Single-step affinity isolation and rapid non-denaturing elution of endogenous protein complexes with subsequent LC - MS characterization

摘要

1. We successfully demonstrate here the combination of a single -step immunopurification under nondenaturing conditions with high-performance liquid chromatography and MS analysis of intact proteins. 2. The use of either the competitive peptide or protease cleavage provides an efficient and rapid means of eluting endogenous protein complexes from small amounts of cryogrindate (less than 1g of grindate processed in less than 2.5 hours.) 3. Ensuing sample handling steps for improved chromatographic protein separation --reduction and alkylation, chloroform -methanol precipitation, and resuspension in LC-compatible solution--were optimized for a system using 60% formic acid in order to minimize polypeptide solubility issues. 4. The components of the affinity -purified protein complex could be separated and identified by our overall LC-MS strategy, with some proteins detected even at 100 -fmol levels (total load). 5. Ongoing work include: a) a strategy for enzymatic or chemical cleavage of the protein samples at the 1- pmol level for middle -down approaches and/or for comprehensive peptide mapping, b) development of a deconvolution algorithm and interface for mass measurements of larger proteins from LC runs, and c) affinity isolation of various endogenous protein complexes from various cellular pathways. We currently have over 20 tagged strains with a dozen of them already in the cryogrindate form, and we are actively engaged in processing the remaining strains into cryogrindate form. d) addressing the fact that a few proteins were not observed in our LC-MS strategy. Although, the physico- chemical properties of those proteins (mass and hydrophobicity) might be enough to explain their absence, we are still pursuing other avenues of investigation.