Proteomics Sample Preparation

Covaris Adaptive Focused Acoustics® (AFA®) technology provides the most accurate and reproducible sample preparation solution for proteins, enabling non-contact, highly efficient extraction and processing for any downstream analysis method.

Featured Article

Fixed tissues (e.g. with Formalin or OCT) display a valuable depository of clinically relevant primary samples, whether they are embedded in paraffin (FFPE) or fresh. These samples provide great information to elucidate proteome changes during different scenarios such as treatment response, disease courses, remission etc.

Reproducible, Hands-free Protein Sample Preparation with Covaris AFA®

Sample preparation can significantly impact the quality of data in both research and clinical fields. This is especially true in relation to proteomics sample preparation, as proteins are an extremely diverse community of macromolecules, present in an extraordinarily wide range of concentrations, and they cannot be amplified.
Many methods and protocols to isolate proteins are available, but none have the same capabilities as AFA for:
  • Covering the diversity of samples (FFPE, LCM, fresh tissue, cells, bacteria, yeast...)
  • Reducing human error
  • Decreasing hands-on time
  • Increasing speed and throughput
  • Significantly improving reproducibility and reliability

“With the Covaris LE220Rsc we can now fully perform sample preparation for challenging samples, such as fresh frozen and FFPE tissue, in 96-well format. This dramatically improves our throughput to hundreds of thousands of samples per year for true large scale proteomics.”
Dr. Lukas Reiter

Chief Technology Officer - Biognosys

Covaris AFA protein sample preparation can accommodate every sample type for any downstream application in a variety of volumes and throughputs, while still leaving the choice of buffers and clean-up methods open like SP3 (single pot solid phase sample preparation, [1]). Many laboratories need automated solutions for protein sample preparation with better efficiency and higher quality results and Covaris Focused-ultrasonicators are proving to be an extremely robust and flexible solution with growing adoption in the proteomics space.

Watch the video testimonial from Dr. Fabian Coscia at the Novo Nordisk Foundation Center for Protein Research to learn how they developed a reproducible & automated sample preparation solution for clinical proteomics with Covaris Focused-ultrasonicator.

Protein Extraction and Processing For Mass Spectrometry

Covaris has developed workflows and protocols with world leading laboratories looking for improved extraction, automated parallel processing and sample input decrease. Mass spectrometry is the downstream analytical method of choice, but the workflows could be applied to other applications such as western blotting, ELISA or multiplex assays.

“We are very impressed with our Covaris AFA systems. AFA has shown to be ideal for our facility as we deal with a very wide range of different sample types. This single technology has been successful at effectively disrupting and solubilising every sample we have given it. They are professionally designed, easy to use and highly controllable equipment and our LE220-plus is now one of the most essential and highly used systems in our facility.”
Dr. David Knight

Biological Mass Spectrometry Facility Manager, The University of Manchester

Single Pot extraction using SP3 clean-up

In this workflow used on mammalian cells (1,000 to 400,000) and fresh frozen tissues below 10mg, samples are collected and transferred into a Covaris vessel where they are lysed in the desired buffer using AFA. Following lysis, alkylation and reduction reagents are added; AFA can be used again for efficient mixing and enhanced preparation for digestion. SP3 (Single Pot Solid Phase Sample Preparation) beads are added for the washing steps[1]. Ultimately, trypsin or any appropriate enzyme can be added for digestion.
The exact protocol described above was developed in the publication Automated sample preparation with SP3 for low‐input clinical proteomics by Mueller et al. [2] on their LE220R-plus. This protocol will work identically on LE220Rsc and LE220-plus [3] for AFA-TUBE strips and plates. It has been adapted for other instruments using the same consumables like the R230 or the ML230. To learn more about developing this protocol, check our proteomics quick guide or contact [email protected].

 

“AFA based sonication with SP3 provides a very streamlined, end-to-end workflow for proteomic sample preparation all the way from tissue to peptides with minimal manual intervention, establishing a really standardized workflow with low CVs. It accepts low amounts of samples producing quite nice mass spec data. This can be done from any type of tissue, either fresh frozen, FFPE or cell pellets, in a reproducible manner.”
Professor Jeroen Krijgsveld Division head - Proteomics of Stem Cells and Cancer

Deutsches Krebsforschungszentrum (DKFZ)

A Workflow for Formalin Fixed Paraffin Embedded Scrolls using AFA and SP3 Magnetic Beads

As in the previous workflow, samples are processed in a single Covaris vessel. This workflow has been designed and optimized in the AFA-TUBE™ TPX plate. It consists of a first step of paraffin removal without any solvent and this unique AFA-assisted emulsification is patented. Then the sample is reverse cross-linked before a second homogenization step under AFA.

 

This workflow uses the TLB buffer and has been specifically developed in collaboration with Matthias Mann's research groups for FFPE scrolls, read more in our Application Note. It works with magnetic beads for downstream clean-up, similarly to the above SP3 protocol. To learn more about developing these protocols, contact [email protected].

Automated Collection and Processing of Laser Capture Microdissection using AFA and Magnetic Beads

AFA can also be used with delicate samples like LCM. It works in AFA-TUBE TPX plate or strips and thus is directly compatible with SP3 if clean-up is used.
When needed, slides will be first deparaffinized with an appropriate method. Then the regions of interest are selected, laser dissected and directly transferred into the Covaris plate. This procedure can be automated and samples collected directly in the plate when working with a Leica LMD6 or 7, using the corresponding rack. Solutions also exist for collection and processing in a strip. Please contact [email protected] for more information.
After cell lysis with AFA treatment, the samples can be cleaned-up using SP3 like in the previous example. Other methods like S-Trap can also be used [4].

 

Adapted from Grosserueschkamp et al., 2017

“We have tested this specific platform extensively in our laboratory at the Max Planck Institute of Biochemistry, Munich. Using our materials and setup, we can now prepare hundreds of biological and clinical samples in a highly streamlined manner.”
Professor Matthias Mann Director at the Max Planck Institute of Biochemistry

Head of the Clinical Proteomics group at the Novo Nordisk Center for Protein Research

Formalin Fixed Paraffin Embedded (FFPE) Scrolls using AFA and S-Trap

Our truXTRAC FFPE products offer solutions for DNA, RNA and protein extraction. For proteomics applications, scrolls (or curls) are lysed in a Covaris tube with 5% SDS. The combination of AFA and SDS will streamline paraffin removal and emulsification while efficiently solubilizing all types of proteins. Decrosslinking is performed in the same tube. When the last AFA step is performed, the sample is transferred onto the an S-Trap column, where proteins are reduced and digested. Due to the particular design of the column, SDS is fully removed prior to MS analysis.

 

“Deparaffinization of FFPE tissue in the Covaris E220 allows us to avoid the use of xylene in our small laboratory space. Paraffin is efficiently removed from FFPE samples, removing any concerns of clogging columns, contaminating instrumentation or suppressing ionization.”
Dr. Jessica Chapman

Laboratory Director, Memorial Sloan Kettering

Enhanced trypsin digestion using AFA® for improved recovery of membrane proteins

AFA can be used for highly controlled dissociation of membranes followed by enhanced digestion of the membrane-associated proteins, as described in this Technical Note. To learn more about developing this protocol, contact [email protected]

 

AFA for other proteomics downstream applications

AFA has shown to be efficient in a wide variety of downstream analytical methods with different starting materials. Plants, bacteria, yeast, and hard mammalian tissue like muscle have been successfully processed with Focused-ultrasonicators. The controlled and precise energy delivery makes AFA perfect for the preparation and preservation of low abundance or fragile subpopulations of proteins like phosphoproteins[68] , or for native protein extraction [9].
Some examples are given below - please refer to our Cell Lysis Brochure for a more comprehensive overview, including extraction of other molecules of interest like metabolites or lipids.

1. Protein Extraction and Processing for SDS-PAGE and Antibody-based Assays

For SDS-PAGE applications, including western blotting, cells can be processed as already described or collected directly in gel loading buffers like Laemmli and transferred to AFA vessels for acoustic disruption. The process can be adapted to work for different volumes based on the starting material (6-well to 96-well plates). The resulting lysate can then be loaded directly onto the gel. For ELISA and other antibody-based multiplexed assays, cells can be lysed in aqueous buffers or detergent free buffers, for full compatibility with the required downstream reagents.

2. Plants, Yeasts, Bacteria and other non-mammalian sample types

Sample preparation is always about optimization and there are a significant number of parameters that can affect the efficiency of protein recovery. In addition, some organisms have very rigid membrane constituents, others can have a cell wall on top of their membrane, and the insolubility of some components can drastically decrease the quantity of desired biomolecules. AFA has been shown efficiently process difficult materials such as plants [7,8,10], bacteria [11,12] or yeast [13,14].

Resources

To learn more about developing proteomics protocols with AFA, please contact Nicolas Autret, [email protected] or [email protected]

References

  1. Ultrasensitive proteome analysis using paramagnetic bead technology. CS Hughes et al., Mol Syst Biol. 2014;10:757. DOI: 10.15252/msb.20145625
  2. Automated sample preparation with SP3 for low-input clinical proteomics. Mueller et al., Mol Syst Biol (2020)16:e9111. DOI: 10.15252/msb.20199111
  3. A paired liver biopsy and plasma proteomics study reveals circulating biomarkers for alcohol-related liver disease, Niu et al., biorxiv 2020. DOI: 10.1101/2020.10.16.337592
  4. Laser capture microdissection coupled mass spectrometry (LCM-MS) for spatially resolved analysis of formalin-fixed and stained human lung tissues. Herrera et al., Clin Proteom (2020) 17:24 DOI: https://doi.org/10.1186/s12014-020-09287-6
  5. HYPERsol: High-Quality Data from Archival FFPE Tissue for Clinical Proteomics, Marchione et al., J. Proteome Res. 2020, 19, 2, 973–983. DOI: 10.1021/acs.jproteome.9b00686
  6. Multi-level proteomics reveals host-perturbation strategies of SARS-CoV-2 and SARS-CoV. Stukalov et al. , bioRxiv 2020 - https://doi.org/10.1101/2020.06.17.156455
  7. Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics. E Werth et al., The Plant Journal (2017) 89, 416–426 DOI: 10.1111/tpj.13384
  8. The phosphorylated redox proteome of Chlamydomonas reinhardtii: Revealing novel means for regulation of protein structure and function. McConnell et al., Redox Biology Volume 17, July 2018, Pages 35-46 DOI: doi.org/10.1016/j.redox.2018.04.003
  9. A high-efficiency cellular extraction system for biological proteomics. Dhabaria et al., J of Proteome Res. 2015 August 7; 14(8): 3403–340 DOI: 10.1021/acs.jproteome.5b00547
  10. Acoustic Technology for High-Performance Disruption and Extraction of Plant Proteins. M Toorchi et al., Journal of Proteome Research 2008, 7, 3035–3041. DOI: 10.1021/pr800012c
  11. The Role of Cadaverine Synthesis on Pneumococcal Capsule and Protein Expression MF Nakamya et al., Med Sci (Basel). 2018 Jan 19;6(1). DOI: 10.3390/medsci6010008
  12. An ultra scale-down approach to study the interaction of fermentation, homogenisation and centrifugation for antibody fragment recovery from rec E. coli. Q Li et al., Biotechnology and Bioengineering, 2013 Aug;110(8):2150-60 DOI: 10.1002/bit.24891
  13. A Microscale Yeast Cell Disruption Technique for Integrated Process Development Strategies. MD Wenger et al., Biotechnol. Prog. 2008, 24, 606−614. DOI:10.1021/bp070359s
  14. Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design. Blaha et al., Biotechnol Prog. 2018 Jan;34(1):130-140. DOI:10.1002/btpr.2555