Proteomics and Advanced Mass Spectrometry

Protein Identification . PTM Discovery . Quantitation . Advanced Mass Spectrometry . SILAC . Cross-linking . Pull-downs .

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Introduction

The University of Leicester Proteomics Facility provides proteomics and mass spectrometry services to the College of Medicine and Biological Sciences, to UK and international academic laboratories and to other external clients. The proteomics facility is located in the Hodgkin Building on the main University of Leicester campus.

Advanced mass spectrometry and proteomics technologies allow identification of proteins from gels or protein mixtures. Identification of sites of post-translational modifications can be determined as can relative quantification of protein amounts between differential samples.


Sample Submission

It is best practice to contact the Proteomics Facility prior to generating samples for analysis, as there is often advice or precautions that can be taken to ensure the very best outcome. Where possible all samples should be brought in person to the PNACL facility. For samples that require shipping, please contact us prior to dispatch. All samples should have a completed sample submission form.

The PNACL proteomics facility provides a clear timeline from sample submission through to data return, with e-mail updates at each stage of analysis. There are two different categories of analysis depending upon sample type:

Standard Services

Bespoke Projects


Protein Identification

Single proteins or simple protein mixtures are analysed using short-gradient LC-MS/MS and the data searched against protein databases to provide an ID. Where the submitted protein is a construct (i.e. non-naturally occurring such as GST-fusion/GFP-trap or other tagged protein) the sequence should be provided by the user.

Complex mixtures (e.g. Interaction pulldowns / cell lysates etc.) will require longer gradients and may require sample fractionation. These will incur additional costs based on the number of LC runs or number of fractions.


Post-translational Modifications

A PTM will result in a change in mass of the modified amino-acid and thus peptide mass. This can be detected by the mass spectrometer, and in an LC-MS/MS experiment the site of modification can be determined. The PNACL Proteomics Facility has experience of analysing a wide variety of modifications - phosphorylation, ubiquitinylation, acetylation, myristoylation and chemical cross-linking amongst others. For certain modifiations enrichment strategies are available, examples being IMAC and TiO for phospho-enrichment, anti-GlyGly for ubiquitinylation or anti-acetyllysine for acetylation.


Protein Quantitation

Changes in protein expression/amount can be determined by a range of methods including Stable Isotope Labelling (SILAC), reporter-ion labelling (iTRAQ/TMT), Selected Reaction Monitoring (SRM) and label-free/spectral counting.

 

Prior to starting your sample preparation, please contact either Dr. Andrew Bottrill or Dr. Sharad Mistry to determine whether it is appropriate. Some simple planning at the outset of a project can expedite the analysis time and optimise the data quality.

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