Comparative Analysis of Proteoform Clean-Up Methods for In-Depth Top-Down Proteomics.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Proteoforms, encompassing genetic and post-translational variations, are crucial for understanding biological mechanisms and disease.
• Top-down proteomics (TDP) analyzes intact proteoforms, often requiring sodium dodecyl sulfate (SDS) for fractionation.
• Efficient SDS removal is critical as it interferes with mass spectrometry analysis.

Purpose of the Study:

• To benchmark four commercial SDS clean-up kits against traditional methanol-chloroform-water (MCW) precipitation for TDP.
• To evaluate SDS removal efficiency, proteoform recovery, post-translational modification (PTM) retention, and ease of use.
• To provide practical guidance for optimizing TDP sample preparation workflows.

Main Methods:

• Comparison of four commercial SDS clean-up kits (DetergentOUT, HiPPR, Minute SDS, SurfactAway) with methanol-chloroform-water (MCW) precipitation.
• Assessment of SDS removal efficacy using mass spectrometry-based techniques.
• Evaluation of proteoform identification numbers, focusing on recovery of small and acidic proteoforms.
• Analysis of PTMs, particularly methylation, across different clean-up methods.

Main Results:

• MCW precipitation resulted in fewer proteoform identifications, especially for small and acidic proteoforms.
• Commercial kits DetergentOUT and HiPPR showed SDS removal comparable to MCW but at a higher cost.
• Minute SDS and SurfactAway kits enabled the identification of a higher number of proteoforms.
• Increased prevalence of methylation modifications was observed post-MCW clean-up.

Conclusions:

• For studies sensitive to the loss of low molecular weight or acidic proteoforms, commercial kits like DetergentOUT and HiPPR may offer advantages.
• Minute SDS provides a cost-effective alternative with sufficient SDS removal and broader proteome coverage.
• These findings support the development of more streamlined, reproducible, and accessible TDP workflows for biological and clinical research.