Overview

Proteomics is the quantitative study of the total expressed protein within a biological environment. This is traditionally within a cell or tissue but it can also be used in other environments such as cell media or enrichments of specific sub families of proteins such as phosho-proteins. These other sample types are often given a separate name ending in ‘ome such as the secretome or the phosphoproteome. The commonality between these approaches is that they are unbiased and can be used for generating new hypotheses. Although proteomics is commonly used in reference to mass spectrometry based approaches any other technology that can quantify lots of proteins in a complex sample could also be used for proteomics. When proteomics is used on this site we will be describing mass spectrometry based proteomics.

Typical uses

Proteomics is use to look at differences between at least two types of sample, a control and another where a change has been made. This change can be anything of biological relevance and typical examples include a different treatment, a mutation, a change in location or a different point in time. By looking at the changes in protein abundances between the conditions you can then infer which proteins are involved and from there infer functional information. These results can then be compared to curated lists of known protein interactions using tools such as Reactome by the EBI to build upon established work.

Difficulty level

This is a well-established area with extensive support and so it can be performed by those with basic lab skills with a high chance of success of obtaining a useful data set providing a list of proteins and their relevant levels. Interpreting the data can be more complicated and may require further experiments for full utility.

Factors effecting success

• The clarity and relevance of the question: Broad questions generally lead to broad answers so the more specifically the question can be defined the more likely the results will be useful
• The relevance of the samples: The samples used for the experiment need to be appropriate for the question asked. The closer they can be to the biology that you are studying the better.
• The amount of sample: Up to a point the more sample you have the more proteins you can quantify, after that we need to start diluting the samples. The challenge is to have enough material to give you the results you need without compromising the relevance of the sample.
• The choice of control: Ideally the only differences between your control and your sample should be due to the biology that you are studying. The base assumption of most proteomics experiments are that the majority of proteins stay the same and therefore if your samples are to different then the assumptions are incorrect and the statistics will be wrong. You may need to refine your question in order to allow the use of a control with a more subtle difference.
• The number of replicates: As proteomics is a quantitative approach you will need to establish the confidence and error in the experiments and so replicates will be needed. The more replicates you have the better the results will be. A bare minimum is 3 replicates but we ideally recommend 5 replicates. In complex systems such as patient samples, even more replicates may be required.
• Availability of a validation method: Most high level publications will require an independent validation of your results. On a basic level this may be a Western blot but ideally you would genetically manipulate your system to create a phenotype demonstrating that the functional interpretation of your data is valid. Your ability to validate the results will determine their impact.

Workflows available

• Covaris – S-Trap – R3 desalt – QE – Proteome Discoverer

Costing

The facility charges by time for both equipment usage and staff support as well as having some consumable charges for items that have a significant cost. The cost for an experiment will therefore depend on the amount of instrument time used, the amount of staff support required and the amounts of chargeable consumable used. We recommend you contact the facility to arrange a meeting with facility staff to discuss your experimental design and from there calculate the costs. A small proteomics experiment will be about £1,000 but large, more complex experiments can cost over £10,000. Discount rates are available for students and faculty subsidy is available for certain types of project.

User Stories

External links

Tags: #introductions, proteomics

Related