Services Provided by the PRF
- Identification of protein bands from Coomassie and mass spectrometry-compatible silver-stained gels: This includes reduction/alklylation of cysteines, in-gel digestion with trypsin, followed by a standard 90 minute nano-RP-LC-MS/MS data acquisition, database search using either X!Tandem/TPP or Proteome Discoverer 2.1 (SEQUEST) and delivery of results (either as an e-mail link or an excel file format).
- Shot-gun proteomic analysis of complex mixtures by tandem mass spectrometry (In-solution digestion followed by Multi-dimensional Protein Identification Technology): This service includes trypsin digestion followed by an extended nano-RP-LC-MS/MS analysis for medium complexity samples (3 hour). For more complex samples, 2D-LC separation can be performed followed by fraction collection, desalting, standard 90 minute RP-LC-MS/MS analysis of each fraction, database search using either X!Tandem/TPP or Proteome Discoverer 2.1 (SEQUEST) and delivery of results (either as an e-mail link or an excel file format). The Investigator can decide the number of fractions to be analyzed after consultation with the laboratory manager. Longer gradients are also available at additional cost.
- Identification of post-translational modifications: As post-translationally modified protein is only a small fraction of the total protein content and the ionization of modified peptide(s) is often suppressed in presence of huge number of unmodified peptides, identification of PTM sites require enrichment of modified peptides. Some well established methods for enrichment of phosphopeptides and glycopeptides are available (see below).
- Phosphorylation - Metal Oxide Affinity Chromatography (TiO2).
- Glycosylation - Based on Solid Phase Extraction of Glycopeptides (SPEG)
- Quantitative proteomic profiling
- MS2-based: (For lysis buffer compositions please read "Protocol" carefully)
- Parallel Reaction Monitoring (PRM) for quantification of proteins and post-translational modifications: PRM approach is employed for validating (eg. a potential biomarker identified during discovery phase) and/or more accurate quantitation of an analyte in a large cohort of samples. The instrument is set to isolate only analyte(s)-of-interest (i.e., specific m/z), fragment (HCD) and acquire MS/MS. This configuration can improve the sensitivity of detection and dynamic range of quantitation of the analyte. For absolute quantification, the experiment involves spiking-in a known amount of heavy-isotope labeled analyte into the peptide mixture derived from relevant sample matrix and monitoring both endogenous and heavy-isotope labeled peptides. Since the heavy-isotope labeled peptide is physico-chemically identical (except mass) to the endogenous peptide, they co-elute and the area under the curve (AUC) can be used to determine the concentration of the endogenous peptide. As PRM process requires optimization of experimental and data analysis pipelines, please contact the PRF beforehand to discuss the feasibility, sample requirements and cost of analysis.
- Data-Independent Acquisition (DIA):
- All our instruments use electrospray ionization (ESI) for introducing the sample into the mass spectrometer and are less tolerant to several buffer components (salts, detergents etc). While some of these can be removed using additional cleaning steps, these will result in loss of sample. To avoid these steps if possible, the Investigator is encouraged to discuss the sample preparation method with the laboratory manager beforehand.
- Most of the above services (except identification of Ubiquitination sites) can be performed using alternative enzyme. If enzymes other than trypsin are needed, the Investigator will provide the appropriate sequencing-grade enzyme.
- All kits/reagents needed for PTM enrichment (such as TiO2 or ZIC-HILIC) or quantitative profiling (such as ICAT, iTRAQ, TMT or heavy labeled synthetic peptides necessary for MRM) will be provided by the Investigator.
Please review the Submission Guidelines for information on sample preparation that is necessary for proteomic analysis by tandem mass spectrometry.