Rationale High‐throughput reliable data generation has become a substantial requirement in many “omics” investigations. In proteomics the sample preparation workflow consists of multiple steps adding more bias to the sample with each additional manual step. Especially for label‐free quantification experiments, this drastically impedes reproducible quantification of proteins in replicates. Here, a positive pressure workstation was evaluated to increase automation of sample preparation and reduce workload as well as consumables. Methods Digested peptide samples were purified utilizing a new semi‐automated sample preparation device, the Resolvex A200, followed by nanospray liquid chromatography/electrospray ionization (nLC/ESI) Orbitrap tandem mass spectrometry (MS/MS) measurements. In addition, the sorbents Maestro and WWP2 (available in conventional cartridge and dual‐chamber narrow‐bore extraction columns) were compared with Sep‐Pak C18 cartridges. Raw data was analyzed by MaxQuant and Perseus software. Results The semi‐automated workflow with the Resolvex A200 workstation and both new sorbents produced highly reproducible results within 10–300 μg of peptide starting material. The new workflow performed equally as well as the routinely conducted manual workflow with similar technical variability in MS/MS‐based identifications of peptides and proteins. A first application of the system to a biological question contributed to highly reliable results, where time‐resolved proteomic data was separated by principal component analysis (PCA) and hierarchical clustering. Conclusions The new workstation was successfully established for proteolytic peptide purification in our proteomic workflow without any drawbacks. Highly reproducible results were obtained in decreased time per sample, which will facilitate further large‐scale proteomic investigations.

Background & Objectives Large-scale proteome analysis by mass spectrometry is commonly preceded by enzymatic digestion of proteins. The conventional protocol for in-solution digest of complex protein mixtures includes trypsin and is performed at room temperature for at least 12h. To improve this time-consuming method we assessed the efficiency of focused ultrasound-assisted enzymatic digest. It has previously been shown that ultrasonication can dramatically reduce digestion time. However, it is not known how this affects protein identification and quantification when applied to complex protein mixtures such as whole cell lysates. Here, we explore in-depth the proteome of embryonic stem cells comparing trypsin-digestion methods with and without ultrasound support side-by-side. Methods In-solution protein digest Protein extracts were digested with sequencing-grade trypsin (Promega) for 1, 5, 10, 20, 30, 40min and 1h in the presence or absence of focused ultrasonication (Covaris). A conventional digestion overnight was included. Reactions were stopped by acidification and heat. Peptides were desalted by reverse phase C18 StageTips. Mass spectrometry Peptides were analyzed on a QExactive (Thermo Scientific) coupled to Easy nLC 1000 (Proxeon). Raw files were processed by MaxQuant v1.3.0.3 software. Peptide searches were performed by Andromeda search engine against the UniProt database. Results and Conclusions Focused ultrasonication clearly enhanced the enzymatic digestion and can be implemented in the daily workflow of mass spectrometry-based proteome analysis.

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