Automated sample prep –the reality of proteomics in the clinic

Automation of protein sample preparation workflows to be able to address clinical questions has come of age: it is both possible and realistic, despite the hurdles. Here, we discuss recent developments in reproducible, high throughput proteomics sample preparation workflows that do not require modification, even when applied to highly disparate sample types. These advances bring proteomics a significant step closer to clinical reality.

Not another ordinary attempt at ‘simplifying proteomics’

There are four major bottlenecks to being able to bring proteomics techniques to the clinic: the ability to analyze enough samples (hundreds to thousands or more), with a simplified workflow that is both time- and cost-efficient. Below, we will describe how new technological developments differ to previous claims and products, and support the bold assertion that they truly bring proteomics in clinical settings closer to reality. While tests developed with such workflows might require FDA regulatory approval, many CLIA LDTs (laboratory derived tests) using the instruments and workflows described here are already in use, and we anticipate that robust automation will ease, and even promote, the development of many new LDTs.

Automation 101 with solid phase sample prep

Automated protein sample preparation must faithfully recapitulate the underlying proteome, regardless of the sample type. Further, interfering substances incompatible with downstream processing – like viral transport media (VTM) detergents, salts, glycerol, PEG, Laemmli loading buffer and biological contaminants

(eg. bile components) – must be fully removed. This step likely requires a solid support for capturing proteins.

While non-solid support solutions for automation exist, including bead- and liquid-based systems, they can require extensive optimization when working with complex biological samples, and often have issues with changes of scale. Ultimately, these limitations add complexity, time and cost. In the end, the stability of a solid phase support system remains unrivaled.

Filter-aided sample preparation (FASP) used to be frequently employed as a membrane support to remove sodium dodecyl sulfate (SDS) prior to mass spectrometry analysis.1 However, FASP protocols are notoriously long, and membranes fail regularly, leading to experimental failure. Such disadvantages led to the development of new solid phase capture strategies. In particular, the widely adopted S-Trap™ technology (ProtiFi) allows the use of high concentrations of SDS in a fraction of the time of FASP, with proteins captured on a true solid phase surface.

Independent research compared the yields from in-solution, FASP, and S-Trap based digestions of proteins extracted in SDS and urea-based lysis buffers. 2 Label-free quantification was performed to analyze the differences in the identified proteome using each method. The results showed that, while the different digestion methods were reproducible within the method type, S-Trap outperformed FASP and in-solution digestions by yielding the most efficient digestion, with the greatest number of unique protein identifications

Uniquely, the S-Trap gives the ability to explore the insoluble portion of the proteome, even from such tough substrates as bone or muscle, by first fully solubilizing the samples and then converting the formerly insoluble proteins into soluble peptides. The product enables the extraction, solubilization and handling of all proteins in high concentrations of SDS, prior to further denaturation via reduction and alkylation, acidification (pH <1) and exposure to high concentrations of organic solvent. This four-stage denaturation ensures complete destruction of undesired enzymatic activity – such as proteases and phosphatases – and maximizes the efficiency of downstream digestions. Reduction and alkylation are performed in five percent SDS, precluding precipitation, or can be performed on-column.

Denatured proteins are captured, concentrated and rapidly cleaned of contaminants in the submicron pores of the S-Trap. Proteins are then digested in situ in a rapid (<1 hour), reactor-type digestion. Capture of protein, washing (SDS and contaminant removal) and protease addition requires less than five minutes. After a one- or two-hour digest at 47 °C, peptides are eluted and ready for downstream processing.

S-Trap sample processing technology is available either as spin columns, or automatable 96-well plates. Both formats enable the use of SDS in proteomics sample preparation. S-Trap technology has been used for samples as diverse as serum and dirt and, more recently, has been extensively used to study SARS-CoV-2 infection in COVID-19 research.

Increasing throughput and efficiency: positive thinking

Simplifying the protein sample prep workflow requires a robust protocol that can automate the protein capture, cleaning and digestion steps. The ideal protocol should be identical for the highly varied sample types of interest, because sample preparation is the largest source of experimental variation – up to 75 percent according to published studies. 3

Complex protein samples can have heterogeneous and viscous consistencies, so automation with more robust positive pressure is preferable: vacuum at a maximum of one atmosphere may not be enough, and centrifugation requires expensive and complex mechanisms. Positive pressure has been shown to be more reproducible than vacuum, 4 and typically operates at a higher pressure than vacuum approaches. Positive pressure can therefore more reliably process all the columns of a parallel set-up of a matrix of samples, such as in a 96-well plate, with constant pressure over a specified period with a defined pressure profile.

A realistic assessment of the requirements of a simplified, automated protein sample prep workflow should include processing captured protein samples using a positive pressure unit with liquid dispensing capabilities, for cleaning and digesting samples prior to analysis. S-Traps are available in a 96-well plate format that is compatible with multiple automated platforms, including the Resolvex® A200 positive pressure workstation.

S-Trap sample preparation in combination with the compact, benchtop Resolvex A200 workstation offers affordable, accessible and high throughput automated proteomics sample preparation to laboratories for the first time. The Resolvex A200 system uses gas-based positive pressure to deliver maximum process reproducibility and uniformity across columns or wells, and automates accurate liquid dispensing for up to 11 protocol solvents, including the S-Trap denaturation, washing, binding and elution buffers. This ensures efficient clean-up, digestion and elution. The standard S-Trap proteomics protocols come preinstalled on the Resolvex A200 workstation, or users can create custom protocols optimized for unique needs, reducing processing times and enhancing analytical performance.

How far can S-Trap take us in the clinic?

Automation of the proteomics sample preparation will make a huge difference to the clinic in many key areas. One such area is in the mining of the millions of archival FFPE (formalin-fixed, paraffin-embedded) samples held in labs around the world. S-Trap has already been successfully used in this area, and the standardized protocol includes the use of the HYPERsol protocol, 5 which has been extended to the world’s only simultaneous 96-well megasonicator, the PIXUL™ (Active Motif). The HYPERsol protocol affords the best correlation ever published between paired FFPE and flash frozen samples, as determined by both proteomics identifications and quantifications.

Having solved the bottlenecks associated with sample prep – being at last able to use the same, scalable protocol from sub-microgram to multiple milligram sample sizes, and having obtained new and standardized -omics data with the implementation of these simplified workflows – the next challenge is what to do with the data.

Making sense of the data

Automated sample processing enables generation of large numbers of samples, which brings with it a new problem: how do we make sense of all the data? The sheer quantity of data – along with the myriad tools and approaches – can lead to significant errors in data analysis, due to faulty assumptions and tools, or analyses being incorrectly applied. Indeed, this problem will only grow with the increasing sample throughput afforded by automation.

An efficient solution to this problem might be an online, cloud-based and browser-accessible -omics analysis engine, which translates data into biological meanings through an easy, accessible and interactive interface. ProtiFi recently developed such software, a world first. Called SimpliFi™, it accepts quantified -omics data of all kinds as input, obligates QC of the data, and then performs many statistical analyses to allow users of all skill levels to explore and visualize their data. The interactive analyses include differentially expressed pathways, GO enrichments, cellular markers, proteinprotein interaction networks and molecular-level classifications, all of which can be performed on a smart phone. Results can be shared or published simply by sending a URL. SimpliFi uses all nonparametric statistics and resampling to provide accurate results and confidence intervals, regardless of the structure of the underlying data.

So, can we automate protein sample prep… or not? To revisit the original set of challenges in bringing proteomics into a clinical research setting:

Can we analyze enough samples, and can we simplify our proteomics workflow, both time-efficiently, and cost-effectively?

Yes, we can. Tecan and ProtiFi have partnered to create an integrated, highly scalable and easy-to-use proteomics workflow powered by the S-Trap sample preparation system and the Resolvex A200 automated positive pressure workstation.

Can we then analyze and understand the data?

Yes, SimpliFi allows non-omics experts to use these powerful tools to interrogate and understand biological questions.

References

1) Wiśniewski, JR et al. Universal sample preparation method for proteome analysis. Nature Methods, 2009, 6(5), 359-362.

2) Ludwig, KR et al. Comparison of in-solution, FASP, and S-Trap based digestion methods for bottom-up proteomic studies. Journal of Proteome Research, 2018, 17(7), 2480-2490.

3) Piehowski, PD et al. Sources of technical variability in quantitative LC-MS proteomics: human brain tissue sample analysis. Journal of Proteome Research, 2013, 12(5), 2128-2137.

4) https://www.americanlaboratory. com/914-Application-Notes/172423Evaluation-of-an-Automated-SolidPhase-Extraction-Method-UsingPositive-Pressure

5) Dylan, M et al. HYPERsol: High-quality data from archival FFPE tissue for clinical proteomics. Journal of Proteome Research, 2020, 19 (2), 973-983.

For research use only. Not for use in clinical diagnostics.

To find out more about Tecan’s positive pressure sample preparation solutions, visit diagnostics.tecan.com/samplepreparation-resolvex-positivepressure-processors

For more information about S-Trap technology and SimpliFi, visit protifi.com