This Is A Combined Synopsis Solicitation For Commercial Items Prepared In Accordance With The Format In Far Subpart 12 6 As Supplemented With Additional Information Included In This Notice The Solicitation Number Is 1026567 And Is Issued As A Request For Quotation Rfq Unless Otherwise Indicated Herein This Announcement Constitutes The Only Solicitation Quotations Are Being Requested And A Written Solicitation Will Not Be Issued The Associated North American Industrial Classification System Naics Code For This Procurement Is 541380 Requirement Usda Ars Pullman Wa 99164 Has A Requirement Wheat Proteomics Research Requirement 42 Samples At 500Mg Each Will Need To Be Processed Per The Below Specifications Wheat Proteomics Research Super Soft Kernel Trait In Soft White Wheat Required Specifications 2 8 21 Craig F Morris Sample Preparation Protein Extraction And Quantification Samples Will Be Homogenized Using Appropriate Mechanical Disruption E G A Bullet Blender 5 Storm Next Advance At Speed 12 For 5 Minutes Followed By Incubation On Ice Addition Of 75Ul Homogenization Buffer And A Second Homogenization At Speed 12 For 3 Minutes Sample Proteins Will Be Extracted E G Homogenates Mixed With Sds To Approximately 2 Final Concentration Followed By Cup Horn Sonication Amplitude 70 10S Pulse Followed By 20S Rest 8 Minutes Total Sonication Followed By Centrifuging At 10 000Xg 4C For 2 Minutes Protein Solution Is Quantified E G Supernatant Aliquots Are Diluted 1 5 And 1 10 In 2M Urea 2 Sds And Subjected To A Bca Protein Assay Pierce Following Manufacturer S Instructions Absorbance At 550Nm Will Be Measured On A Biorad 680 Microplate Reader And Total Protein Concentrations Calculated Based On A Bovine Serum Albumin Standard Curve Fit To A Quadratic Microplate Manager 5 Software Biorad Sample Preparation In Solution Trypsin Digestion Samples Will Be Processed For In Solution Trypsin Digestion E G 50 Ug Protein Will Be Resolubilized In 8M Urea And 0 2 Proteasemaxtm Surfactant Trypsin Enhancer Promega Samples Will Be Reduced And Alkylated With 5Mm Dithiothreitol And 5Mm Iodoacetic Acid Samples Will Be Digested With Trypsin And Quantified E G Trypsin Pierce Ms Grade Thermo Scientific Will Be Added At An Enzyme To Substrate Ratio Of 1 28 And Incubated At 37C For 3 Hours Trypsin Will Be Deactivated With The Addition Of 5 Trifluoroacetic Acid And Desalted Using Pierce C18 Spin Columns Thermo Scientific Using Manufacturer S Instructions Peptide Eluate Will Be Dried In A Vacuum Evaporator And Resuspended In 5 Acetonitrile 0 1 Formic Acid Once Resolubilized Absorbance At 205Nm Will Be Measured On A Nanodrop Thermoscientific And Total Peptide Concentration Will Subsequently Be Calculated Using An Extinction Coefficient Of 31 Mass Spectrometry Analysis Mass Spectrometry Analysis Will Be Performed E G Reverse Phase Chromatography Will Be Performed Using Water With 0 1 Formic Acid A And Acetonitrile With 0 1 Formic Acid B A Total Of 1 g Of Peptides Will Be Purified And Concentrated Using An On Line Enrichment Column Waters Symmetry Trap C18 100 5Um 180 Um Id X 20Mm Column Subsequent Chromatographic Separation Will Be Performed On A Reverse Phase Nanospray Column Waters Peptide Beh C18 1 7Um 75 Um Id X 150Mm Column 45 C Using A 90 Minute Gradient 5 30 Buffer B Over 85 Minutes Followed By 30 45 B Over 5 Minutes At A Flow Rate Of 350 Nanoliters Min Peptides Will Be Eluted Directly Into The Mass Spectrometer Orbitrap Velos Pro Thermo Scientific Equipped With A Nanospray Flex Ion Source Thermo Scientific And Spectra Will Be Collected Over A M Z Range Of 400 2000 Under Positive Mode Ionization Ions With Charge State 2 Or 3 Will Be Accepted For Ms Ms Using A Dynamic Exclusion Limit Of 2
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