- Cell/Tissue Protein Extraction and Trypsin Digestion: The resource has protocols in place for efficient, high-throughput extraction of total protein from a wide variety of cell and tissue sample types by chloroform/methanol extraction or filter-assisted sample preparation. Both of these protocols integrate disulfide reduction, cysteine alkylation, and trypsin digestion into a single-day procedure.
- Plasma/Serum Protein Depletion: Plasma and serum samples are prepared in a high-throughput manner for subsequent processing and analysis using HighSelect Top14 abundant protein depletion spin columns (Thermo Scientific) in order to reduce the dynamic range of protein concentrations in these samples. A variant of this protocol has also been developed by the resource for cerebro-spinal fluid (CSF) analysis.
- Histone Extraction: Staff routinely isolate histone proteins from a wide range of biological samples by acid extraction for identification and quantification of epigenetic histone modifications, including acetylation, methylation, ubiquitylation, and phosphorylation.
- SDS-PAGE and In-Gel Trypsin Digestion: The facility is equipped to run as many as eight SDS-PAGE mini-gels in a single day to prepare protein samples for gel-based analysis. The facility stocks 4-12% and 4-20% gradient gels to allow optimum resolution of different sample types. A high-throughput in-gel trypsin digestion protocol is also in place for rapid processing of gel bands or entire gel lanes.
- Isobaric Labeling: The resource uses tandem mass tags (TMT; Thermo) to label tryptic peptides for quantitative multiplexing. Up to eleven isobaric TMT reagents are routinely used, each of which generates a unique quantifiable reporter ion during MS/MS or MS3 fragmentation of labeled peptide during a single analysis.
- Phosphopeptide Enrichment: The resource has fully developed protocols for enrichment of phosphorylated peptides for analysis of the phospho-proteome. In addition to HighSelect TiO2 and Fe-NTA enrichment columns (Thermo Scientific) routinely used for general phospho-proteomics, a Src SH2 domain super-binder enrichment protocol is also being used for the selective enrichment and identification of tyrosine-phosphorylated peptides.
- Peptide Fractionation: Peptides in complex samples are fractionated by reverse-phase chromatography on an Acquity BEH C18 column under basic pH conditions using a stand-alone UltiMate 3000 UHPLC systems (Thermo Scientific) prior to LC-MS/MS analysis under acidic pH conditions.
Custom service. Often investigators will require some hybrid of the above approaches or completely distinct approaches. These can be customized based on the needs of the user.
- Orbitrap Eclipse Mass Spectrometer
- Orbitrap Fusion Lumos Mass Spectrometer
- Orbitrap Fusion Tribrid Mass Spectrometers (2)
- Orbitrap Exploris Mass Spectrometer
- UltiMate 3000 UHPLC
- Each Fusion-class mass spectrometer is equipped with the following options and accessories:
- UPLC System Interface
- Nanospray Ion Source
- EASY-IC (internal calibration) Ion Source
- Electron-Transfer Dissociation (ETD) Source