observed with the system, which enables assay standardization across multiple testing sites.
With the further validation of CNIA assays and development of the technology, it is expected that a growing number of applications in both discovery research and clinical practice for signaling pathway dissection, proteomic biomarker assessment, and targeted therapy evaluation. The high reproducibility and data precision of the technique mean the technology can be readily adapted for use in CLIA-certified settings. Complemented with other genome, transcriptome and additional protein analysis technologies, the CNIA technology shows great promise for developing pathway specific diagnostics for better treatment group stratification and acceleration of drug development and disease management.
size-based capillary nano-immunoassay
charge-based capillary nano-immunoassay
enzyme-linked immunosorbent assay
coefficient of variation
reverse-phase protein array
multiple reaction monitoring
Miller I, Ashton-Chess J, Spolders H, Fert V, Ferrara J, Kroll W et al (2011) Market access challenges in the EU for high medical value diagnostic tests. Pers Med 8:137–148
Hudis CA (2007) Trastuzumab—mechanism of action and use in clinical practice. N Engl J Med 357:39–51
Plesec TP, Hunt JL (2009) KRAS mutation testing in colorectal cancer. Adv Anat Pathol 16:196–203
Tan EH, Ramlau R, Pluzanska A, Kuo HP, Reck M, Milanowski J et al (2010) A multicentre phase II gene expression profiling study of putative relationships between tumour biomarkers and clinical response with erlotinib in non-small-cell lung cancer. Ann Oncol 21:217–222
Guo NL, Wan YW, Tosun K, Lin H, Msiska Z, Flynn DC et al (2008) Confirmation of gene expression-based prediction of survival in non-small cell lung cancer. Clin Cancer Res 14:8213–8220
Garman KS, Acharya CR, Edelman E, Grade M, Gaedcke J, Sud S et al (2008) A genomic approach to colon cancer risk stratification yields biologic insights into therapeutic opportunities. Proc Natl Acad Sci USA 105:19432–19437
Hudson TJ, Anderson W, Artez A, Barker AD, Bell C, Bernabe RR et al (2010) International network of cancer genome projects. Nature 464:993–998
Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E et al (2012) Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 366:883–892
Din S, Lennon AM, Arnott ID, Hupp T, Satsangi J (2007) Technology insight: the application of proteomics in gastrointestinal disease. Nat Clin Pract Gastroenterol Hepatol 4:372–385
Haab BB (2005) Antibody arrays in cancer research. Mol Cell Proteomics 4:377–383
HUPO (2010) A gene-centric human proteome project: HUPO–the human proteome organization. Mol Cell Proteomics 9:427–429
Gygi SP, Rochon Y, Franza BR, Aebersold R (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 19:1720–1730
Brennan DJ, O’Connor DP, Rexhepaj E, Ponten F, Gallagher WM (2010) Antibody-based proteomics: fast-tracking molecular diagnostics in oncology. Nat Rev Cancer 10:605–617
Kopf E, Zharhary D (2007) Antibody arrays—an emerging tool in cancer proteomics. Int J Biochem Cell Biol 39:1305–1317
VanMeter A, Signore M, Pierobon M, Espina V, Liotta LA, Petricoin EF 3rd (2007) Reverse-phase protein microarrays: application to biomarker discovery and translational medicine. Expert Rev Mol Diagn 7:625–633
Hughes AJ, Lin RK, Peehl DM, Herr AE (2012) Microfluidic integration for automated targeted proteomic assays. Proc Natl Acad Sci USA 109:5972–5977
Wulfkuhle JD, Edmiston KH, Liotta LA, Petricoin EF 3rd (2006) Technology insight: pharmacoproteomics for cancer–promises of patient-tailored medicine using protein microarrays. Nat Clin Pract Oncol 3:256–268
O’Neill RA, Bhamidipati A, Bi X, Deb-Basu D, Cahill L, Ferrante J et al (2006) Isoelectric focusing technology quantifies protein signaling in 25 cells. Proc Natl Acad Sci USA 103:16153–16158
Besong Agbo D, Klafki H, Poschmann G, Seyfarth K, Genius J, Janssen C et al (2013) Development of a capillary isoelectric focusing immunoassay to measure DJ-1 isoforms in biological samples. Anal Biochem 443:197–204
Chen JQ, Heldman MR, Herrmann MA, Kedei N, Woo W, Blumberg PM et al (2013) Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system. Anal Biochem 442:97–103
Harding A (2013) Life science technologies: not your PI’s western blot. Science 339:1100–1102
Fan AC, Deb-Basu D, Orban MW, Gotlib JR, Natkunam Y, O’Neill R et al (2009) Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens. Nat Med 15:566–571
Iacovides DC, Johnson AB, Wang N, Boddapati S, Korkola J, Gray JW (2013) Identification and quantification of AKT isoforms and phosphoforms in breast cancer using a novel nanofluidic immunoassay. Mol Cell Proteomics 12:3210–3220
Klein BY, Tamir H, Hirschberg DL, Glickstein SB, Welch MG (2013) Oxytocin modulates mTORC1 pathway in the gut. Biochem Biophys Res Commun 432:466–471
Kedei N, Telek A, Czap A, Lubart ES, Czifra G, Yang D et al (2011) The synthetic bryostatin analog Merle 23 dissects distinct mechanisms of bryostatin activity in the LNCaP human prostate cancer cell line. Biochem Pharmacol 81:1296–1308
Klamer SE, Kuijk CGM, Hordijk PL, van der Schoot CE, von Lindern M, van Hennik PB et al (2013) BIGH3 modulates adhesion and migration of hematopoietic stem and progenitor cells. Cell Adhes Migr 7:434–449
Gonsalves CS, Crable S, Chandra S, Li W, Kalra VK, Joiner CH (2014) Angiogenic growth factors augment K-Cl cotransporter expression in erythroid cells via hypoxia-inducible factor-1alpha. Am J Hematol 89:273–281
Gordillo GM, Biswas A, Khanna S, Pan X, Sinha M, Roy S et al (2014) Dicer knockdown inhibits endothelial cell tumor growth via microRNA 21a-3p targeting of Nox-4. J Biol Chem 289:9027–9038
Serrano M, Moreno M, Ortega FJ, Xifra G, Ricart W, Moreno-Navarrete JM et al (2014) Adipose tissue mu-crystallin is a thyroid hormone-binding protein associated with systemic insulin sensitivity. J Clin Endocrinol Metab 99:E2259–E2268
Price FD, von Maltzahn J, Bentzinger CF, Dumont NA, Yin H, Chang NC et al (2014) Inhibition of JAK-STAT signaling stimulates adult satellite cell function. Nat Med 20:1174–1181
Bakhsheshian J, Hall MD, Robey RW, Herrmann MA, Chen JQ, Bates SE et al (2013) Overlapping substrate and inhibitor specificity of human and murine ABCG2. Drug Metab Dispos 41:1805–1812
Choi YP, Lee JH, Gao MQ, Kim BG, Kang S, Kim SH et al (2014) Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models. Int J Cancer 135:2024–2033
Beccano-Kelly DA, Kuhlmann N, Tatarnikov I, Volta M, Munsie LN, Chou P et al (2014) Synaptic function is modulated by LRRK2 and glutamate release is increased in cortical neurons of G2019S LRRK2 knock-in mice. Front Cell Neurosci 8:301
Daniels VW, Smans K, Royaux I, Chypre M, Swinnen JV, Zaidi N (2014) Cancer cells differentially activate and thrive on de novo lipid synthesis pathways in a low-lipid environment. PLoS One 9:e106913
Liu SB, Sardi S, Sonom B, Zocco D, McSweeney R, Fraser AD et al (2013) The application of a novel nanovolume capillary electrophoresis-based protein analysis system in personalized & translational medicine research. J Bioanal Biomed S3:004. doi:10.4172/1948-593X.S3-004
Kohn EA, Yang YA, Du Z, Nagano Y, Van Schyndle CM, Herrmann MA et al (2012) Biological responses to TGF-beta in the mammary epithelium show a complex dependency on Smad3 gene dosage with important implications for tumor progression. Mol Cancer Res 10:1389–1399
Sabnis H, Bradley HL, Bunting ST, Cooper TM, Bunting KD (2014) Capillary nano-immunoassay for Akt 1/2/3 and 4EBP1 phosphorylation in acute myeloid leukemia. J Transl Med 12:166
Kedei N, Lewin NE, Geczy T, Selezneva J, Braun DC, Chen J et al (2013) Biological profile of the less lipophilic and synthetically more accessible bryostatin 7 closely resembles that of bryostatin 1. ACS Chem Biol 8:767–777
DeChristopher BA, Fan AC, Felsher DW, Wender PA (2012) “Picolog,” a synthetically-available bryostatin analog, inhibits growth of MYC-induced lymphoma in vivo. Oncotarget 3:58–66
de Kreuk BJ, Anthony EC, Geerts D, Hordijk PL (2012) The F-BAR protein PACSIN2 regulates epidermal growth factor receptor internalization. J Biol Chem 287:43438–43453
Fiscus RR, Leung EL, Wong JC, Johlfs MG (2012) Nitric oxide/protein kinase GIα promotes c-Src activation, proliferation and chemoresistance in ovarian cancer. In Farghaly DS (ed) Ovarian Cancer—Basic Science Perspective. InTech, pp 315–334
Aspinall-O’Dea M, Pierce A, Pellicano F, Williamson AJ, Scott MT, Walker MJ et al (2015) Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines. Nat Protoc 10:149–168
Eldridge S, Guo L, Mussio J, Furniss M, Hamre J 3rd, Davis M (2014) Examining the protective role of ErbB2 modulation in human-induced pluripotent stem cell-derived cardiomyocytes. Toxicol Sci 141:547–559
Frogne T, Sylvestersen KB, Kubicek S, Nielsen ML, Hecksher-Sorensen J (2012) Pdx1 is post-translationally modified in vivo and serine 61 is the principal site of phosphorylation. PLoS One 7:e35233
Huang CC, Weng JH, Wei TY, Wu PY, Hsu PH, Chen YH et al (2012) Intermolecular binding between TIFA-FHA and TIFA-pT mediates tumor necrosis factor alpha stimulation and NF-kappaB activation. Mol Cell Biol 32:2664–2673
Sun Z, Li X, Massena S, Kutschera S, Padhan N, Gualandi L et al (2012) VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd. J Exp Med 209:1363–1377
Guo H, Gao M, Lu Y, Liang J, Lorenzi PL, Bai S et al (2014) Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules. Oncogene 33:3463–3472
Chen JQ, Lee JH, Herrmann MA, Park KS, Heldman MR, Goldsmith PK et al (2013) Capillary isoelectric-focusing immunoassays to study dynamic oncoprotein phosphorylation and drug response to targeted therapies in non-small cell lung cancer. Mol Cancer Ther 12:2601–2613
Icardi L, Mori R, Gesellchen V, Eyckerman S, De Cauwer L, Verhelst J et al (2012) The Sin3a repressor complex is a master regulator of STAT transcriptional activity. Proc Natl Acad Sci USA 109:12058–12063
Tikhanovich I, Kuravi S, Campbell RV, Kharbanda KK, Artigues A, Villar MT et al (2014) Regulation of FOXO3 by phosphorylation and methylation in hepatitis C virus infection and alcohol exposure. Hepatology 59:58–70
Seetharam M, Fan AC, Tran M, Xu L, Renschler JP, Felsher DW et al (2012) Treatment of higher risk myelodysplastic syndrome patients unresponsive to hypomethylating agents with ON 01910.Na. Leuk Res 36:98–103
Fan AC, O’Rourke JJ, Praharaj DR, Felsher DW (2013) Real-time nanoscale proteomic analysis of the novel multi-kinase pathway inhibitor rigosertib to measure the response to treatment of cancer. Expert Opin Investig Drugs 22:1495–1509
Hubbard MA, Khalil AA, Schoeff SS, Allak A, VanKoevering KK, Maxwell AK et al (2014) Nanoimmunoassay to detect responses in head and neck cancer: feasibility in a mouse model. Otolaryngol Head Neck Surg 151:92–99
Siggers P, Carre GA, Bogani D, Warr N, Wells S, Hilton H et al (2014) A novel mouse Fgfr2 mutant, hobbyhorse (hob), exhibits complete XY gonadal sex reversal. PLoS One 9:e100447
Drew DA, Devers T, Horelik N, Yang S, O’Brien M, Wu R et al (2013) Nanoproteomic analysis of extracellular receptor kinase-1/2 post-translational activation in microdissected human hyperplastic colon lesions. Proteomics 13:1428–1436
Goff DJ, Recart AC, Sadarangani A, Chun HJ, Barrett CL, Krajewska M et al (2013) A pan-BCL2 inhibitor renders bone-marrow-resident human leukemia stem cells sensitive to tyrosine kinase inhibition. Cell Stem Cell 12:316–328
Xin HW, Ambe CM, Hari DM, Wiegand GW, Miller TC, Chen JQ et al (2013) Label-retaining liver cancer cells are relatively resistant to sorafenib. Gut 62:1777–1786
Williamson AJ, Pierce A, Jaworska E, Zhou C, Aspinall-O’Dea M, Lancashire L et al (2013) A specific PTPRC/CD45 phosphorylation event governed by stem cell chemokine CXCL12 regulates primitive hematopoietic cell motility. Mol Cell Proteomics 12:3319–3329
Maiso P, Liu Y, Morgan B, Azab AK, Ren P, Martin MB et al (2011) Defining the role of TORC1/2 in multiple myeloma. Blood 118:6860–6870
Kentsis A, Reed C, Rice KL, Sanda T, Rodig SJ, Tholouli E et al (2012) Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia. Nat Med 18:1118–1122
Beurlet S, Omidvar N, Gorombei P, Krief P, Le Pogam C, Setterblad N et al (2013) BCL-2 inhibition with ABT-737 prolongs survival in an NRAS/BCL-2 mouse model of AML by targeting primitive LSK and progenitor cells. Blood 122:2864–2876
Michels DA, Tu AW, McElroy W, Voehringer D, Salas-Solano O (2012) Charge heterogeneity of monoclonal antibodies by multiplexed imaged capillary isoelectric focusing immunoassay with chemiluminescence detection. Anal Chem 84:5380–5386
Rustandi RR, Loughney JW, Hamm M, Hamm C, Lancaster C, Mach A et al (2012) Qualitative and quantitative evaluation of Simon, a new CE-based automated Western blot system as applied to vaccine development. Electrophoresis 33:2790–2797
Loughney JW, Lancaster C, Ha S, Rustandi RR (2014) Residual bovine serum albumin (BSA) quantitation in vaccines using automated capillary western technology. Anal Biochem 461:49–56
Rissin DM, Kan CW, Campbell TG, Howes SC, Fournier DR, Song L et al (2010) Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol 28:595–599
Leng SX, McElhaney JE, Walston JD, Xie D, Fedarko NS, Kuchel GA (2008) ELISA and multiplex technologies for cytokine measurement in inflammation and aging research. J Gerontol A Biol Sci Med Sci 63:879–884
Vignali DA (2000) Multiplexed particle-based flow cytometric assays. J Immunol Methods 243:243–255
Hoheisel JD, Alhamdani MS, Schroder C (2013) Affinity-based microarrays for proteomic analysis of cancer tissues. Proteomics Clin Appl 7:8–15
Rifai N, Gillette MA, Carr SA (2006) Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol 24:971–983
Kyselova Z, Mechref Y, Al Bataineh MM, Dobrolecki LE, Hickey RJ, Vinson J et al (2007) Alterations in the serum glycome due to metastatic prostate cancer. J Proteome Res 6:1822–1832
Parsons DW, Li M, Zhang X, Jones S, Leary RJ, Lin JC et al (2011) The genetic landscape of the childhood cancer medulloblastoma. Science 331:435–439
Mueller C, Liotta LA, Espina V (2010) Reverse phase protein microarrays advance to use in clinical trials. Mol Oncol 4:461–481
Petricoin EF 3rd, Espina V, Araujo RP, Midura B, Yeung C, Wan X et al (2007) Phosphoprotein pathway mapping: Akt/mammalian target of rapamycin activation is negatively associated with childhood rhabdomyosarcoma survival. Cancer Res 67:3431–3440
Zhu H, Cox E, Qian J (2012) Functional protein microarray as molecular decathlete: a versatile player in clinical proteomics. Proteomics Clin Appl 6:548–562
Rapkiewicz A, Espina V, Zujewski JA, Lebowitz PF, Filie A, Wulfkuhle J et al (2007) The needle in the haystack: application of breast fine-needle aspirate samples to quantitative protein microarray technology. Cancer 111:173–184
Pierobon M, Calvert V, Belluco C, Garaci E, Deng J, Lise M et al (2009) Multiplexed cell signaling analysis of metastatic and nonmetastatic colorectal cancer reveals COX2-EGFR signaling activation as a potential prognostic pathway biomarker. Clin Colorectal Cancer 8:110–117
Liotta LA, Espina V, Mehta AI, Calvert V, Rosenblatt K, Geho D et al (2003) Protein microarrays: meeting analytical challenges for clinical applications. Cancer Cell 3:317–325
Ellington AA, Kullo IJ, Bailey KR, Klee GG (2010) Antibody-based protein multiplex platforms: technical and operational challenges. Clin Chem 56:186–193
Weibrecht I, Leuchowius KJ, Clausson CM, Conze T, Jarvius M, Howell WM et al (2010) Proximity ligation assays: a recent addition to the proteomics toolbox. Expert Rev Proteomics 7:401–409
Seidal T, Balaton AJ, Battifora H (2001) Interpretation and quantification of immunostains. Am J Surg Pathol 25:1204–1207
Haussmann U, Jahn O, Linning P, Janssen C, Liepold T, Portelius E et al (2013) Analysis of amino-terminal variants of amyloid-beta peptides by capillary isoelectric focusing immunoassay. Anal Chem 85:8142–8149
Bentley C, Jurinka SS, Kljavin NM, Vartanian S, Ramani SR, Gonzalez LC et al (2013) A requirement for wild-type Ras isoforms in mutant KRas-driven signalling and transformation. Biochem J 452:313–320
Wiegand KC, Hennessy BT, Leung S, Wang Y, Ju Z, McGahren M et al (2014) A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation. BMC Cancer 14:120
Liebler DC, Zimmerman LJ (2013) Targeted quantitation of proteins by mass spectrometry. Biochemistry 52:3797–3806
Addona TA, Abbatiello SE, Schilling B, Skates SJ, Mani DR, Bunk DM et al (2009) Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Nat Biotechnol 27:633–641
JC formulated the structure and drafted the manuscript. LW critically revised the manuscript. JC, LW and DG conceived the concept jointly. All authors read and approved the final manuscript.
The author thanks Drs. Peter Blumberg for critical review of the manuscript and valuable insights and advice. Also thank to Michelle A Herrmann for the contribution on developing and applying the CNIA assays in the PKC isoform and NSCLC research reviewed in this manuscript. The work was supported by the Intramural Research Program of the National Cancer Institute at NIH (Z1C BC 011434).
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
About this article
Cite this article
Chen, JQ., Wakefield, L.M. & Goldstein, D.J. Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics. J Transl Med 13, 182 (2015). https://doi.org/10.1186/s12967-015-0537-6
- Capillary nano-immunoassay
- Simple western
- Cell signaling
- Molecular diagnostic