Abstract
Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.
Highlights
The efficient sample preparation for proteomic analysis of plants represents a real challenge
We evaluated four sample processing methods applied to the model plant A. thaliana; whole leaf lysates are processed by four workflows: the classical filter-aided sample preparation (FASP), two SP3 protocols using carboxylated or HILIC paramagnetic beads, and the suspension trapping method (S-Trap) protocol
We evaluated consecutive digestion with trypsin in our experiment as decreasing activity of the modified trypsin with blocked autolysis was reported being in solution over time (Finehout et al, 2005) and multienzyme digestion was successfully applied on protein extracts mostly from mammalian cell cultures (Wiśniewski and Mann, 2012)
Summary
The efficient sample preparation for proteomic analysis of plants represents a real challenge. Plants contain a low concentration of proteins and high levels of secondary metabolites potentially interfering with proteome analysis (Hussein and El-Anssary, 2018). Usually mechanical force or highly effective sonication, have to be applied to disrupt cell walls. These procedures are often followed by traditional protein extraction protocols (TCA/acetone precipitation or phenol extraction) to concentrate proteins and avoid protein degradation caused by abundant proteases. Sample preparation approaches containing the removal of substances interfering with digestion and MS analysis are of great importance (Song et al, 2018; Wang et al, 2018)
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