Biomedical subjects

K Gevaert

Publications and source records attributed to K Gevaert.

22 records · Page 2Linked to original sources

PubMed1997

Peptides adsorbed on reverse-phase chromatographic beads as targets for femtomole sequencing by post-source decay matrix assisted laser desorption ionization-reflectron time of flight mass spectrometry (MALDI-RETOF-MS).

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.

Acetonitriles↗

PubMed1997

Novel techniques for identification and characterization of proteins loaded on gels in femtomole amounts.

A combination of techniques is presented allowing gel-purified protein identification in the femtomole range using matrix-assisted-laser-desorption-ionization mass spectrometry. The proteins are detected in the primary gel by a sensitive negative staining procedure, transferred, and concentrated in a secondary gel matrix. There, they are digested in the presence of H218O and their sequences are predicted (1) by peptide mass fingerprinting, (2) by comparing the post-source-decay (PSD) spectra with theoretical spectra of candidate isobaric peptides using a computer algorithm called MassFrag, and (3) by a manual readout of the 18O/16O-labeled fragmentation ions in the PSD spectra.

Acrylic Resins↗

PubMed1996

Structural analysis and identification of gel-purified proteins, available in the femtomole range, using a novel computer program for peptide sequence assignment, by matrix-assisted laser desorption ionization-reflectron time-of-flight-mass spectrometry.

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.

Amino Acid Sequence↗

PubMed1995

An agarose-based gel-concentration system for microsequence and mass spectrometric characterization of proteins previously purified in polyacrylamide gels starting at low picomole levels.

An agarose-based concentration gel system is described for eluting and concentrating proteins previously purified either in one-dimensional or two-dimensional gels. Using the technique, proteins can be concentrated from about 1 ml into volumes as small as 10 microliters. After the proteins have been melted out of the agarose gels, they can be digested with proteases, producing peptide patterns similar to those observed with in-solution digestions. The overall peptide recovery, calculated from the amount of protein loaded on the primary separating gel to the collection of fragments after HPLC, is at least 70% of the peptide yields obtained with digests of the same amount of protein in free solution. These results are routinely obtained with 50 pmol amounts (referring to amounts of protein initially loaded on the primary gel). Proteins can also be analysed by a combination of microsequencing and on-line electrospray mass spectrometry, allowing their identification by peptide mass fingerprinting.

Amino Acid Sequence↗