Abstract
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. 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. 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. 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.
Highlights
Purification of proteolytic peptides prior to sensitive nanospray liquid chromatography/tandem mass spectrometry analysis is one important technique in bottom-up proteomics.[1]
The peptide purification is performed via reversed-phase solid-phase extraction (SPE) chromatography[5,6] in a manual tip or cartridge format
To overcome challenges at this step, SPE technology together with a positive pressure workstation turned out to be a powerful tool in proteomic sample preparation
Summary
Purification of proteolytic peptides prior to sensitive nanospray liquid chromatography/tandem mass spectrometry (nLC/MS/MS) analysis is one important technique in bottom-up proteomics.[1]. The positive pressure solid-phase extraction (ppSPE) technology was evaluated by using a new semi-automated sample preparation device with silica (Maestro) and polymeric (WWP2) reversed-phase sorbents. Leachables are a known problem during reversed-phase purification of pharmaceutically relevant proteins.[12] in addition to contamination of industrial-scale preparative workflows, leachables should be considered for proteomic workflows in terms of potential damage to expensive column material, ion suppression or contamination of sensitive MS components.[13,14] the two new sorbents were tested with blank samples to allow assessments about any leaching of nLC/MS/MS-interfering substances. The optimized workflow was used to answer a question important to the application of proteomics in industrial cell culture technology: Does a cellular proteome reproducibly change within three individual and independent fed-batch cultivation processes or does cell heterogenity predominate?
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