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.
Gevaert
K
K
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Belgium. Krgev@gengenp.rug.ac.be
De Mol
H
H
Verschelde
J L
JL
Van Damme
J
J
De Boeck
S
S
Vandekerckhove
J
J
D000180:Acrylic Resins / Q000737:chemistry*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
D000180:Acrylic Resins; D018076:DNA, Complementary; D011506:Proteins; C016680:polyacrylamide gels