Achieving deep proteoform coverage in top-down proteomics critically depends on effective sample pre-fractionation. To address this, we developed PEPPI-MS (Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS) in 2020, leveraging the widely used SDS-PAGE method in biochemistry as a tool for pre-fractionation. PEPPI enables efficient passive extraction of intact proteins...
Electromigrative techniques are powerful tools for the separation of intact proteins and their proteoforms. However, CE-MS is still restricted by the sensitivity and ease-of-use of the interface in conjunction with low injection volumes limiting its application for biological samples. Various solutions will be presented here overcoming these shortcomings.
Initially, the power of CE-MS for the...
Numerous workflows have been developed for top-down proteomics (TDP). We systematically investigated the influence of different sample preparation steps on proteoform and protein identifications, including cell lysis, reduction and alkylation, proteoform enrichment, purification, and fractionation [1]. We found that all steps in sample preparation influence the subset of proteoforms identified...
To analyze tissue molecules accurately, they must be first solubilized. Conventional sampling and homogenization methods disrupt cell compartments, releasing enzymes that can alter proteoforms through proteolysis and modifications of post-translational modifications (PTMs), thereby changing their original composition.
Using nano- or pico-second infrared laser systems (NIRL or PIRL) for...
Introduction
Histones are heavily and variably decorated by PTMs, thereby affecting their binding to chromosomal regions. Top-down proteomics of histones is advantageous in capturing the PTM combination to obtain epigenetic status. Being rich in lysine residues hampers histone separation by RP chromatography. Capillary electrophoresis (CE) – MS has emerged as powerful alternative for...
