Alkaline extraction of human plasma proteins from nondenaturing micro-2-D gels for protein/polypeptide mass measurement and peptide mass fingerprinting using MALDI-TOF MS.
Previously, we have reported a high-efficiency method of protein extraction from CBB-stained polyacrylamide gels for molecular mass measurement with MALDI-TOF MS [1]. In the present work, the alkaline extraction method was applied to CBB-stained 2-DE gels on which human plasma proteins were separated in the absence of denaturant. In order to examine the performance of the method, ten spots with apparent molecular masses (MMapp) in the range of 65 to 1000 kDa were selected and the proteins were extracted from the gel pieces. The extracts were subjected to whole-mass measurement by MALDI-TOF MS, with and without DTT treatment. In addition, the extracts were subjected to in-solution trypsin digestion followed by MALDI-TOF MS and PMF analysis. Successful extraction of proteins from the ten spots, up to MMapp 1000 kDa, has been ascertained by the significant PMF assignment (MASCOT) with high sequence coverage of the respective proteins or polypeptides. When direct mass measurement of the extracted proteins was attempted, three spots in MMapp range 65-100 kDa provided mass peaks. Five spots in MMapp range 150-400 kDa did not give mass peaks of the intact proteins, but showed those of the constituent polypeptides after the DTT treatment. Extraction of proteins prior to trypsin digestion enabled the procedure of PMF analysis to be much simpler than the conventional in-gel digestion method, providing comparable protein scores and sequence coverage. The technique presented here suggests a new strategy for the characterization of proteins separated by nondenaturing 2-DE.
Jin
Ya
Y
Department of Chemistry, Faculty of Science, Ehime University, Matsuyama, Japan.
Manabe
Takashi
T
D000180:Acrylic Resins / Q000737:chemistry; D001798:Blood Proteins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008970:Molecular Weight; D010449:Peptide Mapping / Q000379:methods*; D012972:Sodium Hydroxide / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000180:Acrylic Resins; D001798:Blood Proteins; C016680:polyacrylamide gels; D012972:Sodium Hydroxide