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The Core provides KI and MIT researchers with the equipment, training, and expertise to execute their proteomics experiments via mass spectrometry. Services include protein identification, identification of post translational modifications, identification of binding partners, global phosphotyrosine, global serine/threonine phosphorylation, and global protein expression profiling. Additionally, all of these services can be qualitative or quantitative by using labeling and tagging reagents such as SILAC, iTRAQ, or TMT.
The facility is currently equipped with several mass spectrometers, including a Thermo QExactive and QExactive HFX, and an AB Sciex QSTAR Elite, along with high performance nanoflow HPLCs. Data analysis is performed using the Mascot/Sequest/Byonic database search software and Proteome Discoverer or Scaffold.
We use a Bruker model MicroFlex MALDI-TOF (matrix-absorption laser desorption instrument time-of-flight) to mass analyze peptides, protein, small molecules, polymers, DNA/RNA, conjugates and other biomolecules. The practical mass range is between approximately 70 and 170,000 Da.
We use the Intavis Model MultiPep multiple peptide synthesizer to make up to 192 peptides simultaneously. The synthesis scale is 1-10umol, with 5umol being the most common. Peptides containing rare or expensive residues may be synthesized at relatively low cost because of the small amounts of materials required. This application enables synthesis of peptides in milligram quantity for proof-of-principle experiments and is ideal for peptide library and screening experiments. Applications include protein binding interactions, epitope mapping, enzyme inhibition and immunodiagnostics. Following synthesis, the peptide is cleaved from the resin, yielding approximately 2.5µmoles of crude peptide with 80-85% purity. HPLC purification can be performed if higher purity is desired. Average peptide lengths are 10-20 amino acids, though peptides up to 60 residues in length can be synthesized. Runs start on the 1st and 3rd Thursday of each month.
The Protein Technologies Inc., model Tribute peptide synthesizer offers three scales of synthesis: 0.1, 0.3 and 0.5 mmol. After synthesis, the peptide is cleaved from the resin, yielding the crude form of the peptide. Generally, this type of synthesis yields crude peptides with 70-85% purity, but HPLC purification can be done if higher purity is desired. Typical peptide lengths are 5-40 amino acids. Longer peptides are possible.
We use the Intavis SPOT synthesis peptide arrayer system, which permits the synthesis of a 20 x 30 array (10x15 cm) of up to 600 distinct peptides immobilized on a cellulose solid support. Instrument software allows generation of spot sequences and residue offsets from an intact protein sequence. Modifications such as phosphorylation and acetylation may be included. Such arrays are useful for mapping modification sites, delineating protein-protein binding sites or antibody recognition sequences, and delineating other sequence motifs, such as protease cleavage or DNA- and metal-binding motifs. Most epitopes synthesized are under 30 residues.
High performance liquid chromatography enables high resolution separation of compounds from a complex mixture allowing analysis, isolation and purification of individual analytes (typically peptides, proteins, small molecules, conjugates, RNA/DNA, polymers and dye-labeled compounds). The Facility uses several analytical and preparative scale HPLC instruments (for purification up to 100 mg).