Peptides were sequenced on a Synapt G2 HDMS (Waters, Milfords, MS, EUA) mass spectrometer coupled to a UPLC NanoAcquity system with 1D technology (Waters, Milfords, MS, EUA) and captured by a C18 Symmetry column (5 μm, 180 μm × 20 mm) (Waters, Milfords, MS, EUA). The peptides were separated by using a 2–90% acetonitrile gradient in 0.1% formic acid and an HSS T3 analytical column (1.8 μm, 75 μm × 100 mm) (Waters, Milfords, MS, EUA) with a flow of 300 μL min− 1 for 120 min. The data were acquired on a Waters Synapt G2S Q-TOF mass spectrometer equipped with a NanoLockSpray (Waters, Milfords, MS, EUA). The experiments were performed in the HDMSE mode (data-independent analysis). The mass spectra were processed with the ProteinLynxGlobalServer (PLGS) software version 3.1. The proteins were identified by comparison to the Aspergillus UNIPROT database (207,966 proteins) [42 ]. The defined parameters were automatic tolerance for precursors and ion products, minimum of three corresponding ion fragments per peptide, minimum of seven corresponding ion fragments per protein, trypsin missed cleavage, carbamidomethylation as a fixed modification, oxidation of methionine as a variable modification, and 4% FDR peptide.
Hss t3 analytical column
Manufactured by Waters Corporation
Sourced in United States
The HSS T3 analytical column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a porous silica-based stationary phase with a C18 functionality, providing efficient and reproducible chromatographic separations.
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Lab products found in correlation
Symmetry c18 column, by Waters Corporation (1 mentions)
Nanolockspray, by Waters Corporation (1 mentions)
Synapt g2s qtof mass spectrometer, by Waters Corporation (1 mentions)
Synapt g2 hdms, by Waters Corporation (1 mentions)
Nanoacquity ultraperformance lc system, by Waters Corporation (1 mentions)
Symmetry c18 trap column, by Waters Corporation (1 mentions)
Symmetry c18 20 mm 180 μm trapping column, by Waters Corporation (1 mentions)
Fusion lumos, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap elite mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Proteome discoverer, by Thermo Fisher Scientific (1 mentions)
Nanoacquity lc system, by Waters Corporation (1 mentions)
Trypsin, by Promega (1 mentions)
5 protocols using hss t3 analytical column
1
Aspergillus Proteome Profiling by MS
2
Optimized Proteomic Identification Workflow
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Protein identification was conducted in the same way as described in [8 ]. Measurements were performed using a nanoAcquity UltraPerformance LC system connected to an auto-sampler equipped with a HSS T3 analytical column (1.8-µm particle, 75 µm × 150 mm) kept at 45 °C, and a Symmetry C18 trap column (5-µm particle, 180 µm × 20 mm), all Waters, USA. This setup was connected to an LTQ Orbitrap Elite. A 180-min gradient was used: (0–5 min: 99% buffer A and 1% buffer B, 5–10 min 99–94% A, 10–161 min: 94–60% A, 161–161.5 min: 60–14% A, 161.5–166.5 min: 14–4% A, 166.5–167.1 min: 99% A, 167.1–180 min: 99% A). To optimize the method for small cell numbers, the MS/MS max ion inject time was increased to 400 ms.
3
Quantification of Phenolic Compounds by UPLC
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The identification and quantification of phenolic compounds was performed on a Waters ultra-high-pressure liquid chromatography Acquity system (UPLC, Acquity H-Class) (Milford, MA, USA) equipped with a 2996 PDA detector. Separation was achieved with an ACQUITY HSS T3 analytical column (100 mm × 2.1 mm, 1.8 μm particle size) protected with an Acquity UPLCTM HSS T3 Van GuardTM Pre-column (Waters, Milford, MA, USA). The column oven temperature was kept at 40 °C. A binary mobile phase combining 0.1% aqueous formic acid solution (solvent A) and acetonitrile (solvent B) was used with a gradient program, as follows: 80% A (0 min), 60% A (3 min), 55% A (6 min), 30% A (8–10 min), and 80% A (12–14 min), followed by a re-equilibration time of 2 min prior to the next injection. The flow rate was 250 μL/min and the injection volume of both standards and samples was 2 μL. The UV detection wavelength was set to the maximum of absorbance for the compounds of interest and the Empower 2 software (Milford, MA, USA) was used for chromatographic data gathering and integration of chromatograms. The identification of polyphenols was based on the retention time and UV spectrum.
4
Optimized LC-MS/MS analysis of enriched peptides
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PDQ-enriched peptides were reconstituted in 15 μL of 97/2/1 (v/v/v/) H2O/MeCN/TFA, and 4.5 μL was analyzed in duplicate by LC-MS/MS. Unbound fractions were reconstituted in the same buffer at ~1 μg/μL, and 1.5–2.5 μL was analyzed. LC-MS/MS used a Waters M-Class interfaced to a Thermo Fusion Lumos via a NanoSpray Flex source and CoAnn emitter 20 μm ID, 10 μm Tip ID, 6.35 cm length)). Peptides were trapped on a Symmetry C18 180 μm × 20 mm trapping column (Waters) at 5 μL/min with 99.9/0.1 v/v H2O/MeCN and separated on a 75 μm × 25 cm HSS-T3 analytical column (Waters) at 400 nl/min and 55 °C and a gradient of 5–30% MeCN over 90 min. MS acquisition used a 3 s vendor-supplied synchronous precursor selection (TMTPro-SPS-MS3) template method. Briefly, Precursor scans used 120,000 resolution, standard AGC target and auto maximum injection time (IT). Precursors (scan range 400–1600 m/z, charge state 2–6, intensity threshold 5E3) were selected for collision-induced dissociation using 0.7 m/z, “Turbo” ion trap scan with 25 ms max IT. Finally 10 SPS precursors were subjected to higher-energy collision-induced dissociation with 0.7 m/z isolation, normalized collision energy of 55%, 50,000 resolution, 200% AGC and 200 ms max IT. Analysis of unbound fractions also used a FAIMS-Pro interface with a 3 CV method (−40 CV for 1.2 s, −55 CV for 1.2 s, −70 CV for 0.8 s).
5
Proteomic Analysis of Sertoli, Testis, and Sperm
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For GeLCMS, proteins of Sertoli cells, testis, and sperm were separated on SDS gels and stained with Coomassie. Per lane, 14–17 gel slices were excised, proteins were in-gel digested with trypsin (Promega), peptides were separated in a 90 or 180 min gradient by a nanoAcquity LC System equipped with a HSS T3 analytical column (1.8 μm particle, 75 μm x 150 mm) (Waters), and analyzed by ESI-LC-MS/MS using an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific). All database searches were performed using SEQUEST as well as MS Amanda (Mechtler lab, Vienna, Austria) algorithm, embedded in Proteome Discoverer (Rev. 1.4, Thermo Electron 2008–2011), with a NCBI protein database (mouse, accession number NP_766280.2, accessed June 13, 2013). Only fully tryptic peptides with up to two missed cleavages were accepted. Oxidation of methionine was permitted as variable modification. The mass tolerance for precursor ions was set to 10 ppm; the mass tolerance for fragment ions was set to 0.4 amu. To filter the results, a peptide FDR threshold of 0.01 (q-value) according to Percolator was set in Proteome Discoverer; two peptides per protein and peptides with search result rank 1 were required.
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