High-End Instrumentation (HEI) Grant Program (S10)

Cleveland Clinic Lerner Com-Cwru, Cleveland OH

Investigators

Linked publications, trials & patents

Abstract

Abstract This proposal is for the acquisition of a high-end LC-MS/MS instrument to be placed in the Proteomics Core Laboratory at the Lerner Research Institute at the Cleveland Clinic. The proposal contains projects from nine NIH funded investigators and is asking for a ThermoScientific Fusion Lumos instrument which will be coupled to a Dionex Ultimate 3000 uHPLC. This is a high-end LC-MS/MS system that represents the state-of-the-art in mass spectrometry that is being utilized for proteomics investigations. Currently, the lab is equipped with two LC-MS/MS systems including a twelve year old ThermoScientific LTQ and a four year old ThermoScientific Orbitrap-Elite system. The Elite instrument is the cornerstone of the Proteomics Core and performs 98% of the current experiments and is currently running at capacity. More importantly, several of the projects presented in this proposal require access to an instrument with higher sensitivity and additional functionality not available on the Elite instrument. We performed several preliminary experiments comparing the performance of the Elite to the Lumos. The Lumos instrument has a much higher scan rate and is more sensitive than the Elite resulting in a 2 fold increase in peptide and protein identifications and a 200 fold increase in LOQ for targeted peptide quantitation. This increase in sensitivity will have a positive impact on the identification of low abundant novel proteins derived from alternative poly(A) sites (Ting) and the identification of low abundant oxidized lipid-protein conjugates (Podrez Lab). One of the features of the Lumos instrument that is not available on the Elite is the ability to perform SPS-MS3 fragmentation for isobaric quantitation at both the protein and peptide levels. Several projects in this proposal will be greatly impacted by this functionality including the analysis of the gut microbiome (Hazen), biomarker identification in PAH (Erzurum), identification of the ADAMTS substrates (Apte), and analysis of the sulfhydrome (Hatzoglou). Another feature of this instrument currently not available in the Proteomics core is ETD which is an alternative fragmentation method that can be used for site-specific identification of post-translational modifications (PTM's) on highly charged ions. Several of these projects include analysis of PTM's including phosphorylation (Stark, Erzurum, Lefebvre, Gupta), sulfhydration (Hatzoglou), and oxidized lipid-protein conjugates (Podrez) all of which will be positively impacted by the ability to perform ETD fragmentation. Another feature of the Fusion Lumos instrument not available on the Elite is the ability to perform sensitive large-scale targeted quantitative studies on 100's of peptides simultaneously. This functionality will greatly impact research involving the large-scale quantitation of post-translational modifications (Stark and Gupta), validation of TMA lyases present in the gut microbiome (Hazen), and the validation of protein biomarkers of PAH (Erzurum).