The presence of contaminants in a peptide sample can have a detrimental impact on the LC-MS/MS analyses. Thus, the availability of efficient methods for the removal of contaminants is crucial. Here, we evaluated the efficacy of single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) for the removal of sodium dodecyl sulphate (SDS) or polyethylene glycol (PEG) from Arabidopsis thaliana tryptic peptides. A 40-minute SP2 protocol was proposed and subjected to a comprehensive benchmarking against the ethyl acetate extraction protocol.

Arabidopsis thaliana rosette leaves were harvested after four weeks of growth, and ground in liquid nitrogen. The plant tissue powder was solubilised in SDT buffer (4% SDS, 100 mM DTT; 100 mM Tris/HCl, pH 7.6). The proteins were digested with trypsin (an enzyme-to-protein ratio of 1:50) using the filter-aided sample preparation (FASP) method. The obtained peptides were subsequently contaminated with SDS and/or a mixture of PEGs (PEG 200 to PEG 8000) to various final concentrations. Aliquots of 250 ng and 10 µg of contaminated peptide samples were subjected to either ethyl acetate extraction or the SP2 protocol in order to remove the contamination. The peptide samples were then analysed by LC-MS/MS using the Ultimate 3000 RSLCnano system connected to the Thermo ScientificTM Exploris 480 mass spectrometer operated in data-independent acquisition mode (DIA). The acquired data were processed in DIA-NN software (version 1.8.0).

The efficacy of the proposed SP2 and ethyl acetate extraction protocols was evaluated in the removal of SDS contamination from A. thaliana tryptic peptide samples. The ethyl acetate extraction protocol demonstrated the ability to effectively remove SDS contamination up to 1%. However, our optimized SP2 protocol demonstrated superior performance, efficiently removing SDS contamination up to 5% from 250 ng and 10 µg peptide inputs. The SP2 protocol was also found to be effective in the removal of PEG contamination up to 1%, and even for simultaneous 5% SDS and 1% PEG contamination without significant sample losses. Conversely, incorporating a small amount of PEG into the sample could potentially prevent losses throughout the clean-up process.

The optimised SP2 protocol outperformed the ethyl acetate extraction in terms of the effectiveness of contamination removal and its versatility. The findings indicate that the SP2 method is capable of efficient performance with various bead and peptide ratios, suggesting that our protocol is versatile enough to be applied to a wide range of peptide inputs without requiring adjustments.

The study was supported by European Regional Development Fund—Project "SINGING PLANT" (CZ.02.1.01/0.0/0.0/16_026/0008446), CIISB, Instruct-CZ Centre of Instruct-ERIC EU consortium, funded by MEYS CR infrastructure (LM2023042), and the e-INFRA CZ project (ID:90254), supported by MEYS CR.