Abstract: Seminal plasma is a promising biological fluid to use for non-invasive clinical diagnostics of male reproductive system disorders. To verify a list of prospective male infertility biomarkers, we developed a multiplex selected reaction monitoring (SRM) assay and measured relative abundance of 31 proteins in 30 seminal plasma samples from normal, non-obstructive azoospermia (NOA) and post-vasectomy (PV) individuals. Median levels of some proteins were decreased by more than 100-fold in NOA or PV samples, in comparison to normals. To follow up the most promising candidates and measure their concentrations in seminal plasma, heavy isotope-labeled internal standards were synthesized and used to re-analyze 20 proteins in the same set of samples. Concentrations of candidate proteins in normal seminal plasma were found in the range 0.1-1000 ug/mL, but were significantly decreased in NOA and PV. This data allowed us to select, for the first time, biomarkers to discriminate between normal, NOA and PV (simulated obstructive azoospermia) seminal plasma samples. Some testis-specific proteins (LDHC, TEX101, SPAG11B) performed with absolute or near-absolute specificities and sensitivities. Cell-specific classification of protein expression indicated that Sertoli or germ cell dysfunction, but not Leydig cell dysfunction, was observed in NOA seminal plasma. The proposed panel of biomarkers, pending further validation, could lead to a clinical assay which can eliminate the need for testicular biopsy to diagnose the category of male infertility, thus providing significant benefits to patients as well as decreased costs associated with the differential diagnosis of azoospermia.
Abstract: Prenatal screening test for Down syndrome (DS) can be improved by discovery of novel biomarkers. A multiplex selected reaction monitoring (SRM) assay was developed to test previously identified thirteen candidate proteins in amniotic fluid (AF). One unique peptide was selected for each protein based on discovery data, while three MS/MS transitions were selected based on intelligent SRM results. For one of the candidates, matrix metalloproteinase-2 (MMP2), ELISA was also performed to validate SRM results in AF and to test serum samples. Comparison of AF samples from DS versus controls via SRM assay revealed five proteins that were differentially expressed. Bile salt-activated lipase, mucin-13, carboxypeptidase A1, and dipeptidyl peptidase 4 showed a decrease in DS-affected AF, and MMP2 showed an increase, in comparison to controls (P<0.05). Discovery-based spectral counting ratios and SRM ratios showed a strong correlation, and MMP2 ELISA further confirmed the validity of the SRM data. Potential implications of differentially expressed proteins during fetal development are proposed. Our data also shows that SRM can provide a high-throughput and accurate platform for biomarker verification.
Abstract: Low-abundance proteins present in biological fluids are often considered an attractive source of new disease biomarkers. Since such proteins are poorly observed in proteome-scale discovery experiments due to an overwhelming mass of high-abundance proteins, the development of quantitative multiple reaction monitoring (MRM) assays for low-abundance proteins is a challenging task. Here, we present a strategy that facilitates the development of MRM assays for large numbers of unpurified low-abundance proteins. Our discovery strategy is based on the reduction of the dynamic range of protein concentrations in biological fluids by means of one-bead one-compound combinatorial peptide libraries (CPL). Our 2D-LC-MS/MS approach allowed us to identify a total of 484 unique proteins in ovarian cancer ascites, and 216 proteins were assigned as low-abundance ones. Interestingly, 74 of those proteins have never been previously described in ascites fluid. Treatment with CPL allowed identification of a significantly higher number of unique peptides for low-abundance proteins and provided important empirical fragmentation information for development of MRM assays. Finally, we confirmed that MRM assays worked for 30 low-abundance proteins in the unfractionated ascites digest. Using a multiplexed MRM method, relative amounts of five proteins (kallikrein 6, metalloproteinase inhibitor 1, macrophage migration inhibitory factor, follistatin-related protein, and mesothelin) were determined in a set of ovarian cancer ascites. Multiplexed MRM assays targeting large numbers of proteins can be used to develop comprehensive panels of biomarkers with high sensitivity and selectivity, and to study complex protein networks.
Abstract: Mass spectrometry (MS) is an attractive alternative to quantification of proteins by immunoassays, particularly for protein biomarkers of clinical relevance. Reliable quantification requires that the MS-based assays are robust, selective, and reproducible. Thus, the development of standardized protocols is essential to introduce MS into clinical research laboratories. The aim of this study was to establish a complete workflow for assessing the transferability and reproducibility of selected reaction monitoring (SRM) assays between clinical research laboratories. Four independent laboratories in North America, using identical triple-quadrupole mass spectrometers (Quantum Ultra, Thermo), were provided with standard protocols and instrumentation settings to analyze unknown samples and internal standards in a digested plasma matrix to quantify 51 peptides from 39 human proteins using a multiplexed SRM assay. The interlaboratory coefficient of variation (CV) was less than 10% for 25 of 39 peptides quantified (12 peptides were not quantified based upon hydrophobicity) and exhibited CVs less than 20% for the remaining peptides. In this report, we demonstrate that previously developed research platforms for SRM assays can be improved and optimized for deployment in clinical research environments.
Abstract: The development of drugs and diagnostics with desirable characteristics requires smart small-molecule ligandsligands with predefined binding parameters of interaction with the target. Here, we propose a general approach for selection of such ligands from highly diverse combinatorial libraries of small molecules by methods of kinetic capillary electrophoresis (KCE). We deduct three fundamental requirements for the combinatorial library to suit the KCE-based selection of smart ligands and suggest a universal design of the library for selecting smart small-molecule ligands: every small molecule in the library is tagged with DNA that encodes the structure of the molecule. Finally, we use several DNA-tagged small molecules, which represent a hypothetical library, to prove experimentally selection of smart small-molecule ligands by the proposed approach.
Abstract: A new method called dynamic kinetic capillary isoelectric focusing (DK-CIEF) is presented for the study of protein-DNA interactions. The method is based on CIEF with laser-induced fluorescence-whole column imaging detection in which protein-DNA complexes are separated with spatial resolution while dissociations of the complexes are dynamically monitored using a CCD camera with temporal resolution. This method allows for the discrimination of different complexes and the measurement of the individual dissociation rate constants.
Abstract: Single-nucleotide polymorphisms (SNPs) are widespread genomic variations, which are associated with serious health disorders and drug resistance. Multiple clinical applications and studies of global population genetics require fast and informative analysis of SNPs. Most of conventional methods sense the presence of the SNP but cannot identify the base pair in it. Here we report simple identification of base pairs in SNPs without DNA sequencing. Our approach is based on the unique ability of MutS protein to bind different single-nucleotide mismatches in DNA with different affinities. Conceptually, the DNA in question is mixed with reference DNA, melted, and reannealed. If the DNA in question has an SNP, the products of reannealing will have two different single-nucleotide mismatches, which provide a base-pair-specific signature of the SNP. The products of reannealing are mixed with MutS, equilibrated, and separated by equilibrium capillary electrophoresis of equilibrium mixtures with MutS in the run buffer. The pattern of migration times of DNAs with mismatches is used for unequivocal identification of the base pair in the SNP. In addition to its ability to identify base pairs in SNPs, the new analytical approach is fast, simple, highly sensitive, and requires no quantitation. It will find applications in studies of heterogeneity of base pairs in known SNPs in large human populations.
Abstract: Aptamers are typically selected from libraries of random DNA (or RNA) sequences by SELEX, which involves multiple rounds of alternating steps of partitioning and PCR amplification. Here we report, for the first time, non-SELEX selection of aptamers-a process that involves repetitive steps of partitioning with no amplification between them. A highly efficient affinity method, non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), was used for partitioning. We found that three steps of NECEEM-based partitioning in the non-SELEX approach were sufficient to improve the affinity of a DNA library to a target protein by more than 4 orders of magnitude. The resulting affinity was higher than that of the enriched library obtained in three rounds of NECEEM-based SELEX. Remarkably, NECEEM-based non-SELEX selection took only 1 h in contrast to several days or several weeks required for a typical SELEX procedure by conventional partitioning methods. In addition, NECEEM-based non-SELEX allowed us to accurately measure the abundance of aptamers in the library. Not only does this work introduce an extremely fast and economical method for aptamer selection, but it also suggests that aptamers may be much more abundant than they are thought to be. Finally, this work opens the opportunity for selection of drug candidates from libraries of small molecules, which cannot be PCR-amplified and thus are not approachable by SELEX.
Abstract: Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX), which involves several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. Here we describe a protocol for non-SELEX selection of aptamers--a process that involves repetitive steps of partitioning with no amplification between them. Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), which is a highly efficient affinity method, is used for partitioning. NECEEM also facilitates monitoring of bulk affinity of enriched libraries at every step of partitioning and screening of individual clones for their affinity to the target. NECEEM allows all clones to be screened prior to sequencing, so that only clones with suitable binding parameters are sequenced. The entire protocol can be completed in 1 wk, whereas conventional SELEX protocols take several weeks even in a specialized industrial facility.
Abstract: We coin the term "smart aptamers" -- aptamers with predefined binding parameters (k(on), k(off), Kd) of aptamer-target interaction. Aptamers, in general, are oligonucleotides, which are capable of binding target molecules with high affinity and selectivity. They are considered as potential therapeutic targets and also thought to rival antibodies in immunoassay-like analyses. Aptamers are selected from combinatorial libraries of oligonucleotides by affinity methods. Until now, technological limitations have precluded the development of smart aptamers. Here, we report on two kinetic capillary electrophoresis techniques applicable to the selection of smart aptamers. Equilibrium capillary electrophoresis of equilibrium mixtures was used to develop aptamers with predefined equilibrium dissociation constants (Kd), while nonequilibrium capillary electrophoresis of equilibrium mixtures facilitated selection of aptamers with different dissociation rate constants (k(off)). Selections were made for MutS protein, for which aptamers have never been previously developed. Both theoretical and practical aspects of smart aptamer development are presented, and the advantages of this new type of affinity probes are described.
Abstract: Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K(d), k(off), and k(on) of the aptamer-target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.
Abstract: We coin a term of "smart aptamers", which describes aptamers with predefined binding parameters of their interaction with the target. Here, we introduce a method for selection of smart aptamers with predefined values of Kd: equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM). Conceptually, a mixture of a target with a DNA (RNA) library is prepared and equilibrated. A plug of the equilibrium mixture is injected into a capillary prefilled with a run buffer containing the target at the concentration identical to the target concentration in the equilibrium mixture. The components of the equilibrium mixture are separated by capillary electrophoresis while equilibrium is maintained between the target and aptamers. The unique feature of ECEEM is that aptamers with different Kd values migrate with different and predictable mobilities. Thus, collecting fractions with different mobilities results in smart aptamers with different and predefined Kd values. In this proof-of-principle work, we used ECEEM to select smart aptamers for MutS protein, for which aptamers have never been previously selected. Three rounds of ECEEM-based selection were sufficient to obtain smart aptamers with Kd values approaching theoretically predicted ones. ECEEM is the first method for aptamer selection whose ability to generate smart aptamers has been experimentally proven.
Abstract: It has been recently demonstrated that single-stranded DNA-binding protein (SSB) can facilitate quantitative analyses of DNA, RNA, and proteins in gel-free capillary electrophoresis (CE). Here, we report the application of SSB-mediated gel-free CE for analyses of polymerase chain reaction (PCR) products. The unique ability of SSB to bind ssDNA but not double-stranded DNA (dsDNA) allows efficient separation of three types of DNA molecules in the PCR reaction mixture: primers, products (amplified templates), and by-products, which originate from non-specific DNA hybridization. SSB-mediated gel-free CE analysis of PCR products combines simplicity, high sensitivity, and outstanding quantitative capabilities. The ability of the method to distinguish between products and by-products makes this method an indispensable tool in preparative PCR (e.g., in the development of nucleotide aptamers).
