Capillary liquid chromatography interfaced to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an on-line coupled piezoelectric flow-through microdispenser

Abstract

A piezoelectric flow-through microdispenser interfacing capillary liquid chromatography (LC) with matrix-assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-TOF MS) was developed for the identification of biomolecules. The MALDI target plate was placed on a computer controlled high-resolution x-y stage, on to which the column effluent was deposited as discrete spots, which thereby facilitated tracing of the chromatographic separation. The entire target plate was sprayed with a homogeneous layer of alpha-cyano-4-cinnamic acid mixed with nitrocellulose by using an air-brush. Hence the tedious manual handling of a micropipetter applying matrix solution on top of each fraction collected spot was avoided. The pre-made target plates were stable for at least 3 weeks if kept in darkness at room temperature, which easily allowed re-analysis of dispensed sample spots. The integrated microsystem was characterized and optimized by means of fluidics, dispersion, operational stability and sensitivity parameters. The dispensing unit was developed specifically to match high-resolution capillary LC separations using a dispenser with an internal volume from inlet to the ejecting nozzle of 250 nl. Minimizing dead volumes was crucial in order to maintain the chromatographic resolution. The volume of the ejected droplets was of the order of 60 pl. Successful separations of seven immunoregulating peptides were made: ACTH 1-17, bradykinin, enkephalin, angiotensin III, angiotensin II, angiotensin I and ACTH 18-39. On-line sample dispensing on the target plate in combination with trace enrichment followed by automated MALDI-TOF MS identification is demonstrated, reaching a sensitivity of 100 amol.