Protein identification using LC-ESI-MS/MS

Service description

Massive protein identification and characterization is performed by (mono or multidimensional) nano-HPLC chromatography coupled to a Orbitrap Exploris OE240 high-resolution mass spectrometer. The lenght of the chromatographic gradient employed (tipically, ultra-short short, medium or long) and the type of mass spectrometer used is decided based on the estimated complexity of the sample to be analyzed, as well as the expected number of proteins identified, the resolution level and mass precision required and, finally, the level of sensitivity needed. Service costs are calculated according to these parameters.

Service description

The identification is carried out from a mixture of proteins having a diverse complexity (1D gel bands, immunoprecipitations, complex proteomes, etc.). For this, proteins are digested with an enzyme and the resulting peptides are analyzed by reverse phase liquid chromatography coupled to mass spectrometry (nLC-msms). Enzymatic digestion of proteins is performed either from the cutting of stained band in a gel, or from previously quantified proteins in solution. After digestion and before the injection into the chromatograph, a peptide extraction and / or clean-up (SPE) of the sample is performed by C18 resin. For the chromatographic separation the nano-LC system Easy-nLC II (Proxeon) with a C18 column (15 cm length) is used, and the gradient duration will depend on the complexity of the samples, being 10-20 min for purified proteins, 30-60 min for simple samples (<5 proteins), 90-120 min for medium complexity (<50 proteins) and 150-180 min for complex samples (> 50 proteins). The output of the chromatograph is coupled to Ion Trap mass spectrometer (IT) Amazon Speed ETD, by a nanoelectrospray source (CaptiveSpray). The spectrometric analysis was performed in an AutoMS2 mode, acquiring a full scan (MS) followed by 3-10 MS/MS scans of the most intense signals detected in the MS scan, working with resolutions from 0,3 to 0,5 Da (depending of the scan rates). From the mass data of the fragmented peptides, a search in databases such as -SwissProt, Trembl, NCBI, or others specified by the user- is done using the search engine MASCOT (Matrix Science) that is integrated in other softwares such as ProteinScape and BioTools (Bruker). Standard procedure will include: - Proteins loading in gel, staining with Blue Coomassie, stained bands manual cutting (if on gel digestion is performed). - Proteins reduction and carbamidomethylation. - Proteins trypsin digestion. - Clean-up with C18 reverse-phase resin (SPE) (if solution digestion is performed). - Peptides chromatographic separation. Mass spectra obtainment and peptides fragmentation. - Mass data processing and protein identification in databases. - Results report. INSTRUCTIONS FOR SUBMITTING SAMPLES From proteins in GEL: 1.-The user must send the gel in Milli-Q water (properly identified), stained with Blue Coomassie, Sypro Ruby or Silver compatible with mass spectrometry. Optionally, the user can send proteins cutted bands, in 1.5 ml eppendorf tubes identified with the samples name clearly labeled on the lid. The size of the bands gel should fit the dyed area. 2.-Samples will be delivered together with the corresponding application properly filled out (IDENTIFICATION OF PROTEINS BY MS or MSMS REQUEST). The gel image should be attached indicating the bands to be identified, as well as proteins amount estimation. From proteins in SOLUTION or LYOPHILIZED: 1.-Proteins in solution or lyophilized must be sent in a 0.5 ml tube clearly identified with the sample name labeled on the lid. 2.-Samples will be delivered or sent along with the corresponding application properly filled out (IDENTIFICATION OF PROTEINS BY MS or MSMS REQUEST), where the sample concentration and solvent composition (proteins quantification of dried or lyophilized samples) must be specified. The form should indicate the technique of analysis requested, as well as the degree of sample complexity.

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).