SDS-PAGE-MALDI-TDS: Mass Spectrometric Top-Down Sequencing of Proteins Extracted from Polyacrylamide Gels.

RP-86 MALDI top-down sequencing (TDS) using In-Source-Decay (ISD) is a powerful tool to analyze protein termini without prior tryptic digestion. Sequence calls up to 80 residues of each terminus can be achieved. In addition, the technique is well suited to detect modifications, truncations, and mutations. The analysis requires ∼20 pmol of an isolated protein for each MALDI preparation. When analyzing protein mixtures or proteins that consist of multiple peptide chains such as antibodies, separation techniques such as gel electrophoresis have to be applied prior to TDS analysis. We used a novel chip-based system for improved extraction of proteins from polyacrylamide gels (GPR-800, Protea). (1). Standard proteins such as Ubiquitin and RNAseA from SDS-PAGE spots were extracted and the eluate purified using, e.g., protein precipitation or ultrafiltration. Subsequently, the samples were analyzed by MALDI-TOF-MS. MS spectra of the intact proteins as well as ISD spectra of high quality were obtained that provided direct access to detailed sequence information. Also, a monoclonal antibody was analyzed by SDS-PAGE-MALDI-TDS to obtain N- and C-terminal sequence assignments for both, the light (LC) and the heavy (HC) chain. 24g of the antibody were reduced, chains were separated by SDS-PAGE, extracted and purified from gel spots and analyzed. In the resulting MALDI-TDS spectra, 70 N- and 30 C-terminal aminoacid residues were assigned and the N-terminal pyroglutamylation of the HC was confirmed. The developed protocols seem to be well suited for a general approach to top-down sequencing of proteins after gel-based separation. This approach rapidly and robustly allows for the detailed analysis of terminal sequences. T.T. Razunguzwa, A. Biddle, H. Andersen, D. Zhan, M.J. Powell, Electrophoresis 2009, 30, 4020-4028.