A Sensitive Microbead-Based Organic Media-Assisted Method for Proteomics Sample Preparation from Dilute and Denaturing Solutions.

We developed a robust and sensitive sample preparation method for proteomics termed microbead-based and organic-media-assisted proteolysis strategy (BOPs). BOPs combines two advantages of current techniques, (1) unbiased binding of reversed-phase polymeric microbeads to any type of protein and (2) enhanced trypsin digestion efficiency in CH3CN-aqueous solvent systems, into a single-tube workflow. Compared with conventional techniques, this method effectively concentrates proteins and improves proteolytic digestion, and can be used with submicromolar protein samples in dilute or denaturing solutions, such as 70% formic acid, 8 M urea, or 7 M guanidine hydrochloride without any sample pretreatment. Proteome analysis of single Caenorhabditis elegans organisms demonstrates that BOPs has the sensitivity, reproducibility, and unbiasedness required to characterize worm proteins at a single organism level. We also show that, by simply incorporating an acetone washing step for detergent removal, BOPs is applicable to low concentration samples contaminated with a variety of detergents, including sodium dodecyl sulfate, with negligible protein loss. Moreover, the utility of this modification has also been demonstrated through proteomic characterization of 2000 human (HEK293T) cells lysed using 1% Triton X-100. The simplicity and availability of the present BOPs make it especially attractive for next-stage proteomics of rare and sample-limited systems.