A micromachined chip-based electrospray source for mass spectrometry.

A micromachining process is described for fabricating a mass spectrometry electrospray source on a silicon chip. The process utilizes polymer (parylene) layers to form a system of chambers, filters, channels, and hollow needle structures (electrospray emitters) that extend more than a millimeter beyond the edge of the silicon substrate. The use of photoresist as the sacrificial layer facilitates the creation of long channels. Access to the channel structures on the chip is through a port etched through the silicon substrate that also serves as a sample reservoir. A reusable chip holder consisting of two plastic plates and an elastomer gasket provides the means to mount the chip in front of the mass spectrometer inlet and make electrical and gas connections. The electrospray emitters have tapered tips with 5 microns x 10 microns rectangular openings. The shape of the tip can be varied depending on the shape of the mask used to protect the parylene structures during the final plasma etch. The parylene emitters are physically robust and require only a high electric field to achieve stable electrospray operation over a period of a few hours. Direct comparisons with conventional glass or fused silica emitters indicated very similar performance with respect to signal strength and stability, spectral quality, and endurance. The automated MS/MS analysis of a mixture of tryptic peptides was no more difficult and yielded nearly identical results as the analysis of the same sample using a conventional nanospray device. This work demonstrates that an efficient electrospray interface to mass spectrometry can be integrated with other on-chip structures and mass-produced using a batch process.

A microscale electrospray interface incorporating a monolithic, poly(styrene-divinylbenzene) support for on-line liquid chromatography/tandem mass spectrometry analysis of peptides and proteins.

A methodology is described for creating a monolithic chromatography support within a pulled fused-silica electrospray needle. The monolith was formed from a mixture of styrene, divinylbenzene, 1-dodecanol, and toluene using 2,2'-azobis(isobutyronitrile) as the catalyst. The mixture was loaded into 150-micron-i.d. fused-silica capillary tubing with a pulled 5-10-micron needle tip at one end. Polymerization at 65 degrees C followed by removal of the porogen material yielded a stable, porous, monolithic support which had excellent properties for the separation and on-line, electrospray, mass spectrometry analysis of peptides and proteins. The performance of the monolith-filled electrospray needles was compared with similar needles filled with commercial C18 silica and polymeric particulate supports. Separation efficiencies for both protein and peptide mixtures were generally equal to or better than the particulate supports at comparable pressures and flow rates. The ion chromatograms derived from the on-line MS analysis were remarkably free from chemical background signals that often complicate the LC/MS analysis of femtomole amounts of sample. Good sequence coverage was obtained by LC/MS/MS analysis of the peptide mixture obtained from a protein isolated by silver-stained gel electrophoresis. The capability of the monolith to do peak parking experiments was demonstrated by the characterization of an immunoreactive HPLC fraction. The simple fabrication method, chromatographic performance, and robust nature of these microscale integrated column electrospray sources make them ideally suited for high-sensitivity tandem LC/MS analyses.

Characterization of reaction dynamics in a trypsin-modified capillary microreactor.

Application of mild vibration to an immobilized trypsin capillary microreactor can enhance digestion rates for many globular and glycosylated proteins (12-70-kDa range) without additional sample handling. A sinusoid wave form generator and a simple piezoelectric transducer were used to apply vibration in a wide frequency range to the 50-µm-i.d. enzyme microreactor over its entire length. The mass transport properties of the microreactor were quantitatively examined for protein digestions through the use of an artificial globular protein. This was synthesized by covering the surface of 35-nm-diameter latex beads with a peptide (Leu-Arg-Leu). Capillary electrophoresis analysis of the microreactor products showed there were no mass transport-related effects for vibration of the capillary. Digestions of a range of globular protein structures were performed and the products analyzed by capillary electrophoresis. The rate enhancements were found to be related to the stability of the protein tertiary and secondary structure. Cytochrome c showed a dramatic acceleration in rate of digestion as the vibration frequency increased over a range of 200 Hz to 7.1 kHz. The ability to enhance reaction rates for very stable proteins can be gained by additional means of destabilizing the protein, as shown by removal of calcium from α-lactalbumin. Vibration of the enzyme capillary will have the greatest utility for extremely limited protein samples since chemical modification to completely denature proteins usually requires considerable sample handling.