Magnetic bead-based SPEED enables rapid and high-throughput proteomics sample preparation: A case study in clinical microbiology for species identification and antibiotic resistance detection

The throughput of proteomics is constantly increasing, which enables the development of new applications focusing on large-scale sample cohorts. These applications range from answering fundamental biological questions based on the analysis of gene knockout libraries to the analysis of population-scale plasma cohorts or the diagnostics of clinical bacterial isolates. Upscaling proteomics challenges all stages of the workflow including sample preparation, data acquisition and data analysis.

Initially, comprehensive, accurate and deep proteome analysis relies on the reproducible generation of peptides from the entirety of proteins present in each sample. Most sample preparation protocols employ detergents or chaotropic agents for protein extraction and support sample lysis by physical disruption methods. As many extraction reagents inhibit enzymatic digestion and are incompatible with LC-MS/MS, the idea behind most sample preparation methods is to remove interfering substances before digestion. Recently, we introduced a detergent-free method, named Sample Preparation by Easy Extraction and Digestion (SPEED), which consists of three steps, acidification, neutralization and digestion. SPEED is a universal method for peptide generation from various sources and is easily applicable even for lysis-resistant sample types. As the scale of proteome studies increases, magnetic-based methods (PAC, SP3) are gaining attraction because of their automation potential. This motivated us to develop and evaluate an improved bead-based SPEED protocol to enable high-throughput sample preparation in 96-well plates with efficient and versatile one-step pure chemical-based sample lysis and direct reagent-free protein quantitation.

The performance of magnetic bead-based SPEED was evaluated in an analysis of bacterial clinical isolates using our workflow for concomitant species identification and antibiotic resistance (AMR) detection. Samples were analyzed using a 100 SPD method (Evosep One) and diaPASEF data acquisition (timsTOF HT). The results show, that proteomics provided high specificity and sensitivity for AMR detection, while allowing species identification from very large sequence databases with high accuracy. This workflow has the ability for the successful implementation of proteomics in clinical microbiology. The magnetic bead-based sample preparation (SPEED) is able to prepare all ESKAPE bacteria for measurement in less than 2h without protocol adaptations. The method also has great potential for automation to prepare native samples for mass spectrometry without human intervention in the future.