Due to its very short analysis time, its high sensitivity and ease of automation, matrix-assisted laser desorption/ionization (MALDI)-peptide mass fingerprinting has become the preferred method for identifying proteins of which the sequences are available in databases. However, many protein samples cannot be unambiguously identified by exclusively using their peptide mass fingerprints (e.g., protein mixtures, heavily posttranslationally modified proteins and small proteins). In these cases, additional sequence information is needed and one of the obvious choices when working with MALDI-mass spectrometry (MS) is to choose for post source decay (PSD) analysis on selected peptides. This can be performed on the same sample which is used for peptide mass fingerprinting. Although in this type of peptide analysis, fragmentation yields are very low and PSD spectra are often very difficult to interpret manually, we here report upon our five years of experience with the use of PSD spectra for protein identification in sequence (protein or expressed sequence tag (EST)) databases. The combination of peptide mass fingerprinting and PSD and analysis described here generally leads to unambiguous protein identification in the amount of material range generally encountered in most proteome studies.

We here describe a procedure for concentrating peptides from solutions by adsorbing them onto reverse-phase beads that were added to these solutions. The beads are then transferred to the target disc of the matrix assisted laser desorption ionization-reflectron time of flight (MALDI-RETOF) mass spectrometer. Because of their hydrophobic nature, these beads cluster in a very small area on the target disc assuring an important concentration step. After drying, peptides are desorbed from the beads by adding a small volume of 50% acetonitrile in 0.1% trifluroacetic acid in water containing the matrix components. Hereby we focus the original amount of peptide material on the target disc on a very small surface, producing highly concentrated peptide-matrix mixtures. This permits high yield identification and sequence tagging by post-source-decay analysis on peptides derived from proteins only available in the femtomole range from one-dimensional (1-D) or two-dimensional (2-D) gels. The procedure is illustrated by the identification of 38 proteins from human thrombocyte membrane skeletons.

A procedure is described for structural characterization and identification of proteins, purified by either one- or two-dimensional gel electrophoresis in the low picomole to femtomole range. The purified proteins are first detected in the primary gels by the sensitive reverse staining procedure described by Fernandez-Patron et al. (Anal. Biochem. 1995, 224, 203-211) and consecutively reeluted from combined get pieces and concentrated in the tip of a Pasteur pipette in a secondary gel matrix consisting of either sodium dodecyl sulfate-polyacrylamide or agarose. The concentrated proteins are in-matrix-digested and the resulting peptides are separated by reverse-phase high performance liquid chromatography (HPLC) combined with microsequencing or analyzed by matrix-assisted laser desorption ionization--time of flight--mass spectrometry. Protein identification is based on sequence homology or on the peptide mass pattern. The matching peptide sequences can additionally be verified by matching their measured post-source decay spectra with the calculated fragmentation patterns of the isobaric candidate peptides appearing on the search list. This is done by a computer program referred to as MassFrag, described in this paper. We demonstrate that it is possible to identify protein that are only available in the femtomole range and whose sequences are stored in nonredundant protein databases or nucleotide and expressed sequence tag databases.