This proposal is seeking funds for a Thermo Orbitrap Eclipse Tribrid with ETD and an Ultimate 3000 RSLCnano system to meet increasing demands for proteomic analysis for 10 labs from two departments of Carnegie Institution (nine are funded by NIH) and for three labs at Stanford University ( two are funded by NIH). The CISMS is equipped with one instrument Orbitrap Q Exactive HF. With only HCD mode and lack of MS3 functionality, lack of sophisticated configuration and software, the current instrument can no longer meet the demands of current NIH-funded research at our institution. In particular, the needs of many researchers in the area of posttranslational modifications (such as O-GlcNAcylation, O-fucosylation, prenylation, methylation), cross-linking mass spectrometry, cell-type specific and single-cell proteomics, rRNA modification, protein-RNA interface mapping, and enhanced quantification using TMT multiplexing, are currently not being met with existing instrumentation. Thus, the planned acquisition of Orbitrap Eclipse ETD is crucial for the development and completion of NIH-supported research, and thereby is fully backed by our research community. The proposed instrument is to be housed within Carnegie Institution for Science Mass Spectrometry facility(CIS-MS) which is highly used by researchers throughout the region. The requested Orbitrap Eclipse with ETD not only doubles the capacity of the facility, but also provides several key capabilities not available on the current instrument of the facility. In particular, ETD and hybrid EThcd fragmentation modes, with MS3 and with sophisticated configuration and software, are essential for projects that study site-specific post-translational modification analysis such as O-GlcNAcylation and O-fucosylation, and prenylation, and structural analysis using MScleavable cross-linker, and rRNA post-transcriptional modifications. The ability to perform real-time search, synchronous precursor selection (SPS) and MS3 fragmentation for TMT isobaric quantification is requested from several groups for projects that demand high accuracy, high sensitivity and high throughput for proteomics and phosphoproteomics, cell type proteomics and single-cell proteomics. Operation and maintenance of the instrument will be performed by experienced staff in CIS-MS. The Carnegie institution provides the $567,739.80 match required to meet the full price of the system, and ensures full support for the facility director and two supporting staff, as well as service contract for year 2 and the remaining effective lifetime of the instrument after the one-year warranty.

Project Narrative This proposal is to request funding for a modern mass spectrometer Orbitrap Eclipse with ETD to empower NIH funded and other federally funded investigators at the Carnegie Institution for Science and Stanford University for their bioanalytical applications. The proposed instrument will be housed within Carnegie Institution for Science Mass Spectrometry Facility (CIS-MS). This requested instrument with many key features required will impact diverse research projects ranging from algae, plants, zebrafish, mice, to humans.