Cores

The Proteomics Platform of the Laboratory of Systems Pharmacology (LSP) is part of an inter-institutional effort to improve the science underlying drug development, drug evaluation in clinical trials, and use of new medicines in patients. Our role is to provide collaborative research projects with custom tailored analysis pipelines of highly advanced mass spectrometry (MS) based proteomics. This begins with scientific advice on experimental design, continues with sample preparation of MS samples by our platform, and is followed by support with processing and analysis of collected data. The cornerstone of the platform is a state of the art Orbitrap Lumos Tribrid MS instrument which provides superior resolution and quantification accuracy. By combining isobaric labeling of up to 11 multiplexed experimental conditions with Tandem Mass Tag (TMT) reagents we achieve highly precise relative quantification of thousands of proteins in parallel. Fractionation by alkaline reversed-phase chromatography allows deep analysis of very complex peptide mixtures such as whole proteome digests. Our platform has extensive expertise quantifying phosphopeptides by isobaric tagging (phospho multiplex TMT), iron metal affinity chromatography (IMAC), and antibody-based enrichment of phosphorylated tyrosines (pY). In addition to these systems-wide approaches, our platform applies targeted proteomics using synthesized trigger-peptides or -phospho-peptides. The Proteomics Platform of LSP has successfully applied these technologies to cell culture, tissue, and clinical samples. Statistical analysis is provided to facilitate biological interpretation of large multiplex datasets.

The Multiplex Imaging Platform recently acquired 3 high content imagers (2 Molecular Devices IXM-C and 1 GE INCell 6000) that can continuously process plates from a 110-plate carousel. We provide the unique capability of allowing different multidimensional experiments to be simultaneously conducted in a multi-user environment. We are the first in the world to upgrade both IXM-C microscopes with Lumencor Spectra Gen 3 light sources to extend the number of markers that can be sequentially imaged from 5 to 8. This will also further enhance multiplexing capabilities in developing high-dimensional imaging techniques, such as CyCIF (Project 2). The Spectra Gen 3’s higher output power, through the use of lasers, will allow shorter exposure times and better signal-to-background for more accurate quantification of intensity and morphology in images. The platform has also acquired 2 Rarecyte Cytefinders for 4 to 6-channel rapid multiplex imaging of tissue on slides. These slide scanners were highly instrumental in setting up multidimensional imaging in tissue at LSP. In addition, we also provide image analytical support for processing, identifying, and segmenting tissue samples for downstream quantitative analysis.

The metabolic profiling core uses a targeted LC/MS method, implemented in the Haigis lab, that measures the levels of ~300 metabolites of known identity based on a library of reference compounds. Metabolite detection is performed on a highly accurate QExactive HF-X mass spectrometer. This technique measures water soluble metabolites (central carbon metabolites, amino acids and their derivatives, bile acids, carbohydrates, organic acids, nucleotides, etc.), as well as isotopologues generated from tracing studies utilizing stable isotope labels. Future plans include expansion of our metabolic compound library to >600 compounds, as well as developing methods to detect hydrophobic compounds from biological samples, including several classes of free fatty acids and lipids.

The computational platform focuses on end-to-end pipelines for RNAseq, proteomics, and CyCIF imaging, working closely with the Project leads to integrate analysis methods into the experimental design and execution. The RNAseq pipeline utilizes published methods to align sequence reads against a reference genome, compute differential expression signatures, and perform pathway enrichment analysis. All modules are implemented as ready-to- run scripts with extensive instructions. The pipeline has been successfully used by several trainee scientists with limited computational experience. Together with the Outreach Core, the computation core offers weekly drop-in sessions at the LSP for all CCSP investigators seeking guidance for their computational needs.

The LSP/CCSP Sequencing Platform performs both conventional deep RNA-Seq and the low-cost high-throughput mRNA-Seq method 3’ Digital Gene Expression (DGE). Microfluidic based single cell RNA sequencing (scRNA-Seq) methods are also fully validated and ready for use by Center members. We have expertise and instrumentation for both the commercial 10x Genomics and custom inDrops platform through our partnership with the Single Cell Core at HMS. In another partnership with the nascent transcriptomics core developing out of the laboratory of Karen Adelman at HMS, we are forming a working group of scientists involved in sequencing method development to share insights and methods in order to keep our Platform up-to-date and give us access to more expert knowledge. The nascent transcriptomics core is aimed at providing high-throughput Pro-Seq and Start-Seq. These methods will be useful for all of our projects that involve changes in transcriptional or splicing machinery or looking at drugs that target these core cellular processes. The Sequencing Platform is gearing up for projects that assay epigenetic changes using the Assay for Transposase-Accessible Chromatin (ATAC-Seq).