Urine is a complex fluid, which is thought to contain valuable diagnostic information regarding general health. In particular, there is great diagnostic potential in the peptide and/or protein ...content of urine, but the information is present in low abundance. Most traditional proteomic techniques lack sufficient sensitivity/dynamic range, especially for dilute and/or complex samples. However, orthogonal separation methods can be applied prior to protein/peptide analysis to increase the success rate of urine proteomic studies and access this potentially valuable information. In this chapter, we describe isoelectric focusing (IEF) of intact urine proteins, via free flow electrophoresis (FFE), prior to typical peptide-based mass spectrometry analysis, facilitating the deep analysis of urine protein detection and identification, for biomarker discovery. Our work demonstrates that such an approach can be used as a preprocessing step and can be integrated into a workflow for the successful identification of protein components (biomarkers) from urine.
Re‐sequencing, the identification of the specific variants in a sequence of interest compared with a known genomic sequence, is a ubiquitous task in today’s biology. Universal arrays, which ...interrogate all possible oligonucleotides of a certain length in a target sequence, have been suggested for computationally determining a polynucleotide sequence from its oligonucleotide content. We present here new methods that use such arrays for re‐sequencing. Our methods are applied to data obtained by the polymerase signaling assay, which arrays single‐based primer extension reactions for either universal or partial arrays of pentanucleotides. The computational analysis uses the spectrum alignment algorithm, which is refined and enhanced here in order to overcome noise incurred by the use of such short primers. We present accurate re‐sequencing results for both synthetic and amplified DNA molecules.
Peptide biomarkers in plasma or serum are subject to proteolytic degradation caused by intrinsic peptidase activities, resulting in a potential barrier in translating a discovered biomarker into ...clinical application. This chapter describes a method using time-course MALDI-TOF MS analysis to investigate the stability of a plasma peptide biomarker under a variety of preanalytical situations. A synthesized peptide with the same primary sequence as a potential endogenous biomarker is spiked into a blood sample, and the sample is incubated over time at r.t. (25 ± 1°C) or other preanalytical situations. At a specific period of incubation time, the sample is quenched with the addition of acid with or without an internal control peptide. The spiked peptides in the sample are extracted with one of three procedures for highly soluble, moderately soluble, or essentially insoluble peptides. The peptide samples are then analyzed using MALDI-TOF MS. The abundance changes of the peptide biomarker are monitored by time-course changes of the mass spectra. These changes over-time are measured and fitted to a first-order degradation reaction so that stability of the peptide biomarker (half-life) can be calculated. Kinetics analysis of both parent and shorter (daughter) peptides are also possible by fitting to a sequential multiple-step reaction (SMSR) model. This optimized method facilitates evaluation of biomarker stability, and helps to define sample handling and analytical processing steps that contribute to instability of measured peptide biomarker(s).
The exocyclic base adduct 3,N 4-deoxyethenocytosine (εC) is a common DNA lesion that can arise from carcinogen exposure and/or as a biproduct of cellular processes. We have examined the thermal and ...thermodynamic impact of this lesion on DNA duplex properties, as well as the structural alterations imparted by the lesion. For these studies, we used calorimetric and spectroscopic techniques to investigate a family of 13-mer DNA duplexes of the form (5‘CGCATGNGTACGC3‘)•(3‘GCGTACNCATGCG5‘), where the central N•N base pair represents the four standard Watson−Crick base pairs (corresponding to four control duplexes), and where either one of the N bases has been replaced by εC, yielding eight test duplexes. Studies on these 12 duplexes permit us to assess the impact of the εC lesion as a function of sequence context. Our spectroscopic and calorimetric data allow us to reach the following conclusions: (i) The εC lesion imparts a large penalty on duplex stability, with sequence context only modestly modulating the extent of this lesion-induced destabilization. This result contrasts with our recent studies of duplexes with abasic sites, where sequence context was found to be the predominant determinant of thermodynamic damage. (ii) For the εC-containing duplexes, sequence context effects are most often observed in the enthalpic contribution to lesion-induced duplex destabilization. However, due to compensating entropies, the free energy changes associated with this lesion-induced duplex destablization are nearly independent of sequence context. (iii) Despite significant lesion-induced changes in duplex energetics, our spectroscopic probes detect only modest lesion-induced changes in duplex structure. In fact, the overall duplex maintains a global B-form conformation, in agreement with NMR structural data. We discuss possible interpretations of the apparent disparity between the severe thermodynamic and relatively mild structural impacts of the εC lesion on duplex properties. We also note and discuss the implications of empirical correlations between biophysical and biological properties of lesion-containing duplexes.
The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated ...that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted α-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/61), which is transcriptionally inactive, possesses a secondary structure similar to that of P0. P1, at a protein concentration of 50 μg/ml, elutes from a gel filtration column apparently as a dimer, whereas both P0 and the double serine mutant elute as a monomer at the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine required a high concentration of CKII for optimal phosphorylation. We demonstrate here that phosphorylation of either Ser-59 or Ser-61 is necessary and sufficient to transactivate L polymerase although alteration of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficiently than P0 and the formation of P1 is a prerequisite for the subsequent phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcription reconstitution assay in the presence of wild-type P protein.
Bistrand abasic DNA lesion was thermodynamically characterized. Experiments reveal that, relative to a control duplex, the lesion is both thermally and thermodynamically destabilizing, with this ...lesion-induced destabilization being entirely entropic in origin. The presence of the lesion has no effect on the nature of the duplex melting process or the global conformation of the host duplex. These findings suggest that the lesion forms an extrahelical loop, thereby allowing the two helical halves to collapse on one another to form an uninterrupted B-form duplex structure. This may help explain the biochemical observation that this lesion is less efficiently repaired.
The interaction of the retroviral nucleocapsid protein (NC) with nucleic acids forms the basis of its varied roles in the replication cycle, which include binding and condensing the viral RNA within ...the virion, stimulation of the early steps in reverse transcription, and dissociation from RNA in the replication complex. As part of an investigation of the NC binding site and of the forces that drive its interaction with nucleic acids, the relative affinities of NC from avian myeloblastosis virus were determined for a series of mononucleotides and mononucleotide components using a competitive displacement assay utilizing the extrinsic fluorescent probe bis-ANS Secnik, J., Wang, Q., Chang, C.-M., & Jentoft, J.E. (1990) Biochemistry 29, 7991-7997. The estimated binding affinities were unexpectedly similar for nucleotides, nucleosides, and bases (Ka greater than 10(6) M-1). AMP, UMP, GMP, and CMP bound to NC with essentially equal affinity, indicating that NC does not discriminate between bases. This is consistent with its role in coating, condensing, and packaging the RNA within virions. Nucleosides, bases, riboses, and ribose phosphate bind to NC with 1000-fold higher affinity than inorganic phosphate, indicating that the NC binding site includes elements that recognize nucleotide base and ribose components in addition to phosphate ions. However, the binding affinities of components are not additive, i.e., the Kapp values for adenine and deoxyribose are very similar to that for deoxyadenosine, indicating that the interaction between the NC subsite and the base and the sugar components is complex. The stoichiometry of the complex between bis-ANS and NC was established to be NC.(bis-ANS)3.
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