Cores for Vision Research Administration Bioinformatics Core Ocular Microanatomy Core (OM Core) Visual Function and Non-invasive Ocular Imaging Core (VFNOI Core) Ocular Mass Spectrometry, Lipidomics, and Proteomics Core (OMSLP Core) Ophthalmic Testing Core (OTC) Ocular Mass Spectrometry, Lipidomics, and Proteomics Core (OMSLP Core) Home Research Cores For Vision Research Ocular Mass Spectrometry, Lipidomics, and Proteomics Core (OMSLP Core) Ocular Mass Spectrometry, Lipidomics, and Proteomics Core Cores of Vision Research within the University of California Irvine (UCI) will strengthen and enhance NEI R01-funded research. The project aims to support the major expansion of vision research currently underway at UCI. Our goal is to broaden and optimize research capabilities, support and expand existing research projects, and help establish new studies, ultimately boosting productivity and strengthening vision research at UCI. It is sponsored by the NEI P30 grant, P30EY034070-01. INITIAL SERVICE REQUEST FORM Rachel Martin, PhD Core Director, Ocular Mass Spectrometry, Lipidomics & Proteomics Zane Long, PhD Manager, Ocular Mass Spectrometry, Lipidomics, and Proteomics (OMSLP) Core Major Equipment Thermo QExactive with Vanquish UHPLC This is our newest instrument, offering high-accuracy high-resolution m/z measurements for the full range of small molecules, lipids, peptides, and protein samples analyzed by the core. Complete with autosampler, a binary pump system, and a suite of column chemistries, this instrumentation can analyze complex mixtures of compounds while providing detailed molecular information about as many molecules as possible. Thermo LTQ-XL with Vanquish UHPLC This low-resolution triple-quadrupole ion trap allows the core to provide high-sensitivity quantitation of known target molecules, which when combined with UHPLC chromatography allows quantitation of a wide range of molecules in a single sample with incredibly high sensitivity and specificity. The instrument also allows the collection of MSn-type fragmentation spectra, where precursor ions are isolated, fragmented, and those fragments are again isolated and fragmented, repeating a number of times to yield incredibly informative fragmentation spectra. This allows incredibly high confidence structure identification despite the low resolution of the instrument. Small Molecule HPLCs Four nearly identical Agilent 1260 Infinity II HPLCs dedicated to the core, each equipped with their own quaternary solvent pumps (allowing complex mixed-solvent gradients for specialized separations), autosamplers, and wide-range diode-array-detectors for observation of wavelengths between 190 and 950 nm. These HPLCs offer fast scan rates and configurable setups ranging from low-flow analytical runs to large-scale preparatory collections of clean compounds, providing users with a wide range of separation solutions. These four HPLCs are in a dedicated HPLC dark-room for light-sensitive work. mRNA HPLC An Agilent 1260 Infinity II HPLC equipped with a binary solvent pump, autosampler, and wide-range diode-array-detector for observation of wavelengths between 190 and 950 nm. This HPLC is specifically designated for mRNA analysis and is unique in its included column manager. The mRNA HPLC resides in a dedicated mRNA workroom. EL-406 Washer/Dispenser A liquid handler for 96-, 384-, and 1536-well plates, allowing precise reagent delivery at small volumes across a plate. This machine allows careful and replicable handling of cell-based assays. It includes an integrated ultrasonic bath for cleaning and automated system maintenance. LC-MS Overview and Capabilities Liquid chromatography (LC) mass spectrometry (MS) is a powerful analytical technique which allows us to determine the mass and abundance of molecules in a sample. The mass of a molecule is measured by obtaining the observed mass-to-charge ratio (m/z) and determining the charge state of the ion based on the spacing of the observed isotopologue peaks. For example, caffeine has the molecular formula C8H10N4O2, and it appears as the [M+H]+1 adduct, so its m/z = 194.0804 + 1.0073 = 195.0877, with spacing of 1 Da between isotopologue peaks (e.g. 196.0911, from the population of caffeine molecules bearing one 13C instead of 12C), corresponding to a charge state of z = 1. While not all samples and molecules are amenable to LC-MS analysis, most are, namely small molecules (e.g. caffeine), peptides, proteins (both intact and proteolytic digests), and lipids. The two instruments in the core (QExactive and LTQ XL) offer different functions for the analysis of these molecules. The LTQ XL is a low-accuracy (± 0.5 Da) linear ion trap instrument, with MSn capabilities – this means we can ionize a molecule, isolate it in the trap, fragment it, measure the mass of the fragments, isolate a fragment, fragment it again, measure the fragment masses, isolate a fragment, etc. This allows us to collect incredibly detailed fragmentation trees/spectra offering an abundance of structural information about a molecule. The instrument’s fragmentation capabilities also serve to allow the identification of unknown molecules in a sample by comparison to libraries of spectra collected by others. This instrument can also be used to collect low-accuracy mass data of intact proteins by taking the observed m/z values for a protein’s various charge states and back-calculating the intact mass through a deconvolution algorithm. It is limited to relatively small proteins with dominant charge states z <= 10 or 11 at most, beyond which the QExactive is necessary due to resolution limitations. The QExactive is a high-accuracy high-resolution Orbitrap, allowing us to measure m/z values to within <= 1 ppm, while offering incredible sensitivity, especially for small molecules. This instrument is used primarily for small molecules, protein digests, and lipidomics experiments, where unambiguous molecular formula determination is desired. We can identify and (with calibration curves made with known quantities of clean standards) quantify unknown molecules across an entire set of samples, based on m/z, retention time, and fragmentation spectra. This instrument can also determine intact protein exact mass, with much higher accuracy and resolution than the LTQ, which allows it to work with larger proteins.