Picoliter Sample Preparation in MALDI-TOF MS Using a Micromachined Silicon Flow-Through Dispenser

Abstract

This paper presents a picoliter sample preparation technique utilizing the flow-through principle, allowing on-line coupling of chromatographic systems to be made. The work was performed in order to investigate the characteristics and the physicochemical properties of the sample preparation using typical mobile phase conditions from µ-CLC (column liquid chromatography) separations. The device presented here is a pressure pulse-driven dispenser, formed by two silicon structures processed by conventional micromachining. The pressure pulse is generated in the flow-through channel by a piezoceramic element. Depending on the orifice size, the droplets ejected range between 30 and 200 pL. The maximum ejection frequency is 500 Hz, limited by resonances within the unit. A pyramid-shaped nozzle improves the directivity of the droplets since it reduces the wetting of the orifice front surface area. The risk of particles sticking close to the orifice is also minimized. The analyses of the deposited sample spots were carried out on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer with delayed extraction. It was possible to detect attomole amounts (159-248 amol) of various proteins (cytochrome c, ribonuclease A, lysozyme, and myoglobin) from a single droplet of matrix:analyte 1:1 (drop volume ≈ 110 pL). Additionally, it was found that sample enrichment could be carried out using multiple depositions on the same spot; i.e., 31 nM of insulin was easily detected when more than four depositions were made on the same spot, while no detection was possible without sample enrichment. Size optimization of the MALDI sample spot gave target zones of 100-500-µm diameter that matched the size of the laser focal point and resulted in a considerably increased sample throughput.

Development of new methodologies for the mass spectrometry study of bioorganic macromolecules

“…Using this method, mainly low-charge (usually singly charged) ions are produced. For example, Figure 1 (Onnerfjord et al, 1998) shows a typical MALDI spectrum of a protein mixture that contained cytochrome c, ribonuclease A, lysosyme, and myoglobin. As can be seen, for all of the analyzed proteins the singly charged ion is clearly detected.…”

“…FIGURE 1. MALDI mass spectrum of a protein mixture containing cytochrome c, ribonuclease A, lysozime, and myoglobin. (Reproduced from Onnerfjord et al (1998) with permission from American Chemical Society, copyright 1998. ) The ESI source operates, coupled with various introduction systems [e.g., syringe, liquid chromatography (LC), capillary electrophoresis (CE)] to spray the sample solution.…”

Improving the signal intensity and sensitivity of MALDI mass spectrometry by using nanoliter spots deposited by induction-based fluidics

“…A second approach uses microdispensing, including use of capillary tubes [7][8][9][10], piezoelectric techniques [11][12][13][14], electrodynamic methods [15], and acoustic ejection [16]. This general approach improves MALDI for imaging [14,16], on-line analysis [13,17], and concentration enhancement by microextraction [10]. Common in both approaches [1-12, 15, 17] is the concept that the sensitivity of MALDI-MS increases when small sample spots are used and that the search for "hot spots" within the sample area becomes more productive when solid matrices are used.…”

Microfluidic technologies for MALDI‐MS in proteomics

“…As an alternative to direct spotting, microfluidic piezoelectric dispensers fabricated in bulk-etched silicon [46,47,[49][50][51][52] have been used for delivering subnanoliter volumes of sample or matrix solutions to MALDI targets. Ekström et al [46,50] combined piezoelectric deposition with a microchip-based immobilized enzyme reactor for proteolytic digestion, with the resulting protein digest deposited onto a high-density silicon nanovial target plate for MALDI-MS analysis.…”