RESUMEN
Poly(dimethylsiloxane) (PDMS) has shown considerable promise in the fabrication of microfluidic devices. Surface modification of PDMS by the grafting of perfluorinated alkanes allows the selective adsorption of fluorous-tagged peptides, demonstrating that this material may be used in fluorous affinity tag technology to enrich and separate specific proteins or peptides from complex mixtures. Here, we explore the non-specific adsorption of proteins which may interfere with this process. The desorption of cytochrome c, carbonic anhydrase, insulin and ubiquitin onto the surfaces of unmodified, oxidized and fluorinated PDMS in solutions of varying water/methanol concentration has been studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The interaction forces involving perfluorinated surfaces are probed using chemical force spectrometry. The denaturation of the proteins in solutions of high methanol concentration is followed using electrospray ionization mass spectrometry (ESI-MS) and the adsorption profiles discussed in the context of the surface hydrophobicity of each protein.
Asunto(s)
Dimetilpolisiloxanos/química , Hidrocarburos Fluorados/química , Proteínas/aislamiento & purificación , Adsorción , Marcadores de Afinidad , Diseño de Equipo , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas , Proteínas/químicaRESUMEN
A novel electrospray interface, which has distinct advantages over conventional pulled silica emitters, has been developed. This novel interface can be easily fabricated by forming a porous polymer monolith (PPM) at the end of a fused-silica capillary that facilitates a stable electrospray over a wide range of flow rates with only a modest increase in back-pressure. A comparison was made between the PPM-assisted electrospray and a commercial nanosprayer in terms of sensitivity, stability and robustness. A PPM-filled electrospray tip produced a day-to-day signal variation of 23% relative standard deviation (RSD) over a 3-day period when spraying a 1.0 microM test peptide solution. Furthermore, three different capillaries fabricated by the same process produced a signal variation of 17% RSD, indicating that the fabrication process shows good reproducibility. The multiple flow paths of the PPM function to split the flow and reduce clogging. Even following the accumulation of debris after prolonged use, a stable spray could still be generated with the PPM-filled capillary while the commercial nanosprayer ceased to function properly. In terms of sensitivity, PPM-assisted electrospray showed an enhancement in sensitivity at infusion flow rates between 100 to 1000 nL/min while commercial nanosprayers performed slightly better at flow rates below 100 nL/min. A sample purification step can be combined with the PPM-assisted sprayer, using the PPM as a stationary phase to desalt and preconcentrate samples prior to mass spectrometric detection.