Quantitative Proteomics Core
The Whitehead Institute Quantitative Proteomics Core applies state-of-the-art methods of mass spectrometry to analyze the dynamics of proteins in living systems. We currently operate Thermo Orbitrap Exploris 480 and Eclipse mass spectrometers for cutting-edge proteome analyses. Our facility provides scientific support to members of the Whitehead and MIT communities as well as external academic or industrial collaborators for small to large scale scientific projects. We provide a versatile proteomics platform, aimed at the supervision of scientific projects from start to finish, including the development of new research directions, consulting on experimental design and feasibility, method development and optimization, sample preparation, data collection and analysis, as well as manuscript preparation and grant writing.
The platform uses the shotgun proteomics approach for global proteome profiling. Sample processing starts with cell lysis, protein digestion, enzymatic protein digestion, and solid-phase extraction of resulting peptides. The purified peptide mixture is separated by liquid chromatography in nano- and microflow modes that inject eluting fractions directly into the Orbitrap systems for the collection of mass spectra. For each sample cohort, hundreds of thousands of peptides are detected and this molecular puzzle can be re-assembled and analyzed by specialized proteomics software packages.
The facility performs simple protein identification and mapping experiments, quantitative proteomics analyses, global screenings of post-translational modifications, as well as analysis of protein-protein interactions. We offer stable-isotope labeling methods including SILAC and TMT and routinely perform fractionation for TMT-based samples and other samples as needed.
The Quantitative Proteomics Core is directed by Dr. Fabian Schulte. Interested users should contact the facility here and provide their samples to the core after scientific consultation. The samples are measured based on the experimental design determined in the preliminary meeting, following the facility guidelines.
We determine the identity of proteins that are dissolved in buffer or from cut gel bands. This approach is straightforward and allows the detection of known and unknown amino acid sequences and their modifications, enzymatic digestion sites, as well as the detection of post-translational modifications. Under ideal experimental conditions, we reach a coverage of at least 70 % of the protein sequence. Further improvements in coverage are achieved by the combination of trypsin with other proteolytic enzymes, such as LysN, AspN, GluC, or ArgC.
The total protein inventory in cellular extracts is typically analyzed via trypsination followed by chromatographic separation of purified peptides and collection of data on MS instruments. One of the most common experimental strategies are label-free approaches, where the peak areas of detectable peptides are used to compare their abundance between samples. Depending on the experimental requirements, we offer a versatile set of targeted and untargeted MS detection modes to survey global proteome profiles at different sensitivities and specificities via relative or absolute quantitation. The core is equipped with high-resolution nanoLC-systems that enable deep proteome studies with the highest possible protein coverage and sensitivity. For higher throughput, our research infrastructure also includes a microflow-HPLC systems allowing the collection of data from large sample cohorts with higher throughput, but lower sensitivity and protein coverage than nanoLC.
Labeling of proteins with stable heavy isotopes is a widely used method for quantitative proteomics. SILAC is most commonly based on the direct supplementation of 13C and 15N enriched arginine and lysine isotopes into the cell culture medium. After protein extraction and digestion, up to three protein samples are multiplexed and the isotopic labels are resolved via LC-MS for quantitation. The use of multiplexed samples eliminates problems that are commonly observed in label-free experiments across individually measured samples, such as zero-values and technical variability, which diminish the statistical power of the assays. SILAC has been widely applied to characterize differences in proteome compositions, analyze the dynamics of post-translational modifications or protein-protein interactions, and to monitor the turnover of proteins with high accuracy and reproducibility proteome-wide.
TMT is a class of labelling reagents that is used to detect proteins in multiplexed samples. Unlike SILAC, the labelling with TMT occurs after sample extraction, and targets peptides via a chemical derivatization that is specific for free amines. TMT labels are designed to have an amine-reactive linker group, a spacer arm, and a mass reporter and the labels contain different numbers and combinations of 13C and 15N isotopes resulting in MS2 reporter ions that are unique for each label. After labelling, up to 18 peptide samples are mixed in one sample and identical peptides with different labels show the same elution profile during chromatographic separation. The TMT labels are designed so that the MS2 reporter group is detectable in high-resolution tandem mass spectrometers and the resulting fragment ions are used to determine the peptide abundance of multiple samples in a single LC-MS run. Since the use of TMT allows for the quantitation of peptides with greater accuracy and reproducibility than many other methods, this technology is particularly useful to monitor post-translational modifications or to collect data from very complex experimental designs, such as time-series experiments or sample populations with many experimental groups.
Post-translation modifications are required for controlling almost every biological process to regulate protein functions in a relatively short time scales. The Quantitative Proteomics Core has implemented multiple experimental strategies to enrich and analyze post-translational modifications, such as phosphorylation, acetylation, and ubiquitination for single proteins and complex proteome extracts. We also offer the identification of protease target sites via PALEO (protease activity labeling employing 18O-enriched water). The sample extraction and MS-detection methods for novel or more uncommon post-translational modifications can be implemented if required. We recommend the use of TMT labelling techniques, to maximize the accuracy and reproducibility in the detection of functional groups.
Proteins typically do not act as single players but form complex cellular network of protein interactions that are tightly regulated and adapt dynamically to external and internal signals. The formation of these macromolecular complexes is central for almost every biochemical process, and provides deeper insight into biochemical reactions, regulation of cellular processes, mechanisms of disease and biomarkers, as well as the discovery of drug targets. Consequently, the study of protein interaction networks and their dynamics is a key-factor to define a protein’s function. To survey the total set of protein interactions in biological samples, the Quantitative Proteomics facility employs a variety of techniques, such as: immunoprecipitation, cross-linking mass spectrometry (XL-MS), and proximity-labelling techniques (e.g., APEX2 labelling).
We offer many different sample preparation protocols to extract proteins from a wide range of matrices, such as bacteria, plants, cell cultures, tissues, and biofluids. However, there may always be a need to tailor sample preparation procedures of the individual research project to obtain optimal results. We have 15 years of experience in the optimization of workflows for the isolation of low abundance analytes and the processing of recalcitrant samples. We can also provide assessments for the feasibility of studies and alternative experimental strategies. If you want to establish a method for a particularly difficult sample or simply need an assessment of different available methods, our team is happy to help.
Our facility possesses a versatile set of data-analysis tools to extract and process raw MS1 and MS2 spectra, determine the sequence of proteins, conduct proteome-wide data analyses, identify post-translational modifications, analyze protein interactions, and survey the turnover of proteins. For the processing of highly complex proteome raw data files, we currently employ the Proteome Discoverer 3.0 platform. We also offer the analysis of datasets with MaxQuant, Peaks Studio Xpro, Skyline, DIA-NN, and MSfragger. The Quantitative Proteomics Core uses publicly available R-scripts for the processing of raw data, but also has a very dynamic collaboration with the Whitehead Bioinformatics and Research Computing Core (BaRC) to extract the best possible results from your experiments.
Please contact us for quotes: fschulte at wi.mit.edu
Orbitrap Exploris 480
The Orbitrap Exploris 480 is the most commonly used instrument in our lab, which uses a high-capacity transfer tubes and orbitrap mass analyzer designed to deliver data with high sensitivity, mass accuracy, and resolving power (480,000,000 at m/z 200) across a wide dynamic range. The system is equipped with the FAIMS-PRO interface that increases the selectivity and signal-to-noise ratio of MS detection. For the high-resolving separation of protein digests, we utilize the EASY-nLC 1200 system to collect deep proteome data with the highest possible protein coverage. The Orbitrap Exploris 480 is ideally suited for a variety of proteome experiments, including the sequencing and identification of proteins, as well as quantitative proteomics experiments, such as the detection of post-translational modifications and multiplexing with TMT.
Orbitrap Eclipse Tribrid
The Orbitrap Eclipse Tribrid includes several leading technologies for ion transmission, selection, and fragmentation to achieve the best possible performance in sensitivity, mass resolution, and versatility. Its setup includes the use of a FAIMS-PRO device for optimized selection of ions, a modified dual-pressure linear ion trap for the fragmentation of ions in CID, HCD, and ETD mode, as well as an Orbitrap analyzer with a resolution of 1,000,000 at m/z 200 to maximize the detection of analytes. The TMT SPS MS3 workflow optimizes the protein ID coverage and accuracy of quantitation in TMT-labelling experiments through comparison of reporter ion intensities on the MS3 level. For the highest possible flexibility in experimental design, the system is equipped with a Vanquish Neo chromatography system. This allows us to run the instrument in nanoflow mode using long gradients (60-180 min) for deep proteome experiments with low sample throughput as well as in microflow mode using short gradients (<30 min) for the high throughput of targeted assays or low-complexity protein samples.
The Whitehead Proteomics Facility is accepting samples. Please contact Dr. Fabian Schulte for further information.
If you have any questions about the services and techniques offered by the Quantitative Proteomics core, please email us (preferred) or give us a call. Open office: every Monday 10-11 AM.
Dr. Fabian Schulte
455 Main St
Phone: (Six One Seven) Three Two Four – Zero Seven Two Seven
fschulte at wi.mit.edu