In-Gel Digestion of Proteins Using a Solid-Phase Extraction Microplate

Abstract

We present a new procedure for in-gel digestion of proteins introducing a combination of two different 96-well microplates. The two plates have incorporated small capillaries with a length of 2.4 mm in each well, one of which has 75-microm-inner diameter capillaries, whereas the second plate has reversed-phase-type capillaries fixed to it. The initial steps of the in-gel digestion process, comprising destaining, reduction/alkylation, dehydration, and digestion, was carried out in the plate containing 75-microm capillaries. Capillaries containing C18 reversed-phase modified monolithic silica rods of a 200-microm diameter were used for the second plate in which extraction and cleanup of peptides were carried out. Peptides were eluted directly from the solid-phase extraction plate onto the MALDI sample support. The separation of the process into two plates led to increased process stability, without compromising sensitivity, i.e. peptide recovery, making it suitable for true high-throughput protein identification. The handling of proteinases could easily be optimized, and no restrictions were made on chosen pH range through the absence of the solid phase in the initial steps of the protocol. Efficient binding of peptides to the solid phase and subsequent direct elution onto the MALDI sample support led to sensitivities in the attomole range. Performance of the process was demonstrated with tryptic digests of proteins stained with colloidal coomassie blue, silver, and the fluorescent stain SYPRO Ruby.