Search

The analysis of airway fluid, as sampled by bronchoalveolar lavage (BAL), provides a minimally invasive route to interrogate lung biology in health and disease. Here, we used immunodepletion, coupled with gel- and label-free LC-MS/MS, for quantitation of the BAL fluid (BALF) proteome in samples recovered from human subjects following bronchoscopic instillation of saline, lipopolysaccharide (LPS) or house dust mite antigen into three distinct lung subsegments. Among more than 200 unique proteins quantified across nine samples, neutrophil granule-derived and acute phase proteins were most highly enriched in the LPS-exposed lobes. Of these, peptidoglycan response protein 1 was validated and confirmed as a novel marker of neutrophilic inflammation. Compared to a prior transcriptomic analysis of airway cells in this same cohort, the BALF proteome revealed a novel set of response factors. Independent of exposure, the enrichment of tracheal-expressed proteins in right lower lung lobes suggests a potential for constitutive intralobar variability in the BALF proteome; sampling of multiple lung subsegments also appears to aid in the identification of protein signatures that differentiate individuals at baseline. Collectively, this proof-of-concept study validates a robust workflow for BALF proteomics and demonstrates the complementary nature of proteomic and genomic techniques for investigating airway (patho)physiology.

The electrochemical detection of nucleic acid targets at low concentrations has a number of applications in diagnostics and pharmaceutical research. Self-assembled monolayers of alkanethiol-derivatized oligonucleotides on gold electrodes provide a useful platform for such detectors, and the electrocatalytic oxidation of nucleobases included in the DNA targets is a particularly sensitive method of electrochemical detection. A strategy has been developed for combining these two aspects by substituting either 7,8-dihydro-8-oxoguanine (8G) or 5-aminouridine (5U) into DNA targets. Upon hybridization of targets containing these modified nucleobases, electrocatalytic signals at probe-modified gold electrodes are observed in the presence of Os(bpy)(3)(2+), which oxidizes both 8G and 5U upon oxidation to the Os(III) state. Self-assembled monolayers were prepared on both macro (1.6 mm) and micro (25 microm) gold electrodes using published procedures involving C6-terminated alkanethiol oligonucleotides and mercaptohexanol as the diluent. The extent of electrode modification by the modified probe was assessed using radiolabeling and a standard chronocoulometry method; both approaches gave loading levels within expected ranges ((1-6) x 10(12) molecules/cm(2)). Hybridization of the modified targets where the non-native nucleobase was incorporated by solid-phase synthesis produced electrocatalytic signals from strands that were independently detected using radiolabeling and chronocoulometry. This result was used as a basis to develop an on-electrode amplification scheme where Taq polymerase was used to extend the immobilized DNA probes from solution-phase polymeric templates using modified nucleotriphosphates. This reaction produced an electrode that was modified with extended DNA containing the appropriate modified nucleotide. Radiolabeled nucleotide triphosphates were used to confirm the desired on-electrode DNA synthesis. When these electrodes were cycled in the presence of Os(bpy)(3)(2+), electrocatalytic signals were observed when as little as 40 amol (400 fM) of the desired target was present in the hybridization solution.

The 7-deaza analogues of guanine and adenine were incorporated into polymerase chain reaction (PCR) products by substitution of the appropriate nucleotide triphosphates into the reaction. These PCR products can be immobilized on ITO electrodes and detected by catalytic cyclic voltammetry with ruthenium polypyridyl complexes. Immobilization on indium tin oxide (ITO) electrodes of 330- and 1200-base pair (bp) PCR amplicons from the E. coli dacA gene containing one or both of the 7-deazapurines was effected by precipitation from a 9:1 DMF/acetate solution. Amplicons containing the 7-deazaguanine base were detected by observing current enhancement in the cyclic voltammogram of Ru(dmb)3(3)+/2+ (dmb = 4,4'-dimethyl-2,2'-bipyridine) due to the selective oxidation of the modified base by this mediator. Oxidation of incorporated 7-deazaadenine bases in addition to native guanines gives rise to a higher current enhancement in the cyclic voltammogram of Ru(bpy)3(3)+/2+ (bpy = 2,2'-bipyridine) compared to the enhancement observed in the presence of guanine only. This strategy was employed to simultaneously detect the 330-bp sequence containing 7-deazaadenine and the 1200-bp sequence containing 7-deazaguanine on the same ITO electrode. Such a strategy may provide a means for detecting multiple genes on a single microlocation and may thereby lead to more highly multiplexed gene assays.

Genomic expansion of the triplet repeat sequences 5'-(CTG)n and 5'-(CGG)n leads to myotonic dystrophy and fragile X syndrome, respectively. Methods for determining the number of repeats in unprocessed nucleic acids would be useful in diagnosing diseases based on triplet repeat expansion. Electrochemical reactions based on the oxidation of guanine were expected to give larger signals per strand for expansion of repeats containing guanine. A novel PCR reaction was used to generate fragments containing 150, 230, 400, and 830 repeats of (CTG)n, which codes for myotonic dystrophy, and 130 and 600 repeats of (CGG)n, which codes for fragile X syndrome. These PCR fragments were immobilized to indium tin oxide electrodes, and oxidation of guanine in the fragments was realized using electrocatalysis by Ru(bpy)3(2+) (bpy = 2,2'-bipyridine). The catalytic currents due to oxidation of the immobilized guanines by Ru(bpy)3(3+) increased with the number of repeats and were a linear function of the repeat number when normalized to the number of strands immobilized. These results suggest a sensing strategy for repeat length based on the combination of the electrocatalytic strategy for determining the repeat length combined with existing methods for determining the number of strands.

Addition of oligonucleotides containing 7-deazaguanine (Z) to solutions containing Ru(dmb)3(2+) (dmb = 4,4'-dimethyl-2,2'-bipyridine) produces an enhancement in the oxidative current in the cyclic voltammogram of the metal complex that can be used, through digital simulation, to determine the rate of oxidation of 7-deazaguanine by Ru(dmb)3(3+). The measured rate constants are about 10 times higher than those for oxidation of guanine by Ru(bpy)3(3+), even though the redox potential of Ru(dmb)3(3+/2+) is 200 mV lower. A potential of 0.75 V (vs Ag/AgCl) can therefore be estimated for the oxidation of 7-deazaguanine, which can be selectively oxidized over guanine when Ru(dmb)3(3+) is the oxidant. The rate of oxidation was much faster in single-stranded DNA, and the difference between rates of single-stranded and duplex DNA was higher than for guanine. The oxidation rate was also sensitive to the presence of a single-base mismatch at the 7-deazaguanine in the order Z.C < Z.T < Z.G approximately Z.A < single-stranded. The Z.T mismatch was much more readily distinguished than the G.T mismatch, consistent with the overall greater sensitivity to secondary structure for Z. The oxidation reaction was also probed by monitoring piperidine-labile cleavage at the Z nucleotide, which could be generated by treatment with either photogenerated Ru(bpy)3(3+) or the thermal oxidant Ru(tpy)(bpy)O2+ (tpy = 2,2',2' '-terpyridine). These oxidants gave qualitatively similar selectivities to the electron-transfer rates from cyclic voltammetry, although the magnitudes of the selectivities were considerably lower on the sequencing gels.

The oxidation of guanines in DNA by Ru(III) is investigated by catalytic electrochemistry and stopped-flow spectrophotometry. The reactions of calf thymus DNA (20% guanine) and herring testes DNA (25% guanine) with Ru(bpy)3(3+) (bpy = 2,2'-bipyridine) show biexponential decays in stopped-flow spectrophotometric experiments with the fast and slow components in 2:1 ratios and average rate constants in 880 mM NaCl of = 18,700 M-1 s-1 for calf thymus DNA and = 24,600 M-1 s-1 for herring testes DNA. The higher rate constant for the more guanine-rich DNA is possibly due to a higher density of electron-rich guanine multiplets. The observation of a biexponential decay is incorporated into digital simulations of the catalytic voltammograms observed for Ru(bpy)3(2+) in the presence of DNA. For both DNAs, the rates observed by voltammetry are somewhat slower than those observed by stopped-flow spectrophotometry and the dependence of the rate constants on scan rate using the biexponential model is less pronounced than when only one decay is treated, supporting the notion that the scan rate dependence arises from the multiphasic decay. At low salt concentrations, where binding of the metal complex to DNA increases the effective catalytic rate constant, rates can be measured by stopped-flow spectrophotometry only with a less oxidizing complex, Fe(bpy)3(3+/2+), which yields trends in the rate constants similar to those observed for the case of Ru(bpy)3(3+/2+) at high ionic strength. Oligonucleotides based on the trinucleotide repeat sequences (AGT)n and (GAA)n produce significant catalytic currents, which are readily interpreted in terms of the guanine concentration and the secondary structure discerned from gel electrophoresis experiments. These experiments may provide a basis for sensing secondary structures and repeat numbers in biologically relevant DNAs.