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

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Traditional proteomics sample preparation methods often face challenges with protein loss, low sensitivity, and limited applicability to diverse sample types.
• Existing techniques may require extensive pretreatment for samples in denaturing or dilute solutions, hindering efficient proteomic analysis.

Purpose of the Study:

• To develop a robust and sensitive single-tube sample preparation method for proteomics.
• To enhance protein concentration and proteolytic digestion efficiency for low-input samples.
• To demonstrate the applicability of the method to various challenging sample matrices and biological systems.

Main Methods:

• Developed the microbead-based and organic-media-assisted proteolysis strategy (BOPs).
• Utilized reversed-phase polymeric microbeads for unbiased protein binding.
• Optimized trypsin digestion in CH3CN-aqueous solvent systems.
• Incorporated an acetone washing step for detergent removal.

Main Results:

• BOPs effectively concentrates proteins and improves proteolytic digestion in a single tube.
• The method is applicable to submicromolar protein samples in denaturing solutions (formic acid, urea, guanidine hydrochloride) without pretreatment.
• Proteome analysis of single Caenorhabditis elegans organisms demonstrated high sensitivity, reproducibility, and unbiasedness.
• BOPs, with an added acetone wash, successfully analyzed detergent-contaminated samples (e.g., HEK293T cells lysed with Triton X-100) with negligible protein loss.

Conclusions:

• BOPs is a sensitive, robust, and versatile sample preparation method for proteomics.
• The strategy is particularly advantageous for analyzing rare and sample-limited biological systems.
• BOPs simplifies the workflow and enhances the scope of proteomic investigations.