Microfluidic electrocapture in proteomic sample preparation for mass spectrometry

Abstract

Proteomics is a technology-driven discipline and very labor-intensive. Over the last couple of years, methods with microfluidic systems have been used to simplify analytical procedures via integration of laboratory processes and equipment into miniaturized formats to achieve reproducible and high throughput analyses of biomolecules.

In this thesis work, a microfluidic capture device was used in which charged biomolecules can be immobilized without chemical binding by application of an electric field along a microflow stream. The device was used in combination with either matrix assisted laser desorption ionization (MALDI) or electrospray ionization (ESI) mass spectrometry.

In paper I, different applications of the electrocapture technique in preconcentration, sample clean-up and separation were presented. In this demonstration initial work of the present thesis study were included.

In paper II, the electrocapture device was used for separation of proteolytic peptides and detergent removal. Capture takes place at an initial, high voltage which is then gradually reduced. Peptides were found to be released in fractions based on their electrophoretic behavior, and their analysis by MALDI-MS was successful. The mechanism and principles which form the basis of the electroimmobilization were investigated in paper III. A modified device with four electric junctions allowed measurements of the electric field values between the junctions. Application of a potential, leads to zones of different electric field strengths: low strength upstream (first junction) and high strength downstream (fourth junction). These zones occur because of ionic rearrangements which are based on the interactions between the channel and the electrode chambers via cation-selective membranes. It was shown that charged molecules can be immobilized in the channel by a stacking mechanism between the zones of different electric field strengths.

A functional interface between the outlet of the electrocapture cell and an electrospray source of an ESI mass spectrometer was established in paper IV. A fused silica capillary of appropriate dimensions allowed peptide transfer to the emitter tip and successful on-line analysis of peptides.

In paper V, an enrichment strategy for peptides with the electrocapture device was developed. Proteolytic mixtures were first pre-concentrated in the device and then released for identification by on-line ESI mass spectrometry. At low concentrations, the enrichment step proved to be necessary for successful precursor ion selection and identification. Also, improved signal-to-noise-ratios and limit-of-detection values in the low femtomolar range were observed both are crucial factors for the analysis of low abundance samples.

In an ongoing study, the electrocapture technology is used for a membrane protein screening project. A shotgun digest of membrane proteins is separated by a novel 2-D approach (first dimension electrocapture, second dimension LC).