Information for Grant Writers

Performance (chromatography, mass accuracy, and sensitivity) of high-resolution, high-mass accuracy LCMS/MS Orbitrap instrumentation is assessed prior to sample analysis by Mass Spectrometry Facility staff using commercially available peptide standards and Hela cell digests (ThermoScientific). The instrumentation is cleaned and calibrated weekly or prior to large-scale, time-intensive experiments. Identification of peptides and protein groups is based on forward and reversed database searches using a false positive rate of <1% at the protein, peptide, and modified peptide levels. Due to the propensity for false positives and ambiguous site assignment of post-translational modifications, the fragmentation patterns (MS/MS) of modified peptides of interest are manually confirmed prior to site-directed mutagenesis, antibody generation, or publication. For global identification of post-translationally modified peptides, the search results are ranked by score, and the tandem mass spectra are inspected to determine the threshold for identification of modified peptides. The probability of site assignments within a peptide is reported for each potential site of modification.

For large-scale proteomic experiments requiring database searching, normalization, and quantitative processing across many LC-MS/MS runs simultaneously, the MaxQuant platform is employed. Raw LCMS/MS data uploaded into MaxQuant are searched to provide peptide identification, inference of the proteins or protein groups present, assignment of sites of post-translational modifications, and extraction of quantitative information at the peptide, protein, and modification-site levels (Nat Biotechnol. 2008;26(12):1367-72. PMID: 19029910; Mol Cell Proteomics. 2014;13(9):2513-26. PMC4159666). The resulting text files are processed and evaluated using the Perseus computational platform, which provides visualization of run-to-run reproducibility, distribution of the quantitative data, statistical tests, and a mechanism for bioinformatic interrogation of the functional relevance of regulated sites of post-translational modification (Nat Methods. 2016;13(9):731-40 PMID: 27348712). The MaxQuant text files are also loaded into programs, such as PTXQC, to rapidly evaluate the reproducibility, efficiency, and robustness of the chromatography and data acquisition (J Proteome Res. 2016;15(3):777-87. PMID: 26653327). These ancillary programs quickly reveal the presence of contaminants (including mycoplasma), technical errors that may have occurred during sample preparation, poor chromatography in one or more analyses, inefficient instrument parameters, and systematic errors that can impact the reproducibility of quantitative measurements. These features are not typically evident in the simplified reports given to investigators.

For label-free quantitation based on chromatographically resolved peak intensities, a minimum of 4 biological replicates are recommended per treatment condition. Replicates are analyzed in a block-randomized format with intervening blanks to prevent carryover. Application of label-free proteomics to identify protein interactions by affinity enrichment-MS (after BioID, immunoenrichment, drug pulldowns) is performed under the assumption that most of the proteins identified are non-specific binding proteins which form the baseline to distinguish proteins that are enriched with the bait (Mol Cell Proteomics. 2015 Jan;14(1):120-35. PMC4288248). For SILAC-based quantitation, three biological replicate experiments included a label-swap control are recommended. Efficient metabolic incorporation of isotopically labeled amino acids (>95%) is confirmed by LC-MS/MS prior to performing the experiment. For TMT-based quantification, instrument parameters and operation are assessed using the triple KO TMT standard (J Am Soc Mass Spectrom. 2016 Oct;27(10):1620-5. PMC5018445). Personnel in the Facility are attuned to looking for and addressing any discrepancies in the performance of the autosamplers, nanoLC systems, mass spectrometers, nitrogen generators, and other components necessary for the proper functioning of the instrumentation.

Authentication of antibodies used to enrich post-translationally modified peptides for subsequent modification-specific proteomic experiments is the responsibility of the project PI. For large-scale experiments, the purchase of the same lot of antibody is recommended to avoid batch effects.

Custom, isotopically-labeled, synthetic peptides with or without post-translational modifications are ordered from Sigma or JPT Peptide Technologies with the following specifications: >98% purity as determined by analytical HPLC, amino acid analysis for lyophilized peptide concentration, and aliquots of a specified quantity in one-time use vials. Mixtures of isotopically labeled synthetic peptides and phosphopeptides for spiking into samples as internal controls and evaluating reproducibility are purchased from ThermoScientific.

The Mass Spectrometry Facility, a University Research Resource Facility, resides within the MUSC Proteomics Center, 4200 sq. ft. of laboratory space on the 3rd floor of the Darby Children’s Research Institute that houses state-of-the-art instrumentation and expertise for proteomics and imaging mass spectrometry.

Mass Spectrometry Facility staff is comprised of the Scientific Director, Lauren E. Ball, Ph.D., Facility Manager, Jennifer Bethard, M.S., and three research assistants with expertise in protein chemistry, LC-MS/MS, and quantitative proteomics who provide consultation and assistance with experimental design, sample preparation, data acquisition, and data interpretation. Data archiving and computing infrastructure are maintained by Mr. Anthony Scott, Information Resources Consultant II. The Facility is supported, in part, by the Vice President for Research at MUSC, the College of Medicine, the Hollings Cancer Center, and NIH grants (S10 OD010731, S10 OD025126, P20 GM103542, and P30 CA138313).

Quantitative proteomic experiments for differential protein expression regulated post-translational modifications, and protein/drug interactions using label-free (MaxLFQ), stable isotope labeling (SILAC), or isobaric tagging (TMT/iTRAQ) approaches are performed on the ThermoScientific Orbitrap Fusion Lumos ETD/UVPD or Orbitrap Elite ETD LC-MS/MS instrumentation. Protein identification and quantification are determined using database searching algorithms within MaxQuant (Max Planck Institute) or Proteome Discoverer 2.3 (Thermo Scientific) software platforms. For the detection of unanticipated or complex post-translational modifications, Protein Prospector (University of California, San Francisco), Byonic (Protein Metrics), BioPharma Finder (ThermoScientific) are also available. Large-scale proteomic experiments are searched using 36 core 128 GB RAM high-performance servers (2) and processed using Perseus (Max Planck Institute), Proteome Discoverer, or Skyline (University of Washington). Statistical analyses (both parametric and non-parametric) can be performed within most of these programs, as well as R-packages from Bioconductor. Data are temporarily stored on high-speed NAS servers with automated backup and archived off-site through CommVault. The Mass Spectrometry Facility shares a license for Advaita Bioinformatics software with the MUSC Bioinformatics Core for downstream data analysis.

Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer with segmented quadrupole, dual pressure ion trap, and high mass accuracy orbitrap mass analyzer with CID, HCD, ETD, EThcD, and UVPD (213 nm) fragmentation capabilities interfaced to an Easy 1200 nano-UHPLC. Thermo Scientific™ Orbitrap Elite™ Hybrid Ion Trap-Orbitrap Mass Spectrometer with CID, HCD, and ETD fragmentation capabilities interfaced to a Dionex UltiMate U3000 nanoLC system for 1D- or 2D-LC. Advion TriVersa Nanomate that can be interfaced with the Orbitrap Elite for direct infusion. Bruker Daltonics AutoFlex III linear MALDI TOF Mass Spectrometer. Waters Xevo TQ-S triple quadrupole mass spectrometer interfaced to a Waters Acquity UPLC M-Class System. Agilent OFF-GEL fractionator. LC Packings Ultimate and Hewlett Packard 1100 HPLC systems.

Bruker Scientific tims-TOF fleX™ Mass Spectrometer capable of trapped ion mobility equipped with sources CaptiveSpray ESI and MALDI using a smartbeam 3D™ laser. Bruker Scientific solariX™ Legacy 7 Tesla FT-ICR Mass Spectrometer with a dual ion source for ESI or MALDI. Dionex Ultimate nanoLC system that can be interfaced to the SolariX via the CaptiveSpray ESI source. Bruker Scientific rapifleX™ MALDI Tissuetyper is equipped with a smartbeam 3D™ laser capable of a 5-micrometer laser spot size with high throughput acquisition rates of up to 50 pixels a second. Two HTX Technologies TM-Sprayer™ Tissue MALDI Sample Prep System, an M3 model, and a high throughput M5 model. Bruker Scientific ImagePrep MALDI matrix sprayer. One Hamamatsu Nanozoomer for high-speed tissue scanning up to 40X resolution. One sublimation apparatus for submicrometer MALDI matrix coating. ThermoScientific HM 550 cryostat. Shandon Finesse microtome AS325 with a heated water bath. Beckel Industries adjustable temperature oven. One Biocare Decloaking Chamber™ for antigen retrieval.

Four -80°C freezers, two -20°C freezers, two 4°C refrigerators, and one cold room with 80 square feet of space for sample and solution storage. Two analytical Mettler Toledo balances ATL160 and Acculabs AL64, one Leica Diavert microscope, one Fisher Scientific sonic dismembrator Model CL-18, and three Savant vacuum concentrators.