A new matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometer with the novel "LIFT" technique (MALDI LIFT-TOF/TOF MS) is described. This instrument provides high sensitivity (attomole range) for peptide mass fingerprints (PMF). It is also possible to analyze fragment ions generated by any one of three different modes of dissociation: laser-induced dissociation (LID) and high-energy collision-induced dissociation (CID) as real MS/MS techniques and in-source decay in the reflector mode of the mass analyzer (reISD) as a pseudo-MS/MS technique. Fully automated operation including spot picking from 2D gels, in-gel digestion, sample preparation on MALDI plates with hydrophilic/hydrophobic spot profiles and spectrum acquisition/processing lead to an identification rate of 66% after the PMF was obtained. The workflow control software subsequently triggered automated acquisition of multiple MS/MS spectra. This information, combined with the PMF increased the identification rate to 77%, thus providing data that allowed protein modifications and sequence errors in the protein sequence database to be detected. The quality of the MS/MS data allowed for automated de novo sequencing and protein identification based on homology searching.

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).

We present a new MALD1 sample preparation technique for peptide analysis using the matrix alpha-cyano-4-hydroxy-cinnamic acid (CHCA) and prestructured sample supports. The preparation integrates sample purification, based on the affinity of microcrystalline CHCA for peptides, thereby simplifying the analysis of crude peptide mixtures. Enzymatic digests can thus be prepared directly, without preceding purification. Prepared samples are homogeneous, facilitating automatic spectra acquisition. This method allows preparation of large numbers of samples with little effort and without the need for automation. These features make the described preparation suitable for cost-efficient high-throughput protein identification. Performance of the sample preparation is demonstrated with in situ proteolytic digests of human brain proteins separated by two-dimensional gel electrophoresis.