Mass Spectrometry

Protein ID

Protein identifications are made by collecting LC-MS-MS/MS data on the peptide mixtures generated by proteolytic digestion of the samples. The obtained mass spectra are searched against existing databases using the appropriate software. The experimental steps are as follows:

1. Sample digestion with trypsin (prior to the digestion, proteins will be reduced using dithiothreitol (DTT) and alkylated with iodoacetamide).
2. LC-MS-MS/MS analysis.
3. Search of the experimental data against the standard protein databases.

Results will be provided in the electronic format, including the protein hits and supporting data. In addition, ESI-MS data from QC runs of 200 fmol enolase will be provided.

Protein Modifications

Modifications of proteins such as phosphorylations, acetylation etc.

This type of analysis aims at complete (or at as high as possible) sequence coverage of the analyzed proteins. In this case, digestions with multiple enzymes (up to three) are performed. The LC-ESI-MS-MS/MS experiments are performed separately for samples digested with different proteases. Those are often followed by repeated runs with exclusion lists of already identified peptides. For this analysis to be effective proteins should be purified and ideally embedded in SDS-polyacrylamide gel bands.

Samples that can be analysed include purified or semi-purified proteins in gel pieces or in solution. In this case the samples are digested by trypsin (following S-S bond reduction and Cys residue alkylation). The proteolytic digests are analysed by LC-MS-MS/MS and the experimental data will be searched against protein databases with the modifications of interest being specified.
In the case of phosphorylation analysis, enrichment of phosphorylated peptides (using titanium dioxide or immobilized metal affinity chromatography) may be performed prior to the MS experiments to increase the efficiency of the phosphopeptides detection.
The results are provided in electronic format, including the protein hits, sequences of the corresponding peptides and the location of the modification sites.

Mapping of protein phosphorylation

The approach is similar to that described in the Section 3, although several proteases are used in this case, in order to reach fuller sequence coverage of the examined proteins.

Quantification

Relative quantification of proteins

If relative quantification of the sample proteins is required, the mass spectral data is processed by dedicated software tools and the results contain fold values for every individual protein in the analysed samples.
The data is provided in .csv and .htm formats. Samples that can be analysed include complex mixtures of proteins such as entire cell lysates or biological fluids; depending on the nature of the sample, up to 300 proteins may be detected and quantified with this approach (less for biological fluids such as plasma, serum or urine). For a more in-depth analysis please contact the MS laboratory.

Relative quantification of selected (up to ~ 10) protein phosphorylation sites

In cases of proteins being present in mixtures of low complexity (e.g., in SDS-polyacrylamide gel bands), relative quantification of specific phosphorylation sites identified in the analysed samples can be performed.

Molecular weight determination of intact proteins, peptides and small molecules

Accurate molecular weights of purified proteins, peptides and small molecules can be determined using the MS lab equipment. The exact protein molecular weight is a useful parameter for characterisation of protein covalent modifications.

Metabolomics

Metabolomics / Peptidomics

1. Metabolite/peptide profiling (untargeted analysis)
2. Targeted analysis of metabolites, drugs and peptides by LC-MS/MS (SRM)
3. Metabolite/peptide characterisation/identification