Integrated system for high-throughput protein identification using a microfabricated device coupled to capillary electrophoresis / nanoelectrospray mass spectrometry

Abstract

An integrated microsystem providing rapid analyses of trace-level tryptic digests for proteomics application is presented. This modular microsystem includes an autosampler and a microfabricated device comprising a sample introduction port and an array of separation channels together with a low dead-volume facilitating the interface to nanoelectrospray mass spectrometry. Sequential injection and separation of peptide standards and tryptic digests was achieved with a throughput of up to 30 samples per hour with less than 3% sample carryover. Replicate injections of peptide mixtures indicated that reproducibility of migration time was typically better than 2.3% relative standard deviation (RSD) whereas RSD values of 3.7-11.8% were observed on peak height. Mass spectral detection of submicromolar protein digests (< 7 femtomoles/injection) was achieved using a quadrupole/time of flight instrument in less than 2 min/per sample with peak widths of 1.8-7.0 s. The analytical potential of this integrated device for the identification of gel isolated proteins from Neisseria meningitidis immunotype L3 has been demonstrated using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry.

Application of Microfluidic Devices to Proteomics Research

“…When coupled to an autosampler, this system provided sequential injection (1.5 l per injection) and separation of in-gel tryptic digests with a throughput of up to 25 samples/per h with less than 3% sample carryover. The concentration detection limit for peptide analysis was in the low micromolar range comparable with microfluidic devices of similar configuration (21,25). Although 1-2 l of sample was introduced to the large chip channel the overall sensitivity of this approach was limited by the actual injection volume on the chip device (typically Ͻ5 nl).…”

“…Obviously, significant advances in automation are still needed to provide the required throughput and ruggedness sought for the rapid and positive identification of proteins. To this end, efforts are also devoted to microfabricated chip designs that enable sample introduction from autosampler or microwell plates (24,25).…”

“…The previous chip design (21,25) was modified to include a port of low flow resistance enabling the introduction of sample on and off the chip without perturbing the fluids in the analysis manifold. Such design was used previously to conduct on-chip tryptic digestion and provided a convenient format to conduct rapid in-solution digest (Ͻ10 min) (26).…”

“…1a was fabricated at the University of Alberta Microfab laboratory (Edmonton, Alberta, Canada), as described previously (25,26). Channels were etched on Corning 0211 glass (Corning Glass) using standard photolithography and wet chemical etching techniques.…”

Recent Developments in Capillary Electrophoresis–Mass Spectrometry of Proteins and Peptides

“…Most reports on MCE-ESI-MS have focused on developing designs for facile interfacing of microfabricated devices with MS. [92][93][94]113,[126][127][128][129][130][131] Wen et al 126 demonstrated direct microchannel isoelectric focusing (IEF)-MS using a chip constructed on polycarbonate plates for the characterization of protein mixtures. Zhang et al 93 designed a microdevice from glass wafers, which integrated a guiding channel for insertion of the electrospray capillary.…”