Identification of a Purified Protein
A highly purified protein, submitted either in-gel or in-solution, is digested with trypsin then analyzed by LC-MS/MS. Data is searched against a publicly available protein database using either Mascot or Sequest search engines. Data is processed in Proteome Discoverer.
Identification of Proteins in Complex Mixtures
A "Shotgun Proteomics" approach is used to identify proteins in a complex mixture. Briefly, the proteins are isolated from the sample and digested with trypsin, which cleaves at Lys (K) and Arg (R) residues. The resulting peptides are separated by HPLC (High Pressure Liquid Chromatography) and sprayed into the mass spectrometer. The mass spectrometer is run in a data-dependent mode. First, a full scan is performed to identify the mass-to-charge ratio (m/z) of the peptides being eluted at any given time. Then, fragmentation spectra for these various peptides are generated. Typically 1D chromatography is used and hundreds to a few thousand proteins are identified from a typical sample. This is highly dependent on the complexity and dynamic range of a sample. For deeper protein coverage of the sample, 2D chromatography is performed.
Relative Quantitation of Complex Protein Mixtures
Relative Quantitation of Complex Protein Mixtures can be performed in a number of ways. These include label-free quantitation (LFQ), using isobaric tags such as TMT or iTRAQ, and labeling cells in culture (SILAC). This is also the type of analysis for identifying binding partners.
- Stable Isotopic Labeling by Amino Acids in Cell Culture (SILAC). Customers are required to purchase SILAC reagents and perform the SILAC incorporation themselves. Prior to large-scale experiments, incorporation testing by LC/MS/MS analysis must be performed to determine the incorporation efficiency.
- Isobaric tagging (iTRAQ or TMT). The facility provides the labels and will perform the labeling step for the customer.
- Label-free Quantitation (LFQ) using area under the curve or spectral counting.
Quantitative data is generated using Proteome Discoverer.
Absolute Quantitation of Protein(s)
Absolute Quantitation of Proteins in Complex Mixtures is performed by PRM (Parallel Reaction Monitoring).
- If the purified protein is available, it is digested using trypsin and analyzed by LC-MS/MS. If a purified protein is not available but the protein is in a complex protein mixture, the sample is digested with trypsin and analyzed by LC-MS/MS.
- Design of potential target peptides is performed. This uses information including that obtained by the LC-MS/MS analysis of the protein, publically available spectral library databases, NCBI sequence searches, and structural analysis to predict peptides which will have the highest potential for success.
- Heavy version of potential target pepetides are commercially synthesized. Typically, standard peptides are labeled by incorporation of [13C6,15N2]lysineand/or [13C6, 15N5]arginine. Three or four unique peptides are synthesized for each protein of interest. This process takes between 4 and 6 weeks. The client is responsible for these costs (~$400 per peptide).
- The quantitation of the protein of interest is performed by adding the heavy peptide to the complex protein mixture. The mixture is digested with trypsin and LC-MS/MS is performed in a data-independent analysis (DIA).
- Data is analyzed using Skyline software (created by MacLean and MacCoss at Univ. of Washington) to generate quantitive results.
Identification/localization of modifications on highly purified proteins, which can be submitted either in-gel or in-solution. Modifications may include:
- Post-translational modifications (PTMs) such as phosphorylation, ubiquitination, acetylation, and glycosylation. If you're interested in a specific modification, please contact us so that we may direct you to sample preparation protocols that could be used to optimize its detection.
- Covalent modifications of a protein. The customer should provide the expected modification(s).
- Cleavage Site Determination.
Proteins are typically digested with trypsin then analyzed by LC-MS/MS. Data is searched against a publicly available or user-provided protein database using either Mascot or Sequest search engines. Data is processed in Proteome Discoverer.
Intact Mass Measurement
Protein or peptides are submitted in-solution for high-resolution mass Analysis. This requires the protein to be relatively pure and free of detergents and glycerol.
Currently, we specialize in phosphopeptide enrichment and analysis using either titanium oxide (TiO2) or IMAC approach. We are able to detect 1000s or phosphopeptides and localize many of the phosphorylation sites. This approach identifies mostly phospho-Serine (pSer) and phospho-threonine (pThr). Normally, 100:10:1 is the ratio of pSer:pThr:pTyr-containing peptides detected from most biological samples.
Other commonly used enrhichment approaches include:
2) PhosphoTyrosine (pTyr) Kit.
3) Ubiquitin (Remnant Motif (K-ε-GG) Kit.
Other types of enrichment (e.g. Succinyl-Lysine Motif [Succ-K] Kit, Pan-Methyl Lysine Kit and Acetyl-Lysine Motif [Ac-K] Kits are available and compatible with our current technology but would require additional development time.