Single-molecule dataset (SMD): a generalized storage format for raw and processed single-molecule data

Background

Single-molecule techniques have emerged as incisive approaches for addressing a wide range of questions arising in contemporary biological research [Trends Biochem Sci 38:30–37, 2013; Nat Rev Genet 14:9–22, 2013; Curr Opin Struct Biol 2014, 28C:112–121; Annu Rev Biophys 43:19–39, 2014]. The analysis and interpretation of raw single-molecule data benefits greatly from the ongoing development of sophisticated statistical analysis tools that enable accurate inference at the low signal-to-noise ratios frequently associated with these measurements. While a number of groups have released analysis toolkits as open source software [J Phys Chem B 114:5386–5403, 2010; Biophys J 79:1915–1927, 2000; Biophys J 91:1941–1951, 2006; Biophys J 79:1928–1944, 2000; Biophys J 86:4015–4029, 2004; Biophys J 97:3196–3205, 2009; PLoS One 7:e30024, 2012; BMC Bioinformatics 288 11(8):S2, 2010; Biophys J 106:1327–1337, 2014; Proc Int Conf Mach Learn 28:361–369, 2013], it remains difficult to compare analysis for experiments performed in different labs due to a lack of standardization.

Results

Here we propose a standardized single-molecule dataset (SMD) file format. SMD is designed to accommodate a wide variety of computer programming languages, single-molecule techniques, and analysis strategies. To facilitate adoption of this format we have made two existing data analysis packages that are used for single-molecule analysis compatible with this format.

Conclusion

Adoption of a common, standard data file format for sharing raw single-molecule data and analysis outcomes is a critical step for the emerging and powerful single-molecule field, which will benefit both sophisticated users and non-specialists by allowing standardized, transparent, and reproducible analysis practices.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0429-4) contains supplementary material, which is available to authorized users.     

Constitutive plasma membrane targeting and microdomain localization of Dok5 studied by single-molecule microscopy

In the present study, single-molecule fluorescence microscopy was used to examine the characteristics of plasma membrane targeting and microdomain localization of enhanced yellow fluorescent protein (eYFP)-tagged wild-type Dok5 and its variants in living Chinese hamster ovary (CHO) cells. We found that Dok5 can target constitutively to the plasma membrane, and the PH domain is essential for this process. Furthermore, single-molecule trajectories analysis revealed that Dok5 can constitutively partition into microdomain on the plasma membrane. Finally, the potential mechanism of microdomain localization of Dok5 was discussed. This study provided insights into the characteristics of plasma membrane targeting and microdomain localization of Dok5 in living CHO cells.     

Microarrays based on affinity-tagged single-chain Fv antibodies: sensitive detection of analyte in complex proteomes

Protein-based microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform global proteome analysis. However, the process of designing adequate protein microarrays is a major inherent problem. In this study, we have evaluated a protein microarray platform based on nonpurified affinity-tagged single-chain (sc) Fv antibody fragments to generate proof-of-principle and to demonstrate the specificity and sensitivity of the array design. To this end, we used our human recombinant scFv antibody library genetically constructed around one framework, the n-CoDeR library containing 2 x 10(10) clones, as a source for our probes. The probes were immobilized via engineered C-terminal affinity tags, his- or myc-tags, to either Ni(2+)-coated slides or anti-tag antibody coated substrates. The results showed that highly functional microarrays were generated and that nonpurified scFvs readily could be applied as probes. Specific and sensitive microarrays were obtained, providing a limit of detection in the pM to fM range, using fluorescence as the mode of detection. Further, the results showed that spotting the analyte on top of the arrayed probes, instead of incubating the array with large sample volumes (333 pL vs. 40 microL), could reduce the amount of analyte required 4000 times, from 1200 attomole to 300 zeptomole. Finally, we showed that a highly complex proteome, such as human sera containing several thousand different proteins, could be directly fluorescently labeled and successfully analyzed without compromising the specificity and sensitivity of the antibody microarrays. This is a prerequisite for the design of high-density antibody arrays applied in high-throughput proteomics.     

Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections

We developed a protein microarray methodology that has the ability of serodiagnosis of IgM antibodies directed against TORCH pathogens. Six chemical surface modifications were validated by a dimension atomic force microscope (AFM) and contact angle measurement, agarose modified surface of which offered an appropriate platform for detecting IgM antibody. Further, signal amplification sensitivities on agarose modified microarrays were detected by Cy3-labeled biotin-streptavidin and immunogold-based assays. The detection limits of IgM antibody on the microarrays were 0.48 and 0.24mug/ml, quantitatively equal to 0.25 and 12.5pg, respectively, on each spot as ascertained by the two assays. Satisfactory linear correlations between the signal intensity and the logarithm of the IgM concentration were obtained. Finally, 60 serum samples characterized by a commercial ELISA were evaluated by the protein microarray. There were good concordances between the results of the protein microarray and ELISA assay for sorting of the TORCH infected sera (95.0% by fluorescence-based assay and 96.7% by immunogold-based assay). Clearly, the potential application of this protein microarray format facilitates clinical detection of not only the antibodies directed against TORCH pathogens but also other autoimmune diseases.     

A simple, rapid, and sensitive integrated protein microarray for simultaneous detection of multiple antigens and antibodies of five human hepatitis viruses (HBV, HCV, HDV, HEV, and HGV)

Protein microarrays for parallel detection of multiple viral antigens and antibodies have not yet been described in the field of human hepatitis virus infections. Here, we describe a simple, rapid and sensitive integrated protein microarray with three different reaction models. The integrated protein microarray could simultaneously determine in human sera two viral antigens (HBsAg, HBeAg) and seven viral antibodies (HBsAb, HBcAb, HBeAb, HCVAb, HDVAb, HEVAb, HGVAb) of human hepatitis viruses within 20 min. The results of the protein microarray were assessed directly by the naked eye but can also be analyzed by a quantitative detector. The detection limit of this protein microarray was 0.1 ng/ml for HBsAg. Overall, >85% concordance was observed between the integrated protein microarrays and an enzyme-linked immunosorbent assay for above hepatitis viral antigen and antibody detections in human sera. This integrated protein microarray can be easily optimized for clinical use and epidemiological screening for multiple hepatitis virus infections.     

Ultrasensitive detection of Shiga toxin 2 and its variants in Shiga toxin‐producing Escherichia coli strains by a time‐resolved fluorescence immunoassay

A rapid and sensitive two‐step time‐resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin‐producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu³⁺) chelate was then used as a detector, followed by fluorescence measurements using time‐resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1–1000 ng/ml). The intra‐ and inter‐assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2‐specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2‐specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation‐based platform and aid in the accurate and prompt diagnosis of STEC infections.     

Two steps forward,one step back: Determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers

XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5′–3′ polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interactions within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein–DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).     

An accurate proteomic quantification method: Fluorescence labeling absolute quantification (FLAQ) using multidimensional liquid chromatography and tandem mass spectrometry

A facile proteomic quantification method, fluorescent labeling absolute quantification (FLAQ), was developed. Instead of using MS for quantification, the FLAQ method is a chromatography-based quantification in combination with MS for identification. Multidimensional liquid chromatography (MDLC) with laser-induced fluorescence (LIF) detection with high accuracy and tandem MS system were employed for FLAQ. Several requirements should be met for fluorescent labeling in MS identification: Labeling completeness, minimum side-reactions, simple MS spectra, and no extra tandem MS fragmentations for structure elucidations. A fluorescence dye, 5-iodoacetamidofluorescein, was finally chosen to label proteins on all cysteine residues. The fluorescent dye was compatible with the process of the trypsin digestion and MALDI MS identification. Quantitative labeling was achieved with optimization of reacting conditions. A synthesized peptide and model proteins, BSA (35 cysteines), OVA (five cysteines), were used for verifying the completeness of labeling. Proteins were separated through MDLC and quantified based on fluorescent intensities, followed by MS identification. High accuracy (RSD% < 1.58) and wide linearity of quantification (1-10(5) ) were achieved by LIF detection. The limit of quantitation for the model protein was as low as 0.34 amol. Parts of proteins in human liver proteome were quantified and demonstrated using FLAQ.     

Improvements and simplifications in in-gel fluorescent detection of proteins using ruthenium II tris-(bathophenanthroline disulfonate): the poor man's fluorescent detection method

Fluorescent detection of proteins is a popular method of detection allying sensitivity, linearity and compatibility with mass spectrometry. Among the numerous methods described in the literature, staining with ruthenium II tris(bathophenanthroline disulfonate) is particularly cost-effective, but slightly cumbersome owing to difficulties in the preparation of the complex and complexity of staining protocols. We describe here the modifications on both aspects that allow to perform a higher contrast staining and offer a more robust method of complex preparation, thereby maximizing the advantages of the method.     

Visualization and force measurement of branching by Arp2/3 complex and N-WASP in actin filament

To determine whether the Arp2/3 complex activated by N-WASP (VCA) branches actin filaments at the side (side branching), or at the barbed (B-)end (end branching) of the mother filaments, we have directly observed the branching process of actin filaments and examined single-molecule unbinding under optical microscope. We found that side branching was predominant, though not exclusive. At the initial stage of polymerization, the branching at the B-end occurred and subsequently the side branching started to occur. In either type of branching, the mother and daughter filaments elongated at nearly the same rate (growing type). Independently of the stage of polymerization, branching due to the direct coupling of filaments with an acute angle to the mother filaments (a coupling type) occurred. Phalloidin suppressed the growing type of branching but not the coupling type, implying that actin monomers are required for the former but not the latter. We found, by single molecule measurements using optical tweezers, that the Arp2/3 complex attaches to the side of actin filaments and the N-WASP appears to detach from the actin-Arp2/3 complex at 6-7 pN.     

A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens

A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.     

Single-molecule fluorescence characterization in native environment

Single-molecule detection (SMD) with fluorescence is a widely used microscopic technique for biomolecule structure and function characterization. The modern light microscope with high numerical aperture objective and sensitive CCD camera can image the brightly emitting organic and fluorescent protein tags with reasonable time resolution. Single-molecule imaging gives an unambiguous bottom-up biomolecule characterization that avoids the "missing information" problem characteristic of ensemble measurements. It has circumvented the diffraction limit by facilitating single-particle localization to ~1 nm. Probes developed specifically for SMD applications extend the advantages of single-molecule imaging to high probe density regions of cells and tissues. These applications perform under conditions resembling the native biomolecule environment and have been used to detect both probe position and orientation. Native, high density SMD may have added significance if molecular crowding impacts native biomolecule behavior as expected inside the cell.     

Pentazocine monitoring in rat hair and plasma by HPLC-fluorescence detection with DIB-Cl as a labelling reagent.

Pentazocine (PZ) in rat hair and plasma was determined by HPLC-fluorescence detection with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a labelling reagent and cyclazocine (CZ) as an internal standard (IS). PZ and IS extracted from hair or plasma sample were derivatized with DIB-Cl and the resulted solution was cleaned up with solid phase extraction. The isocratic separation of DIB-PZ and -CZ within 20 min could be achieved by a Wakopak Handy-ODS column (250 x 4.6 mm i.d.) using a mobile phase composed of 0.1 mol/L acetate buffer (pH 6.2):acetonitrile (25:75, v/v). The detection limits of PZ at a signal-to-noise ratio of 3 for rat hair and plasma were 0.18 ng/mg and 0.57 ng/mL, respectively. Reproducible and precise results could be obtained by an IS method with RSD values less than 6.6% for within- and between-day measurements. The method was successfully applied for the monitoring of PZ levels in Zucker rat hair and plasma samples after a single administration of 25 mg/kg PZ. Moreover, incorporation rates of PZ into black and white hair of Zucker rat were evaluated.     

An ELISA based on a truncated soluble ORF2 protein for the detection of PCV2 antibodies in domestic pigs

Postweaning multisystemie wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA)based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore ,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.     

Immuno-qPCR detection of the tandem affinity purification (TAP)-tag as a sensitive and accurate tool suitable for large-scale protein quantification

The tandem affinity purification (TAP)-tag has rapidly gained a wide popularity, mostly in studies on protein interactions, but lately also in large-scale protein quantification studies. We have developed an immuno-quantitative real-time PCR (qPCR) method to achieve rapid, sensitive and accurate quantification of TAP-tagged (and protein A-tagged) proteins in yeast with a detection range between 10(7) and 10(10) molecules. The immuno-qPCR protein quantification showed an excellent correlation to the published in vivo fluorescent protein (GFP)-based large-scale protein quantifications, but allowed for a much higher sensitivity. The correlation with published data from the large-scale Western blotting-based quantification of the TAP-tag was lower, but the sensitivity of detection was on roughly the same level. The practical use of the immuno-qPCR approach was demonstrated by analysis of osmo-regulated proteins, where the 2000-fold increase in expression of Catalase (Ctt1p), from an extremely low basal expression, could be accurately quantified. All steps of the method, from cell growth, to protein extraction and determination and the immuno-qPCR reaction itself are potentially amenable to automatization. Therefore, since the TAP-tag and protein A are useful in most model organisms, the immuno-qPCR method is both generic and suitable for large-scale studies.     

Conformational selection in protein binding and function

Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may “select” protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational‐selection and induced‐change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational‐selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced‐change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on‐ and off‐rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or single‐molecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational‐selection and induced‐change processes in both binding and unbinding direction.     

An internally eGFP‐tagged α‐adaptin is a fully functional and improved fiduciary marker for clathrin‐coated pit dynamics

Clathrin mediated endocytosis (CME) has been extensively studied in living cells by quantitative total internal reflection fluorescence microscopy (TIRFM). Fluorescent protein fusions to subunits of the major coat proteins, clathrin light chains or the heterotetrameric adaptor protein (AP2) complexes, have been used as fiduciary markers of clathrin coated pits (CCPs). However, the functionality of these fusion proteins has not been rigorously compared. Here, we generated stable cells lines overexpressing mRuby‐CLCa and/or μ2‐eGFP, σ2‐eGFP, two markers currently in use, or a novel marker generated by inserting eGFP into the unstructured hinge region of the α subunit (α‐eGFP). Using biochemical and TIRFM‐based assays, we compared the functionality of the AP2 markers. All of the eGFP‐tagged subunits were efficiently incorporated into AP2 and displayed greater accuracy in image‐based CCP analyses than mRuby‐CLCa. However, overexpression of either μ2‐eGFP or σ2‐eGFP impaired transferrin receptor uptake. In addition, μ2‐eGFP reduced the rates of CCP initiation and σ2‐eGFP perturbed AP2 incorporation into CCPs and CCP maturation. In contrast, CME and CCP dynamics were unperturbed in cells overexpressing α‐eGFP. Moreover, α‐eGFP was a more sensitive and accurate marker of CCP dynamics than mRuby‐CLCa. Thus, our work establishes α‐eGFP as a robust, fully functional marker for CME.     

Rapid specific detection and quantification of bacteria and archaea involved in mineral sulfide bioleaching using real-time PCR

A SybrGreen real-time PCR assay was developed to detect and quantify both total and selected 16S rDNA species of bacteria and archaea involved in the bioleaching of metals from sulfide ores. A set of specific and universal primers based on 16S rDNA sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of Acidianus brierleyi, Sulfolobus sp., Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus, Acidithiobacillus caldus, and Leptospirillum ferrooxidans. An artificial sequence based on 16S rDNA was constructed to quantify total 16S rDNA in mixed DNA samples. The real-time PCR assay was further validated using a mixture of 16S rDNA amplicons derived from the six different species, each added at a known amount. Finally, the real-time PCR assay was used to monitor the change of 16S rDNA copies of four bioleaching strains inoculated into chalcopyrite airlift column reactors operated at different temperatures. The growth dynamics of these strains correlated well with the expected effects of temperature in the chalcopyrite-leaching environment. The suitability of this method for monitoring microbial populations in industrial bioleaching environments is discussed.     

In vitro reconstitution of a transport complex containing Rab27a, melanophilin and myosin Va

Rab27a and melanophilin (Mlph) are required in vivo to form a melanosome receptor for myosin Va in which Rab27a anchored in the melanosome membrane recruits Mlph, which in turn recruits myosin Va. Here, we show by reconstitution using purified proteins that Rab27a and Mlph are sufficient to form a transport complex with myosin Va in vitro. These results suggest that additional proteins are not required in vivo for assembly of the myosin Va receptor, although other proteins may associate with this tripartite complex to regulate its activity and/or to assist Rab27a in anchoring the complex to the melanosome membrane.     

A rhodamine‐triazole fluorescent chemodosimeter for Cu2+ detection and its application in bioimaging

A rhodamine‐based fluorescent chemodosimeter rhodamine hydrazide‐triazole (RHT) tethered with a triazole moiety was developed for Cu²⁺ detection. In aqueous medium, the RHT probe exhibited high selectivity and sensitivity toward Cu²⁺ among other metal ions. The addition of Cu²⁺ triggered a fluorescence emission of RHT by 384‐fold (Φ = 0.33) based on a ring‐opening process and a subsequent hydrolysis reaction. Moreover, RHT also showed a selective colorimetric response toward Cu²⁺ from colorless solution to pink, readily observed with the naked eye. The limit of detection of RHT for Cu²⁺ was calculated to be 1 nM (0.06 ppb). RHT was successfully demonstrated to detect Cu²⁺ in Chang liver cells by confocal fluorescence microscopy.