Cell/Tissue Protein Extraction and Protease Digestion
The facility 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 procedure. In addition, both protocols have been adapted to allow processing of large numbers of samples simultaneously.
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 resin (Thermo Scientific) in order to reduce the dynamic range of protein concentrations in these samples. Depletion of abundant proteins from these types of samples dramatically increases the number of lower-abundance proteins that can be identified and quantified by mass spectrometry.
Histone proteins can be isolated 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 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.
Tandem mass tags (TMT; Thermo Scientific) are used to label tryptic peptides for quantitative multiplexing. Up to sixteen isobaric TMT reagents are routinely used, each of which generates a unique quantifiable reporter ion during MS/MS or MS3 fragmentation of labeled peptides during a single analysis. Multiplexing of samples allows a several-fold increase in the efficient use of mass spectrometer time and improves data quality by eliminating variability resulting from analyzing samples from the same experiment at different times.
The facility has fully developed protocols for enrichment of phosphorylated peptides from a variety of sample types for analysis of the phospho-proteome. HighSelect TiO2 and Fe-NTA enrichment columns (Thermo Scientific) are routinely used for general phospho-proteomics, with analysis of both enriched and un-enriched samples to allow differences in protein abundance to be distinguished from changes in phosphorylation.
Peptides in complex samples are fractionated by reverse-phase chromatography under basic pH conditions using a stand-alone UltiMate 3000 UHPLC system (Thermo Scientific) prior to LC-MS/MS analysis under acidic pH conditions. The pH differential between the off-line and in-line separations results in an even distribution of peptides across up to 36 hours of mass spectrometer time, maximizing the number of identifiable peptides in each sample and allowing efficient use of instrument time.
The facility has implemented a DIA workflow harnessing the unparalleled performance of the Orbitrap Exploris 480 mass spectrometer to acquire high-quality data for large sample sets. This approach is particularly well-suited to analysis of serum and plasma samples. DIA collects data for all peptides within defined mass-to-charge windows rather than detecting each peptide in a sample prior to fragmentation.