Prestructured MALDI-MS sample supports have been developed that simplify high-throughput analysis of biomolecules and improve the detection sensitivity. The mass spectrometric sample support is coated with a thin layer of hydrophobic Teflon that carries an array of 200-microm gold spots, which provide hydrophilic sample anchors. Each transferred sample droplet contacts one anchor, on top of which, after solvent evaporation, the sample is exclusively deposited due to the strongly water repellent nature of the Teflon surface. The initial matrix concentration is kept low, enabling sample up-concentration by more than 2 orders of magnitudes before crystallization commences. As a result, the detection sensitivity is improved as documented by mass spectra recorded from 100 amol of various peptides, 1 fmol of a DNA 20 mer, and 5 fmol of a 130 bp PCR product. Size and spacing of the hydrophilic anchors are optimized for MALDI-MS performance (sample spot size approximately = laser irradiation spot size), for short analysis times (predetermined sample coordinates), and for high throughput sample preparation (sample anchor array according to the 1536 microtiter plate format).

A new strategy for identifying proteins in sequence data-bases by MALDI-MS peptide mapping is reported. The strategy corrects for systematic deviations of determined peptide molecular masses using information contained in the opened database and thereby renders unnecessary internal spectrum calibration. As a result, data acquisition is simplified and less error prone. Performance of the new strategy is demonstrated by identification of a set of recombinant, human cDNA expression products as well as native proteins isolated from crude mouse brain extracts by 2-D electrophoresis. Using one set of calibration constants for the mass spectrometric analyses, 20 proteins were identified without applying any molecular weight restrictions, which was not possible without data correction. A sequence database search program has been written that performs all necessary calculations automatically, access to which will be provided to the scientific community in the Internet.

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