Protein identification using LC-ESI-MS/MS

Service description

Mass spectrometry-based methods for the identification of proteins have become a standard platform in proteomics. The most popular MS-based strategies rely on proteolytic digestion of proteins into peptides before introduction into the mass spectrometer. Digestion of proteins into similar sized peptides helps to overcome the solubility and handling problems associated with proteins and creates peptide fragments which are easily ionized in the mass spectrometer. Peptide ions are first measured as intact fragment ions, then selected based on their m/z and subject to collisionally induced dissociation (CID), in a process known as tandem mass spectrometry (MS/MS). Under the low-energy conditions employed for CID, peptide ions fragment in predictable patterns. Because fragmentation patterns are predictable, theoretical spectra can be constructed for sequences in protein or nucleotide databases. Computer algorithms use the CID fragmentation patterns of sample peptides to determine the sequence of the peptide, and this sequence information is used to search against theoretical spectra generated from protein and nucleotide databases. Protein identifications are made by finding the best correlation between experimentally derived sequence information and sequences in the database. User Instructions: 1. Run a SDS-PAGE with the aim of estimate the amount of protein present in the sample, the protein complexity, the dynamic range and the level of contaminants. 2. The sample can be in solution or gel. 3. The minimal amount of sample depends on mass spectrometer we use. Procedure: Protein Digests are analyzed using LC-MS/MS on either a Thermo Scientific LTQ-Velos or a Thermo Scientific LTQ-Orbitrap-Velos-Pro mass spectrometer. LTQ-Velos system is equipped with Agilent 1100 high pressure liquid chromatograph (HPLC) and LTQ-Orbitrap-Velos-Pro with Proxeon Easy-nLC II high pressure liquid chromatograph (HPLC) for peptide separation. The instrument choice (determined by the Proteomics Staff) depends on the project and the level of sample available. Typically, the gradient used for low complexity mixture (< 5 proteins) is 90 min, but as complexity increases, gradient times increase proportionally. Peptide identification from raw data is carrying out using PEAKS Studio Xpro (Bioinformatics Solutions).