Cell/Tissue Protein Extraction and Trypsin Digestion
We routinely analyze a variety of sample types: tissue (fresh frozen, FFPE, OCT, laser micro-dissected), cells (pellets, flow-sorted), extracellular matrix (soluble and crosslinked ECM components, exosomes), fluids (plasma, serum, urine, cerebro-spinal), and organisms (bacteria, yeast). The resource has protocols in place for efficient, high-throughput extraction of total protein from a wide variety of sample types by chloroform/methanol extraction, filter-assisted sample preparation, or single-pot, solid-phase-enhanced sample preparation (SP3).
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. A variant of this protocol has also been developed by the resource for cerebro-spinal fluid analysis.
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 or 18 TMTpro 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 phosphoproteome.
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 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.
DDA Data Acquisition
Following sample preparation, desalted tryptic peptides in aqueous solution are injected for data acquisition from 96-well plates. Up to 1 ug of each peptide sample is drawn into a 20 ul sample loop by the UltiMate 3000 autosampler and loaded onto a 0.5 cm x 75 um trapping column (Waters XSelect CSH C18 3.5 um resin). Peptides are then separated by an acetonitrile gradient on a 15 cm x 75 um analytical column (Waters XSelect CSH C18 2.5 um resin) over a 60-90 min data-dependent acquisition. MS data are acquired at 120,000 resolutions in the Orbitrap mass analyzer. Following HCD fragmentation, MS/MS data are acquired in the linear ion trap analyzer. Data files are backed up from the instrument computer to NAS and Google Cloud storage and processed through MaxQuant or Mascot and in-house bioinformatics analysis software. Typical proteomic depth for complex samples is up to 3,000 quantifiable proteins.
TMT Data Acquisition
Following sample preparation, desalted tryptic peptide fractions in aqueous solution are injected for data acquisition from 96-well plates. For the standard TMT workflow, 18 super fractions consolidated from 46 off-line high-pH reverse-phase peptide fractions are injected successively. For phospho-TMT projects, a similar number of enriched phospho-peptide fractions are also analyzed. Typical proteomic depth for complex samples is up to 8,000 quantifiable proteins and up to 15,000 phosphorylation sites for phospho-TMT projects.
DIA Data Acquisition
Following sample preparation, including depletion of abundant proteins from serum or plasma samples, desalted tryptic peptides in aqueous solution are injected for data acquisition from 96-well plates. Up to 1 ug of each peptide sample is drawn into a 10 ul sample loop by the UltiMate 3000 autosampler and loaded onto a 0.5 cm x 75 um trapping column (Waters XSelect CSH C18 3.5 um resin). Peptides are then separated by an acetonitrile gradient on a 15 cm x 75 um analytical column (Waters XSelect CSH C18 2.5 um resin) over a 60 min data independent acquisition. The Exploris 480 is configured to acquire a precursor scan at 60,000 resolutions, followed by 50x 12 m/z DIA spectra (12 m/z precursor isolation windows at 15,000 resolution) using a staggered window pattern with optimized window placements. Data files are backed up from the instrument computer to NAS and Google Cloud storage and processed through EncyclopeDIA or Spectronaut database search and in-house bioinformatics analysis software. Typical proteomic depth for complex samples is up to 6,000 quantifiable proteins.
