ABSTRACT
Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.
Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/genetics , Gene Expression Profiling/methods , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Proteins/analysis , Biomarkers, Tumor/genetics , Breast Neoplasms/diagnosis , Breast Neoplasms/metabolism , Cell Line, Tumor , Chromatography, Liquid/methods , Female , Gene Expression Regulation, Neoplastic , Humans , Mass Spectrometry/methods , Microfluidic Analytical Techniques/economics , Proteins/geneticsABSTRACT
8-hydroxy-deoxyguanosine (8-OH-dG) DNA adduct is one of the most frequently used biomarkers reporting on the oxidative stress that leads to DNA damage. More sensitive and reliable microfluidic devices are needed for the detection of these biomarkers of interest. We have developed a capillary electrophoresis (CE)-based microfluidic device with an electroplated palladium decoupler that provides significantly improved detection limit, separation efficiency, and resolving power. The poly(dimethylsiloxane) (PDMS)/glass hybrid device has fully integrated gold microelectrodes covered in situ with palladium nanoparticles using an electroplating technique. The performance and coverage of the electrodes electroplated with palladium particles were evaluated electrochemically and via scanning electron microscope (SEM) imaging, respectively. The performance of the device was tested and evaluated with different buffer systems, pH values, and electric field strengths. The results showed that this device has significantly improved resolving power, even at separation electric field strengths as high as 600 V cm-1. The detection limit for the 8-OH-dG adduct is about 20 attomoles; the concentration limit is on the order of 100 nM (S/N=3). A linear response is reported for both 8-OH-dG and dG in the range from 100 nM to 150 microM (approximately 100 pA microM-1) with separation efficiencies of approximately 120,000-170,000 plates m-1.
Subject(s)
DNA Adducts/isolation & purification , Deoxyguanosine/analogs & derivatives , Microfluidic Analytical Techniques , Palladium/chemistry , 8-Hydroxy-2'-Deoxyguanosine , DNA Adducts/chemistry , Deoxyguanosine/chemistry , Deoxyguanosine/isolation & purification , Dimethylpolysiloxanes/chemistry , Electrochemistry , Electrophoresis, Capillary/instrumentation , Electrophoresis, Capillary/methods , Glass/chemistry , Metal Nanoparticles/chemistry , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Microscopy, Electron, Scanning , Oxidation-Reduction , Sensitivity and Specificity , Silicones/chemistry , Surface PropertiesABSTRACT
The development of novel and reliable technologies for the analysis of proteins and their post-translational modifications, in particular, has recently received much attention and interest. The implementation of a fully integrated microfluidic device interfaced with MS detection for the analysis of phosphoproteins is presented in this paper. The microfluidic platform (3''x1.5'') comprises two individual sample processing systems: one for performing direct sample infusion and one for performing microfluidic LC separations. Various MS detection strategies, specific for the study of post-translational modifications, were conducted using alpha-casein as a model protein. Neutral loss ion mapping, data-dependent triple-play and neutral loss analysis, and in situ dephosphorylation followed by LC separation and MS detection were performed. Consistent results in identifying phosphopeptides with conventional and microfluidic instrumentation have been obtained. Unlike with conventional instrumentation, however, the microfluidic device enabled the completion of each analysis from only a few microliters of sample, in approximately 10-15 min, and on a bioanalytical platform that facilitates multiplexing and disposability, and thus high-throughput, contamination-free analysis.