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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: 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 1μg of
peptides will be purified and concentrated using an on-line enrichment column (Waters Symmetry Trap
C18 100Å, 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 MS/MS spectra of a given m/z value for 30 s (exclusion duration of 90 s). The instrument will be
operated in FT mode for MS detection (resolution of 60,000) and ion trap mode for MS/MS detection
with a normalized collision energy set to 35%).
• Compound lists of the resulting spectra will be generated (e.g., using Xcalibur 3.0 software (Thermo
Scientific) with a S/N threshold of 1.5 and 1 scan/group).
Data Analysis
• Data will be analyzed (e.g., tandem mass spectra will be extracted, charge state deconvoluted and
deisotoped by ProteoWizard MsConvert (version 3.0). Spectra from all samples will be searched using
Mascot (Matrix Science, London, UK; version 2.6.0) against the Uniprot reference proteome for Triticum
aestivum (UP000019116) and/or other appropriate database(s) assuming the digestion enzyme trypsin.
Mascot will be searched with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 20
PPM. Carboxymethylation of cysteine will be specified in Mascot as a fixed modification. Deamidation of
asparagine and glutamine and oxidation of methionine will be specified in Mascot as variable
modifications).
• Proteins will be identified (e.g., search results from all samples will be imported and combined using
the probabilistic protein identification algorithms implemented in the Scaffold software (version
Scaffold_4.11.0, Proteome Software Inc., Portland, OR). Peptide thresholds will be set such that a
peptide FDR of less than 0.1% will be achieved based on hits to the reverse database. Protein
identifications will be accepted if they can be established at greater than 95.0% probability and contain
at least 2 identified peptides. Protein probabilities will be assigned by the Protein Prophet algorithm.
Proteins that contain similar peptides and cannot be differentiated based on MS/MS analysis alone will
be grouped to satisfy the principles of parsimony).
• Relative quantitation will be determined (e.g., using spectral counting (SpC); a student’s t-test (at p ≤
0.05) will be applied to determine protein species that are significantly different in abundance between
groups (p-value Benjamini-Hochberg-Corrected). Pseudo values will be added (+0.5) prior to fold change
calculations to eliminate zero values).
Instrument Suitability and Quality Control
• Instrument suitability will be monitored (e.g., through analysis of commercially purchased BSA
standard digest and automated monitoring using PanormaQC. Metrics (e.g. mass accuracy, peak area,
retention time, etc.) will be monitored and flagged as outliers if results are outside +/- 3 standard
deviations of the guide set (i.e. optimal operation). Values for all metrics will be within normal limits
throughout the duration of the experiment indicating instrument stability and data robustness.
1. Read the solicitation in its entirety (including the Specifications
documents).
2. The quote should include any other documentation deemed necessary
such as references.
3. Quotes must be submitted via email by 12:00 PM Pacific Standard Time,
March 9th, 2021, by email to [email protected].
4. This is a Lowest Price Technically Acceptable requirement.
5. USDA-ARS is Tax Exempt. Tax Exemption Certificate: 720564834F.
6. Contractor must be registered at SAM.gov (System for Award
Management) at https://www.sam.gov/portal/public/SAM/ prior to quote
submission.
The Government will award a contract resulting from this solicitation to the
responsible offeror whose ffer conforming to the solicitation will be most
advantageous to the Government. All quotes must be FOB Destination.
Please respond with a firm fixed price quote to the email address below.
Offerors are encouraged to provide discounts in their pricing. The
Government will evaluate prices offered for reasonableness in accordance
with FAR 13.106-3(a) - Basis for Award.
The clause 52.214-4, Contract Terms and Conditions--Commercial Items and
52.214-5, Contract Terms and Conditions required to implement Statues or
Executive Orders--Commercial Items, apply to this acquisition. Commercial
Items Clauses are attached.
SUBMIT QUOTE TO: [email protected]