A peptide concentration and purification method for protein characterization in the subpicomole range using matrix assisted laser desorption/ionization-postsource decay (MALDI-PSD) sequencing.
We here describe the use of added reversed-phase chromatographic beads to concentrate peptides from highly diluted solutions. In the procedure developed, peptide-bead suspensions are dried under vacuum to complete dryness; peptides are subsequently eluted in a small volume of matrix-assisted laser desorption/ionization (MALDI)-matrix containing organic/aqueous solvent and transferred to a MALDI-target for mass analysis. We show that by using this bead-peptide concentration procedure, low femtomole amounts of peptides are efficiently concentrated, up to 1000 times, to volumes smaller than 0.7 microL. We have used this concentration procedure in combination with MALDI-post-source decay analysis to identify subpicomole amounts of proteins present in polyacrylamide gels. Furthermore, we show that the bead-peptide concentration method can be elegantly used to clean up samples contaminated with high concentrations of substances normally deleterious to MALDI-mass spectrometry (MS) experiments. We have found additionally that the bead-peptide concentration procedure can be successfully used to store low femtomole amounts of peptide for prolonged periods of time without severe losses of peptide material. This bead-peptide concentration procedure therefore seems to be a simple and convenient step in the MALDI-MS sample preparation process.
Gevaert
K
K
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Belgium. krgev@medusa.rug.ac.be
Demol
H
H
Sklyarova
T
T
Vandekerckhove
J
J
Houthaeve
T
T
D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry; D008863:Microspheres; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods; D010455:Peptides / Q000032:analysis* / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D017421:Sequence Analysis / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods*
D010455:Peptides; D011506:Proteins