A positive pressure workstation for semi-automated peptide purification of complex proteomic samples

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 nLC-ESI-Orbitrap 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 well as the routinely conducted manual workflow with similar technical variability in MSMS-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 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.


| INTRODUCTION
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 Especially for preparing and interpreting quantitative data, highly reproducible methods are required. [2][3][4] However, the high manual workload in a proteomic sample preparation workflow has several drawbacks in terms of contamination risk, time needed, costs and introduced bias to the sample. Routinely, 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. Positive pressure systems are well known and suitable for simultaneous preparations. 7 In addition, workstations including solvent dispensing and computer-controlled pressure gradients are highly valuable in saving time, consumables and costs while maintaining reproducible parallel preparation of up to 96 samples. As several deep proteome investigations have been published recently, 8,9 it is evident that the general need for high-throughput methods has greatly increased also for proteomic investigations over recent years.
Here, 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. For comparison, a routinely performed manual peptide purification workflow using Sep-Pak C18 cartridges was conducted. Sep-Pak cartridges contain C18-modified, silicabased sorbent, which is additionally endcappedthus supporting hydrophobic characteristics. 10 In contrast to this endcapped chemistry, the Maestro sorbent is unendcapped and characterized by a much smaller particle size of 10 μm than Sep-Pak, with a particle size specified in a range of 55-105 μm. Another type of sorbent is polymeric WWP2 with a particle size of approx. 30 μm.
Both sorbents tested here are available in conventional column design (similar to Sep-Pak cartridges) and in a special dual-chamber design, called Narrow Bore Extraction™ (NBE™). NBE columns are characterized by an airlock technology for in-cartridge-based sample preparations, which is similar to the well-established in-StageTip method. 11 The use of this feature possibly reduces the need for consumables and at the same time should minimize sample loss through decreased sample transfer.
Whenever the workflow of sensitive nLC/MS/MS analysis is changed, several aspects need to be considered, for example, the introduction of leaching and/or interfering substances. Leachables are a known problem during reversed-phase purification of pharmaceutically relevant proteins. 12 However, in addition to contamination of industrial-scale preparative workflows, leachables should also be considered for proteomic workflows in terms of potential damage to expensive column material, ion suppression or contamination of sensitive MS components. 13,14 Therefore, the two new sorbents were tested with blank samples to allow assessments about any leaching of nLC/MS/MS-interfering substances.
Two different set-ups were used to evaluate the advantages and limitations of the workstation and the two sorbents, Maestro and WWP2, in the proteomic sample preparation workflow. First, the manual workflow with Sep-Pak cartridges was performed in three technical replicates and the automated workflow was carried out in comparison using two different input samples for the two new sorbents: Maestro and WWP2 ( Figure 1A). In a second set-up, varying amounts of peptides were purified using the semi-automated

| Tryptic digest
The reduction and alkylation of cysteines were performed with the appropriate amount of starting material (10-300 μg protein) and

| Establishment of semi-automated peptide purification and general comparison with manual workflow
Peptides purified with both new sorbents, Maestro and WWP2, in NBE column format using the Resolvex A200 workstation showed high Pearson correlation of 99.3% in nLC/MS/MS measurements (triplicates each, Figure 2A). In addition, the manual workflow with

| Scale down to 10 μg of starting material
In a scale-down approach the recovery of samples was evaluated, which is of particular importance for low-input clinical proteomics. 8,16 Here, Sep-Pak cartridges were tested for the first time directly within the Resolvex A200 workstation (100 μg of starting material,

| CONCLUSIONS
The new semi-automated positive pressure workstation Resolvex ® A200 is of great benefit for proteomic sample preparation approaches such as the peptide purification techniques tested here. The use of the workstation drastically reduces the time required per sample and the amount of consumables. The evident benefits in combination with the investigated high reproducibility and ease of use make the workstation a optimal solution for most (proteomic) laboratories with increased sample throughput for sample preparations.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/rcm.8873