Overview

This workflow is preparing global proteomics samples in a generic easy to use way. It uses focussed ultrasonication in the Covaris systems in the facility using highly aggressive buffers and this approach has been demonstrated to work with every sample type tested so far include many cultures cell types, tissues and microbial systems. This is followed by S-Trap analysis which is used to concentrate, clean and digest the sample and again this has been demonstrated to work on every sample type that has been tested. The next step is sample clean up using a plate based method incorporating R3 beads as a reversed phase support. This method removes any remaining contamination that would lead to long term contamination of the system.  The samples are analysed by our flagship proteomics focussed mass spectrometry systems and the data analysed using Proteome Discoverer software. This is our preferred data analysis solution as it is a comprehensive application that can provide many of the statistical tests you need as well as providing highly comprehensive results and uses robust statistics to provide reliable quantification.

Typical uses

This workflow can be used for any proteomics project and should be considered the default approach unless there is a significant reason not to.

Expertise required

There are extensive protocols for all lab methods so the workflow can be performed by those with basic lab skills e.g. you must be able to use a pipette. Performing the data analysis yourself can be complicated and is best performed by those experienced in using data analysis tools. Data analysis can be supplied as a service if required at extra cost.

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 in compliance with the Facility’s sample submission guidelines 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 analytical systems are functioning correctly and that the data produced is an accurate representation of the sample provided.
• Providing feedback on the results, identifying issues if they occur and suggesting ways to address the issues if possible.
• Providing access to the relevant data analysis software and providing training and advice where required.
• Providing data analysis services where available and required (at extra cost)

Factors effecting success

• The samples have an unusual composition. This may be the presence of a high level of contamination such as lipids or polymers, a high level of a subset of proteins or unusual mechanical properties such as a high fibrous content.
• Inadequate choice of control whereby it is significantly different to the samples it compares to.
• The availability of a reference protein sequence library. You ideally need to working in a biological system that has a full protein library or has a closely related species that does. Please note we require a protein sequence library (proteome) – a sequenced genome is not sufficient as the translation from genetic sequence to protein sequence is non-trivial.

Protocol

The latest comprehensive protocol is available in the facility.

Methods involved

Covaris disruption of protein samples before S-Trap sample preparation

S-Trap based sample preparation for protein analysis

Mass spectrometry based quantitative analysis of peptides samples

Quantitative analysis of peptide based mass spectrometry data using Proteome Discoverer

 [RO1]Does this comment refer to R3? Overall I would bet that it is the S-trap method that removes the bulk non-protein derived material, most other labs inject directly after S-trap processing, we do it as a polishing step, and I guess because we do not have a trap on our LC systems.

 [RO2]I would emphasis this aspect of sample submission.

For this I would say that the samples on submission must meet the minimum criteria for acceptance, these include PPMS order number, samples dried down in vials, and a sample manifest number, with a manifest that makes sense. The organism from which the smaples are to be searched against, any consideration of run order, carry-over and so on.

 [RO3]Following on from this, we would then ensure that Upon arrival to the laboratory, the samples and their accompanying request form are checked to ensure they meet the minimum criteria for acceptance. Staff ensure that the names on form and LCMS vials match and that the correct type of vial is received

Tags: #workflows, s-trap

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