Studying the proteome

There are a multitude of experimental strategies for proteome analysis and the choice is dependent on the experimental question, sample type and availability. However they can be broadly classified into two groups:

Global proteomics compares the expression of many proteins (thousands) between samples in a non-targeted manner. These are ‘hypothesis-generating’ experiments.

Targeted proteomics measures the levels of a smaller number of specific proteins between samples in a targeted manner. These are ‘hypothesis-testing’ experiments.

Global proteomics

These experiments are performed to study the changes that result from an intervention (e.g. expression of a gene, or addition of a drug or growth factor) in a model system, or the differences between types of sample (e.g. diseased vs. healthy).

These experiments allow an unbiased assessment of what proteins and pathways are implicated in a particular process. Protein from the sample of interest is digested into peptides using an enzyme such as trypsin. Protein levels are then compared, either by analysing each sample sequentially and comparing the signal from each peptide (‘label-free’ quantification) or by chemical labelling using isobaric tags such as iTRAQ (isobaric Tags for Relative and Absolute Quantitation) , where samples are pooled and analysed simultaneously. If there is a specific population of proteins that are of interest e.g. nuclear proteins, phosphoproteins, it is possible to enrich these and quantify a ‘sub-proteome’.

These experiments generate a list of peptide identifications along with the relative quantity of each peptide in each sample. Sophisticated bioinformatics tools are then used to generate a relative protein ratio (based on the ratio of peptides from that protein) between samples and allow the identification of protein ‘changes’.

Targeted proteomics

This type of experiment is aimed at a specific protein of interest, either determining its quantity, post-translational modification status or both. Immunochemical methods for protein analysis e.g. western blotting or immunohistochemistry, are a type of targeted proteomics. In CADET, while we use antibody-based methods where appropriate, we can also use mass spectrometry (MS) to specifically detect proteins of interest. The advantage of using MS is that there is no requirement for antibodies and as such it is suitable for investigating post-translational modifications, protein isoforms and poorly characterised proteins. An additional advantage of using MS methods such as Selected Reaction Monitoring (see Methods for more detail), is that they can be multiplexed, where several peptides/proteins are measured simultaneously in a single sample. Examples of experiments would be the quantification of specific proteins in plasma samples from patients and normal individuals, the identification of post-translationally modified peptides in a protein, or the quantification of modified peptides either between samples or over a timecourse.

 

Methods

Staff

Instrumentation