SWATH enables precise label‐free quantification on proteome scale

MS‐based proteomics has emerged as a powerful tool in biological studies. The shotgun proteomics strategy, in which proteolytic peptides are analyzed in data‐dependent mode, enables a detection of the most comprehensive proteome (>10 000 proteins from whole‐cell lysate). The quantitative proteomics uses stable isotopes or label‐free method to measure relative protein abundance. The isotope labeling strategies are more precise and accurate compared to label‐free methods, but labeling procedures are complicated and expensive, and the sample number and types are also limited. Sequential window acquisition of all theoretical mass spectra (SWATH) is a recently developed technique, in which data‐independent acquisition is coupled with peptide spectral library match. In principle SWATH method is able to do label‐free quantification in an MRM‐like manner, which has higher quantification accuracy and precision. Previous data have demonstrated that SWATH can be used to quantify less complex systems, such as spiked‐in peptide mixture or protein complex. Our study first time assessed the quantification performance of SWATH method on proteome scale using a complex mouse‐cell lysate sample. In total 3600 proteins got identified and quantified without sample prefractionation. The SWATH method shows outstanding quantification precision, whereas the quantification accuracy becomes less perfect when protein abundances differ greatly. However, this inaccuracy does not prevent discovering biological correlates, because the measured signal intensities had linear relationship to the sample loading amounts; thus the SWATH method can predict precisely the significance of a protein. Our results prove that SWATH can provide precise label‐free quantification on proteome scale.     

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.     

Monitoring protein modification with allosteric ribozymes

An allosteric ribozyme is an RNA-based enzyme (ribozyme) whose catalytic activity is modulated by molecular recognition of a protein. The direct coupling of a detectable catalytic event to molecular recognition by an allosteric ribozyme enables simple assays for quantitative protein detection. Most significantly, the mode of development and molecular recognition characteristics of allosteric ribozymes are fundamentally different from antibodies, providing them with functional characteristics that complement those of antibodies. Allosteric ribozymes can be developed using native proteins and, therefore, are often sensitive to protein conformation. In contrast, antibodies tend to recognize a series of adjacent amino acids as a consequence of antigen presentation and typically are not sensitive to protein conformation. Unlike antibody development, the development of allosteric ribozymes is a completely in vitro process that allows the specificity of an allosteric ribozyme to be tightly controlled. These significant differences from antibodies allow the pre-programmed development of conformation-state-specific protein detection reagents that can be used to investigate the activation-state of signal transduction components.     

Quantitative protein profiling using antibody arrays

Traditional approaches to microarrays rely on direct binding assays where the extent of hybridisation and the signal detected are a measure of the analyte concentration in the experimental sample. This approach, directly imported from the nucleic acid field, may fail if applied to antibody-antigen interactions due to the shortage of characterised antibodies, the significant heterogeneity of antibody affinities, their dependence on the extent of protein modification during labelling and the inherent antibody cross-reactivity. These problems can potentially limit the multiplexing capabilities of protein affinity assays and in many cases rule out quantitative protein profiling using antibody microarrays. A number of approaches aimed at achieving quantitative protein profiling in a multiplex format have been reported recently. Of those reported, the three most promising routes include signal amplification, multicolour detection and competitive displacement approaches to multiplex affinity assays. One in particular, competitive displacement, also overcomes the problems associated with quantitation of affinity interactions and provides the most generic approach to highly parallel affinity assays, including antibody arrays.     

Label-free multiplexed virus detection using spectral reflectance imaging

We demonstrate detection of whole viruses and viral proteins with a new label-free platform based on spectral reflectance imaging. The Interferometric Reflectance Imaging Sensor (IRIS) has been shown to be capable of sensitive protein and DNA detection in a real time and high-throughput format. Vesicular stomatitis virus (VSV) was used as the target for detection as it is well-characterized for protein composition and can be modified to express viral coat proteins from other dangerous, highly pathogenic agents for surrogate detection while remaining a biosafety level 2 agent. We demonstrate specific detection of intact VSV virions achieved with surface-immobilized antibodies acting as capture probes which is confirmed using fluorescence imaging. The limit of detection is confirmed down to 3.5 × 10(5)plaque-forming units/mL (PFUs/mL). To increase specificity in a clinical scenario, both the external glycoprotein and internal viral proteins were simultaneously detected with the same antibody arrays with detergent-disrupted purified VSV and infected cell lysate solutions. Our results show sensitive and specific virus detection with a simple surface chemistry and minimal sample preparation on a quantitative label-free interferometric platform.     

Rapid detection of “Candidatus Phytoplasma mali” by recombinase polymerase amplification assays

Isothermal recombinase polymerase amplification (RPA) assays for the specific detection of “Candidatus Phytoplasma mali (Ca. P. mali),” the causal agent of apple proliferation, were developed. The assays amplify a fragment of the imp gene and amplimers were detected either by fluorescence in real‐time mode (TwistAmp^®exo assay) using a fluorophore‐labelled probe or by direct visualization employing a lateral flow device (TwistAmp^®nfo assay/Milenia^®HybriDetect). The RPA assays specifically amplified DNA from “Ca. P. mali” strains, and cross‐reactivity with other phytoplasmas or plant DNA was not observed. The limit of detection was determined with a cloned imp standard, and positive results were obtained down to 10 copies with both RPA assay formats. In comparison with a TaqMan real‐time PCR assay based on the same target gene, the RPA assays were equally sensitive, but results were obtained faster. Simplified nucleic acid extraction procedures from plant tissue with Tris‐ and CTAB‐based buffers revealed that crude Tris–DNA extracts were a suitable source for RPA tests while larger concentrations of CTAB were inhibitory. This is the first report of RPA‐based assays for the detection of “Ca. P. mali”. The assays are suitable for high‐throughput screening of plant material and point‐of‐care diagnostic and can be potentially combined with a simplified DNA extraction procedure.     

Measurement of cytokine antibodies. Test development

Several assays have been used for detection of antibodies against cytokines. The choice of assay is greatly dependent on the intended goal, e.g. detection of naturally occurring antibodies or therapy induced antibodies. The different assays can be grouped in 2 categories. The interference or indirect assays are based on the detection of the test sample interference with the biological activity, with detection of the cytokine in EIA or with binding to cellular receptors. In direct assays cytokine antibodies are detected by binding to solid phase fixed cytokines, followed by incubation with a secondary enzyme-labelled anti-human Ig antibody or by binding to¹²⁵I-labelled cytokines in RIA.     

Highly sensitive SERS detection of cancer proteins in low sample volume using hollow core photonic crystal fiber

Enzyme-linked immunosorbent assays (ELISA) are commonly used for detecting cancer proteins at concentration in the range of about ng-μg/mL. Hence it often fails to detect tumor markers at the early stages of cancer and other diseases where the amount of protein is extremely low. Herein, we report a novel photonic crystal fiber (PCF) based surface enhanced Raman scattering (SERS) sensing platform for the ultrasensitive detection of cancer proteins in an extremely low sample volume. As a proof of concept, epidermal growth factor receptors (EGFRs) in a lysate solution from human epithelial carcinoma cells were immobilized into the hollow core PCF. Highly sensitive detection of protein was achieved using anti-EGFR antibody conjugated SERS nanotag. This SERS nanotag probe was realized by anchoring highly active Raman molecules onto the gold nanoparticles followed by bioconjugation. The proposed sensing method can detect low amount of proteins at ～100 pg in a sample volume of ～10 nL. Our approach may lead to the highly sensitive protein sensing methodology for the early detection of diseases.     

Improving the limit of detection in portable luminescent assay readers through smart optical design

Critical biomarkers of disease are increasingly being detected by point‐of‐care assays. Chemiluminescence (CL) and electrochemiluminescence (ECL) are often used in such assays due to their convenience and that they do not require light sources or other components that could complicate or add cost to the system. Reports of these assays often include readers built on a cellphone platform or constructed from low‐cost components. However, the impact the optical design has on the limit of detection (LOD) in these systems remains unexamined. Here, we report a theoretical rubric to evaluate different optical designs in terms of maximizing the use of photons emitted from a CL or ECL assay to improve the LOD. We demonstrate that the majority of cellphone designs reported in the literature are not optimized, in part due to misunderstandings of the optical tradeoffs in collection systems, and in part due to limitations imposed on the designs arising from the use of a mobile phone with a very small lens aperture. Based on the theoretical rubric, we design a new portable reader built using off‐the‐shelf condenser optics, and demonstrate a nearly 10× performance enhancement compared to prior reports on an ECL assays running on a portable chip.     

A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard

The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.     

Antibody array analysis of labelled proteomes: how should we control specificity?

Researchers who use protein binders in multiplexed assays can be divided into two camps. One believes that arrays with proteome-wide coverage will become a reality once we have developed binders for all proteins. The sceptics claim that detection with immobilized protein binders and sample labelling will not provide the required specificity. In this article, we review the evidence showing that antibody array analysis of labelled samples can provide meaningful data and discuss the issues raised by the sceptics. We argue that direct the evidence for monospecificity has yet to be published. This will require assays designed to resolve the proteins captured by each binder. One option is to combine array measurement with protein separation. We have developed an assay where labelled sample proteins are separated by size exclusion chromatography (SEC) before contact with microsphere-based arrays (Size-MAP; size exclusion chromatography-resolved microsphere-based affinity proteomics). The effect is an 'antibody array Western blot' where reactivity of immobilized binders is resolved against the size of the proteins in the sample. We show that Size-MAP is useful to discriminate monospecific- and polyreactive antibodies and for automatic detection of reacting with the same target. The possibility to test specificity directly in array-based measurement should be useful to select the best binders and to determine whether the DNA microarray for the proteome is a realistic goal or not.     

Characterization of antibodies for quantitative determination of spiggin protein levels in male and female three-spined stickleback (Gasterosteus aculeatus)

Spiggin is an adhesive glycoprotein produced in the kidney of sticklebacks during the breeding season and is subsequently secreted into the urinary bladder from where it is employed for nest building. Since the production of the protein has been shown to be under androgenic control, spiggin has been suggested to be a useful biomarker for androgenic substances in the environment. In this study, two polyclonal spiggin antibodies based on synthetic peptides and one polyclonal antibody directed against native spiggin have been characterized. The antibodies ability to identify spiggin was investigated by quantitative immunoassay. For both peptide antibodies the quantification range was determined to be between 1 and 80 ng spiggin and determination of renal spiggin levels from immature and mature males displayed a 15-fold increase in total spiggin content of the kidney resulting in a 6-fold increase in male kidney weight due to hypertrophy. The kidney somatic index (KSI) was found to correlate well with the total renal spiggin content and therefore it appears that KSI in sticklebacks could be used as an initial method to identify substances displaying androgenic effects. Furthermore, western blot analysis revealed that the polyclonal antibodies recognize different spiggin isoforms and that spiggin can be detected in the urinary bladder and kidney of both males and female sticklebacks. In order to develop a quantitative detection method for native spiggin it is necessary to produce a standard that can be used in a bioassay. Due to the adhesive and polymerization characteristics of spiggin the protein is difficult to use as a standard in bioassays. So far spiggin has been shown to exist in at least 14 isoforms, all of which contain polymerization domains. To overcome the solubility problem we have produced recombinant spiggin gamma, with only one polymerization domain, that can be expressed in E. coli. Western blot analysis demonstrated that the polyclonal antibodies were able to detect recombinant spiggin gamma protein in bacterial cell lysate, suggesting that it may be developed into a useful source of standard spiggin to be used for quantitative determination of androgen induced spiggin production in sticklebacks.     

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.     

Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication.

The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation.     

Evaluation of blood collection tubes using selected reaction monitoring MS: Implications for proteomic biomarker studies

An emphasis of current proteomic research is the validation of plasma protein biomarkers. The process of blood collection itself is critical to the accuracy and reproducibility of quantitative biomarker assays. We have developed selected reaction monitoring (SRM) assays to analyse thirteen abundant plasma proteins and evaluated the impact of three different blood collection tubes on the levels of these proteins. We also assessed the implications of the time taken to analyse plasma samples by evaluating the recovery of these proteins. We showed that SRM detects minor differences in the levels of some proteins which can be attributed to collection tube type. The average recovery for 12 of 18 assays was higher for proteins that were collected in tubes containing protease inhibitors compared to conventional collection tubes. For five of the assays, the differential recovery was statistically significant. Delaying MS analysis of a freeze‐thawed sample for 1 hour showed greatly reduced recovery of these analytes; however differences attributed to tube type were only evident at the baseline timepoint. Finally, we assessed the natural variation of circulating levels of these proteins in a cohort of seven healthy individuals. This study provides useful information for researchers contemplating blood collection for undertaking protein biomarker studies.     

Improved Methods for Capture,Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.     

Ratiometric assay of epidermal growth factor receptor tyrosine kinase activation

Activation of cells is frequently followed by tyrosine phosphorylation of proteins. To quantify this process, we developed a ratiometric enzyme-linked immunosorbent assay (ELISA) using epidermal growth factor receptors (EGFR) as a model. Microtiter dishes were coated with anti-EGFR monoclonal antibodies to capture the receptor followed by parallel detection of receptor and phosphotyrosine content with secondary antibodies. The ratio of these two parameters was found to directly reflect EGFR activation and was insensitive to the effect of receptor downregulation. Our assay could resolve differences in EGFR activation due to small changes (less than 1 ng/ml) in ligand. We found that phosphotyrosine detection by ELISA was 8- to 32-fold more sensitive than Western blot detection and could be reliably detected using as little as 4 ng of cellular lysate. Detection of EGFR levels by ELISA was 30 times more sensitive than Western blot analysis and was reliable for as low as 8 ng of cellular lysate per well. Because of the wide linear range of the ELISA, we could directly compare receptor activation in cell types with different EGFR expression levels. Our assay provides a rapid and sensitive method of determining EGFR activation status and could be easily modified to evaluate any tyrosine-phosphorylated protein.     

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA– protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measure ments were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level ~10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.