Overview

The purpose of this method is to effectively disrupt samples to solubilse proteins ready for clean-up and digestion before mass spec analysis. The benefits of using the Covaris systems are as follows:

• High energy disruption to ensure physical structures are broken down.
• Highly controlled application of energy to ensure results have batch to batch reproducibility.
• Ability to process up to 96 samples at a time to ensure within batch reproducibility and to save time.
• A controlled temperature environment that ensures minimum heat related sample degradation.

The Covaris systems uses Adaptive Focussed Acoustics (AFA) technology which consists of high frequency/low wavelength acoustic energy that can be focused into a single point within the sample vial. This concentrates all the energy into a single point thereby increasing the efficiency of the process and minimising temperature increases.

The buffers used in this process are 5% SDS which is very disruptive by themselves but when combined with the physical disruption using the AFA leads to our most effective means of sample disruption and protein solubilisation. We generally do not see a non-soluble pellet after disruption of cultured cells anymore.

Typical uses

This method can be used to solubilise proteins from any biological sample before sample preparation using S-Trap devices. It can be adapted to other sample preparation methods by adjusting the lysis buffer used but buffers must be checked for compatibility before using.

Expertise level required

The method uses pre-existing methods and is easy to use with minimal risk to user injury and therefore can be performed by those with basic lab skills.

Responsibilities

Your responsibilities:

• Ensuring the sample is as you have described in any previous meeting.
• Following the provided protocol as described without deviation.
• Informing facility staff if there are any issues when following the protocol or if there are any other deviation.
• Respecting facility equipment and environment, ensuring all equipment is replaced after usage and the workspace is left clean and clear on completion.
• Respecting facility staff and appreciating their workload so ensuring any ad hoc disruption is necessary and concise.
• Using PPMS software correctly for requesting the work including the addition of any associated notes and the inclusion of a manifest.

Our responsibilities:

• Providing you with comprehensive advice before you start your experiment, including clear assessments of costs and any risks associated with the analysis.
• Giving facility inductions and training where required.
• Providing a comprehensive protocol and all relevant reagents and consumables (some are chargeable).
• Providing support during the experimental procedures where required.
• Ensuring that equipment is functioning correctly.

Factors effecting success

• Control of the sizes of the particles in the vials before disruption. Care should be taken with tissue samples to ensure you have some control over the sizes of the pieces placed in the vial. Techniques used in the control of the particle size include manual dissection or the use of a Covaris CryoPrep system – see facility staff for details.
• The number of cycles used. If samples are not completely disrupted then the disruption method on the AFA system can be repeated until disruption is complete.
• The amount of sample used. There is a maximum amount of sample that can be applied to each vial size. Please check with facility staff to ensure you are using the correct vial size.
• Using the correct amount of buffer/avoiding foaming. Foaming dampens the acoustic waves and therefore needs to be avoided. Consult facility staff and follow the protocol in order to avoid foaming.

Protocol

The latest protocol is available in hard copy in the facility. Protocols are not distributed electronically to ensure version control so that the most up to date protocol is always used.

Links

Description of AFA technology https://covaris.com/pre-analytical/

LE220+ manual https://covaris.com/wp-content/uploads/pn_010398.pdf

Publication information

The following text is a guide to how to describe your experiments in reposts or publications. If in doubt please consult with facility staff as it all our responsibilities to ensure that experiments are properly described and reproducible by others.

XXmg of XX were transferred to a XXul AFA tube (Covaris Inc, Woburn, USA, part number XX) and XXul of S-trap solubilisation buffer (5% SDS in 7.55 TEAB) using AFA sonication in a Covaris LE220+ (Woburn, Massachusetts). Sample were disrupted for XXmin, with XX cycles per burst, a duty factor of XX% and a peak incident power of XXW.

Options:

130ul tube part number 520045

500ul tube part number 520185

Acknowledging the facility

Please include the following in your acknowledgements:

[Sample prep/mass spectrometry] work was supported by the Biological Mass Spectrometry (BioMS) facility in the Faculty of Biology, Medicine and Health of the University of Manchester.

Recognition:

The charges within the facility are a mechanism of evenly distributing the cost of running the facility and do not invalidate any intellectual input from members of the facility into your research. Please consider facility staff in the same way you would any other scientific contributor when publishing your work and comply with established guides for designating authors (see https://www.staffnet.manchester.ac.uk/rbe/ethics-integrity/author-guidance/ for the University of Manchester’s policy).

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