LC-MSMS is the gold standard approach for global high-throughput analysis of complex proteomes. DC Biosciences offer a range of proteomic services to meet all of your proteomic requirements.
In a standard, so-called “bottom-up” LC-MSMS experiment, the proteins in biological samples (1) are extracted (2), and can be further fractionated if required (3).
The proteins are digested into smaller components called peptides (4). The peptides can be further separated by chromatographic methods such as high-pH C18 reversed-phase, strong cation exchange (SAX) or hydrophilic interaction liquid chromatography (HILIC). In addition, certain classes of peptides, e.g., phosphopeptides (5) can be specifically-enriched,
Following purification and concentration (6), the peptides are analysed by liquid chromatography tandem mass spectrometry (LC-MSMS) (7). Raw data files are created that contain several thousand individual spectra. These are then analysed (8) using specific software (e.g., MaxQuant, Proteome Discoverer) to match spectra to expected peptide sequences.
Data can then be further analysed and annotated using publicly-available databases, e.g., GO, KEGG etc. (9)
The commonly-used acquisition mode in mass spectrometry is known as data-dependent acquisition (DDA). Here, a specified number of the most abundant peptide precursor ions are selected by the mass spectrometry method for fragmentation.
Data-independent acquisition (DIA) is becoming increasingly popular. With this approach, all peptides within a specified mass range are selected and simultaneously fragmented to generate extremely complex spectra. More about acquisition methods.
DC Biosciences offers a range of relative quantitative proteomic services including Tandem Mass Tags (TMT), Stable Isotope Labelling with Amino acids in Cell culture (SILAC), label-free and data-independent acquisition (DIA).