Services & Costs
Here are some examples of what we can do for you:
- Protein Identification / Partial Sequencing
- Identification and Localization of Post-Translational Modifications
- Quantitative comparison of protein abundances in complex mixtures using 10-plex TMT labeling
- Identification of protein/protein interactions by analysis of affinity purified complexes
- Targeted MRM analysis on known proteins
- Determination of whole protein mass
Protein Identification / Partial Sequencing
Identification of unknown proteins is the bread-and-butter analysis for a Proteomics Facility. Proteins are enzymatically digested into peptides, which are then introduced into a mass spectrometer via a liquid chromatography system. Once inside the mass spectrometer we fragment the peptides to produce MS/MS spectra. These fragmentation patterns are matched against theoretical spectra from a protein database. Scoring algorithms and statistical tools are then used to determine the identity of the proteins in the sample.
Samples can be submitted as either a liquid solution of the protein(s), or as a band from a gel. Liquid samples may also by lyophilized or taken to dryness before submission.
Identification and Localization of Post-Translational Modifications
This type of analysis is a step beyond simple protein identification. Post-Translational Modifications (PTMs) that result in a simple mass addition to the peptide can be detected using the software present in our lab. A good example of this type of modification is phosphorylation. The mass shift of the potential PTM is entered into the protein identification software, and the software performs multiple searches of potentially modified peptides looking for forms both with and without the PTM.
This analysis can take considerably longer because the search time increases geometrically with each potential modification added. Also the algorithms for identifying PTMs are less well developed then those for protein identification, meaning every potential PTM must be verified by hand! This adds considerably to the amount of time it takes to analyze the data, and drives up the overall cost of the experiment.
As with protein identification, samples can be submitted as either a liquid solution of the protein(s), or as a band from a gel. Liquid samples may also by lyophilized or taken to dryness before submission.
Label-Free Relative Quantitative Analysis
Relative Quantitation of the same protein in different samples is usually performed via spectral counting in our laboratory. This type of analysis is simple and easy to perform, as the number of times MS/MS spectra matches to a protein entry in the database has been shown to be roughly proportional to the amount of protein present.
Though the analysis of this data is relatively straight forward it requires that more effort is spent on preparing the samples. Errors introduced in the sample preparation phase can result in serious problems with the analysis.
Samples for this kind of analysis are usually submitted as either a liquid solution, or lyophilized powder.
Single gel bands are not generally compatible with the procedure however, as the counting statistics are affected by the nature of the sample. In this case the abundance difference apparent on gel is a more useful then the spectral counting numbers.
Targeted MRM Analysis on Known Proteins
Multiple Reaction Monitoring (MRM) Analysis is a way to do good quantitation on proteins with a relatively low amount of background interference. However a lot of groundwork must be completed before a MRM Analysis can take place. A MRM experiment involves tracking the abundance of chosen ions that result from MS/MS fragmentation in the mass spectrometer. The masses chosen for monitoring must be determined before the experiment, so this process usually requires several mass spectrometry runs to even gather a list of ions to attempt the experiment on. Software for analysis of MRM data is in its infancy, so the data analysis portion of the experiment is still very lengthy as well.
If you'd like to attempt an MRM Experiment, close consultation with the Proteomics Shared Resource is necessary. These experiments usually take significant amounts of time and resources, but in the end can yield a very nice dataset. If you'd like to perform this type of analysis please contact PSR, so that we can assist you in this process.
Determination of whole protein mass
Depending on the nature of the experiment a hypothetical sequence or other information may be required. Samples for this kind of analysis are usually submitted as either a liquid solution, or lyophilized powder.
Whole mass determination of a protein is often a relatively straight-forward analysis. When the protein is introduced into the mass spectrometer a series of charge states are produced. De-convolution software can interpret this complex spectrum, and reconstruct the mass of the protein.