CHARACTERIZATION AND LABELING EFFICIENCY OF 13C1-ALA

University Of Georgia, Athens GA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sample preparation One hundred fifty micro grams of the 13C1-Ala FucT3 and 12C-AlaFucT3 were mixed and dried in speed vac. The mixed sample was dissolved in 100 [unreadable]L of 50 mM ammonium bicarbonate buffer. The sample was immediately reduced with 25 mM dithiothreitol (45 min at 50oC) and carboxyamidomethylated with 90 mM iodoacetamide (45 min at room temperature in the dark). The sample was digested with sequence grade typsin (Promega, Madison, WI) overnight at 37[unreadable]C. After protease digestion, the sample was treated with 1% formic acid to deactivate the enzyme and then was passed through a C18 reversed phase cartridge. The sample was dried down in a speed vac. Analysis of peptides of TEP1 The peptides were resuspended in 39 [unreadable]L of mobile phase A (0.1% formic acid) and 1 [unreadable]L of mobile phase B (80% acetonitrile, in 0.1% formic acid) and then filtered with 0.2 [unreadable]m filters (Nanosep, PALL). The sample was loaded by off- line onto a nanospray tapered capillary column/emitter (360 [unreadable] 75 [unreadable] 15 [unreadable]m, PicoFrit, New Objective) self-packed with C18 reverse phase (RP) resin (10.5 cm, Waters) in a Nitrogen pressure bomb for 5 min at 1000 psi (~5 [unreadable]L load) and then separated via a 160 min linear gradient of increasing mobile phase B at a flow rate of ~200 nL/min directly into the mass spectrometer. The separated peptides were analyzed by nanoelectrospray ionization mass spectrometry (NSI-MS) using an LTQ Orbitrap XL mass spectrometer (Thermofisher, San Jose, CA). A full FTMS (Fourier Transform Mass Spectrometry) spectrum at 60, 000 resolution was collected at m/z 300 to 2000 followed by 6 data dependent MS/MS spectra of ITMS (Ion Trap Mass Sepctrometry) in the most intense ion peaks following CID (36 % normalized collision energy). Dynamic mass increases of 15.9949 Da and 57.0215 Da were allowed from oxidized methionine and alkylated cystenine, respectively. The parent mass width was set up [unreadable] 20.0 ppm and reject mass list was applied to remove the contaminant peaks from column. The resulting data was searched against the FucT3 database using the TurboSequest algorithm. DTA files were generated for spectra with a threshold of 15 ions, a TIC of 1e3 and a range of [MH]+ 500[unreadable]5000 m/z. The SEQUEST parameters were set to allow [unreadable] 30.0 ppm of precursor ion mass tolerance and 0.5 Da of fragment ion tolerance with monoisotopic mass for LTQ Orbitrap XL data searching.