Specific Genetic Analysis

THIS IS A NOTICE OF INTENT TO AWARD A SOLE SOURCE CONTRACT AND IS NOT A REQUEST FOR COMPETITIVE QUOTES.The USDA/ARS intends to award a Firm fixed price sole source contract under the authority of 41 U.S.C 253(c)(1) and FAR 6.302-1(a)(2), only one responsible source, to:Proteomics and Metabolomics FacilityColorado State UniversityC121 Microbiology-Campus 2021Fort Collins, CO 80523-2021Description of Requirement: The objective of this sole source award is to procure the following items:These are the exact procedure that need to be completed in this exact order.Non‐targeted GC‐MS and LC‐MS and targeted fatty acid methyl ester GC‐MS.Biphasic Extraction: Homogenize mature oat seed tissue. Extract with 1 mL MTBE/MeOH/H2O (6/3/1 v/v/v) under constant vortexing at 4C for 60 min. Add 350 μL of water added and vortex at 4C for an additional 30 minutes before centrifugation at 4C for 30 minutes at 3000 xg.GC‐MS analysis of fatty acid methyl esters (FAME): An aliquot of 70 μL of the upper MTBE

layer from biphasic extraction. FAME separation on a 30m DB‐WAXUI column (J&W, 0.25 mm ID, 0.25 μm film thickness). Oven temperature held at 200°C for 1 min, ramped at 10°C/min to 250°C and held for 1 min. Mass detector operated under full scan mode (50‐650 m/z, 5 scans per sec) and electron impact ionization. Calibration curves prepared by a series of dilutions of authentic standards of derivatized fatty acids.GC‐MS analysis of small polar molecules: 40 μL of the lower phase extract dried under nitrogen, re‐suspended in 50 μL of pyridine containing 25 mg/mL of methoxyamine hydrochloride, incubated at 60oC for 45 min, sonicated for 10 min, and incubated for an additional 45 min at 60°C. Metabolites detected using a Trace 1310 GC coupled to a Thermo ISQ mass spectrometer (Thermo Scientific). Samples injected in a 1:10 split ratio twice in discrete randomized blocks. Separation occurred using a 30 m TG‐5MS column (Thermo Scientific, 0.25 mm i.d., 0.25 μm film thickness) with a 1.2 mL/min helium gas flow rate, with a program of 80 oC for 30 sec, a ramp of 15°C per min to 330°C, and an 8 min hold. Masses between 50‐650 m/z scanned at 5 scans/sec after electron impact ionization.UPLC‐MS Phenyl Hexyl Column Analysis: 2 μL of lower aqueous phase extract injected onto a Waters Acquity UPLC system in discrete, randomized blocks with a pooled QC injection after every 6 sample injections, and separated using a Waters Acquity UPLC CSH Phenyl Hexyl column. The column eluent infused into a Waters Xevo G2 Q‐TOF‐MS with an electrospray source in positive mode, scanning 50‐2000 m/z at 0.2 seconds perscan, alternating between MS (6 V collision energy) and MSE mode (15‐30 V ramp).Data Analysis: For each sample, raw data files converted to .cdf format, and matrix of molecular features as defined by retention time and mass (m/z) generated using XCMS software in R [1] for feature detection and alignment. Raw peak areas normalized to total ion signal in R, outlier injections detected based on total signal and PC1 of principle component analysis, and the mean area of the chromatographic peak calculated amongreplicate injections (n=2). Features grouped using RAMClustR [2], which groups features into spectra based coelution and covariance across the full dataset, whereby spectra are used to determine the identity of 2 observed compounds in the experiment. Compounds annotated based on spectral matching to in‐house, NISTv14, Golm, Metlin, Massbank, and 1‐SToP [3] metabolite databases. Annotations are classified based on MSI standards described [4].REFERENCES[1] Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G (2006) XCMS: Processing Mass Spectrometry Data for Metabolite Profiling Using Nonlinear Peak Alignment, Matching, and Identification. Analytical Chemistry 78: 779‐787.[2] Broeckling, CD, Afsar, FA, Neumann, S., Ben‐Hur, A., Prenni, JE. (2014). RAMClust: A novel feature clustering method enables spectral‐matching‐based annotation for metabolomics data. Analytical Chemistry. DOI: 10.1021/ac501530d.[3] Broeckling, C. D., Ganna, A., Layer, M., Brown, K., Sutton, B., Ingelsson, E., ... Prenni, J. E. (2016). Enabling Efficient and Confident Annotation of LC-MS Metabolomics Data through MS1 Spectrum and Time Predic􀆟on. Analytical Chemistry, 88(18), 9226-9234. https://doi.org/10.1021/acs.analchem.6b02479[4] Sumner LW, Amberg A, Barrett D, Beale MH, Beger R, et al. (2007). Proposed minimum reporting standards for chemical analysis. Metabolomics 3:211-21The NAICS code for this requirement is 541714; the small business size standard is 1000 employees.This notice of intent is not a request for competitive quotes; however, interested persons may identify their interest and capability to respond to this requirement. All information received by 3:00 PM Central Time on August 31, 2018, will be considered by the Government. Responses shall be submitted to Lynn Hults at lynn.hults@ars.usda.gov.The e-mail subject line shall state: 1232SC18P-931243 Notice of Intent to Sole Source. A determination by the Government not to compete this proposed contract based on responses to this notice is solely within the discretion of the Government.NO TELEPHONE REQUESTS WILL BE HONORED.