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TripleTOF 5600

Manufactured by AB Sciex
Sourced in United States, Canada, Singapore, Germany, Japan, United Kingdom, Spain

The TripleTOF 5600 is a high-resolution mass spectrometer designed for advanced analytical applications. It combines a triple quadrupole front-end with a time-of-flight (TOF) mass analyzer, providing high sensitivity and accurate mass measurements. The instrument is capable of performing various modes of operation, including full-scan acquisition, targeted data-dependent acquisition, and independent data acquisition.

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455 protocols using TripleTOF 5600

Each sample was resuspended in 20 μL buffer A and analyzed both on an Orbitrap Elite (Thermo Scientific) online coupled to an Acquity nano UPLC (Waters) and a TripleTOF 5600 (AB SCIEX) coupled to an Eksigent ekspert nanoLC 400 cHiPLC system. Orbitrap Elite: Peptides were separated on a nano Acquity UPLC system (Waters) supplemented with a 25 cm BEH130 C18 column, 1.7‐mm particle size using a linear gradient from 8 to 35% buffer B in buffer A at a flow rate of 250 nL/min for 60 min. Peptides were introduced to an Orbitrap Elite mass spectrometer using a nanoESI source. Subsequent isolation and collision‐induced dissociation was induced on the 20 most abundant ions per full MS scan using an isolation width of 1.5 amu. All fragmented precursor ions were actively excluded from repeated selection for 15 s. TripleTOF 5600: Peptides were separated on an ekspert nanoLC 400 cHiPLC system (Eksigent) supplemented with a 15 cm x 75 μm ChromXP C18‐CL, 3 μm particle size using a linear gradient from 8% buffer A to 35% buffer B at a flow rate of 300 nL/min for 60 min. Peptides were introduced to TripleTOF 5600 mass spectrometer and collision‐induced dissociation fragmentation using ramped collision energy was induced on the 30 most abundant ions per full MS scan using unit isolation width 0.7 amu. All fragmented precursor ions were actively excluded from repeated selection for 15 s.
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Triple TOF 5600 (AB SCIEX, Framingham, MA, United States) + liquid chromatography-mass spectrometry (AB SCIEX, Framingham, MA, United States) was used for mass spectrometry (MS) data acquisition, and the samples were analyzed using a Triple TOF 5600 plus mass spectrometer coupled with an Eksigent nanoLC system (AB SCIEX, Framingham, MA, United States). The polypeptide sample was dissolved in 2% acetonitrile and 1% formic acid and eluted with a time gradient of 90 min and a flow rate of 300 nl/min. The two mobile phases were buffer A (2% acetonitrile, 0.1% formic acid, and 98% H2O) and buffer B (98% acetonitrile, 0.1% formic acid, and 2% H2O), and finally freeze-dried. The mass-charge ratio ranges of primary and secondary scanning were 350–1,500 and 100–1,500 m/z, respectively. Each process was performed in triplicate.
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Ten microliter extracted sample was injected into LC‐ESI‐MS/MS mass spectrometer (TripleTOF™ 5600+, AB SCIEX, USA) under negative ESI MRM to obtain the relative area ratio of the ALA and the internal standard and calculate the amount of ALA in the sample. Liquid chromatography was performed by HPLC (SHIMADZU, LC20A) with Poroshell 120 LC column (Bonus‐BP 3.0 × 100 mm, 2.7 μm; Agilent). Mobile phase A, methanol: 0.1% glacial acetic acid (add ammonia to pH 4.5) = 55: 35. Mobile phase B, 100% acetonitrile. Mobile phase A: mobile phase B = 90: 10. Flow rate, 0.25 ml/min with 5 mM ammonium formate for and 100% methanol for mobile phase B. Mass spectrometry was performed by a LC‐ESI‐MS/MS mass spectrometer (TripleTOF™ 5600+, AB SCIEX, USA) under negative ESI MRM using parameters for ALA (m/z 205.0‐>171.0). Mass spectrometer were as follows: ion source gas1 55 psi; ion source gas2 55 psi; curtain gas 35 psi; temperature 500°C; collision energy 40 V; collision energy spread 20 V and declustering potential 80 V, ion spray voltage floating 5,500 V.
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All the results are expressed as mean ± S.D of three replicates. The EC50 values were calculated using the non-linear regression for determining DPPH and ABTS assays. The HR-MS peak integration was performed using the AB Sciex Triple TOF 5600, Peak View 2.1 Software (AB Sciex Triple TOF 5600, Singapore), and the NMR spectra were processed using the Brüker TOPSPIN 2.1 software.
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5

Shotgun Proteomics by Information-Dependent Acquisition

Samples were analysed on an AB Sciex 5600 TripleTOF (ABSciex) in information-dependent acquisition (IDA) mode. Peptides were fractionated by liquid chromatography (nanoLC Ultra 2D, Eksigent) on a MicroLC column ChromXPTM C18CL reverse-phase column (300 µm ID×15 cm length, 3 µm particles, 120 Å pore size, Eksigent) at 5 µl min−1 and eluted into the mass spectrometer with an acetonitrile gradient in 0.1 % FA (2 –30 % ACN, in a linear gradient for 30 min), using an electrospray ionization source (DuoSprayTM Source, ABSciex) with a 50 µm internal diameter (ID) stainless steel emitter (New Objective). For information-dependent acquisition (IDA) experiments, the mass spectrometer was set to scanning full spectra (350–1250 m/z) for 250 ms, followed by up to 80 MS/MS scans (100–1500 m/z from a dynamic accumulation time – minimum 30 ms for precursor above the intensity threshold of 1000 – in order to maintain a cycle time of 2.7 s). Candidate ions with a charge state between +2 and +5 and counts above a minimum threshold of 10 counts s–1 were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 15 s (mass spectrometer operated by Analyst TF 1.6, ABSciex). Rolling collision was used with a collision energy spread of 5.
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Samples were analyzed using an ESI-LC-MS/MS system in a nano-spray configuration (ABSciex 5600 TripleTOF®, AB SCIEX, Framingham, MA, USA) coupled with an ultra-nano-HPLC with a cHiPLC system (Eksigent, Dublin, CA, USA). Samples were loaded onto a 0.5 mm C18 CL 3 μm 120 Å trap column (Eksigent, Dublin, CA, USA), washed with 98:2 HPLC water with 0.1 % formic acid: ACN with 0.1 % formic acid for 10 min and then eluted through a 15 cm C18 CK 3 μm 120 Å ChromXP column (Eksigent, Dublin, CA, USA) with 98:2 HPLC water with 0.1 % formic acid: ACN with 0.1 % formic acid using a 60-minute linear gradient of 0–60 % ACN with 0.1 % formic acid. The instrument was calibrated every 5–6 samples using 25 fmol of β-galactosidase standards provided by the manufacturer. The acquisition method was in the data dependent mode with one full scan followed by fragmentation of the 50 most abundant peaks. Precursor peaks with a minimum signal count of 100 were dynamically excluded after two selections for 6 s within a range ± 25 mDa. Charge states other than 2–5 were rejected. Rolling collision energy was used.
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Thirty pairs of tear samples selected randomly for proteomics analysis. Tear proteins from finely cut Schirmer’s strip were extracted by ammonium bicarbonate (50 mM, 100 μl per strip; Sigma-Aldrich, St. Louis, MO, USA) at 1300 rpm, room temperature (23 °C) for 1 h, and the protein concentration was measured. Protein samples (100 μg) were applied for SWATH-MS and a spectral library was established as previously described [17] (link), [18] (link). The digested samples were reconstituted in loading buffer (0.1% formic acid, 2% acetonitrile in water), and iRT standard (Biognosys, Switzerland) was spiked. Sample analysis was performed using an Ultimate 3000 nanoLC system (Dionex, Thermo Fisher Scientific, MA, USA) coupled with AB Sciex 5600 triple TOF (AB Sciex, Framingham, MA, USA) as previously reported [18] (link), [19] (link).
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MS and MS/MS data for five independent experiments (biological replicates) (*.wiff, 1 per fraction, 12 files per experiment) were submitted to protein identification using the Paragon algorithm as implemented in the ProteinPilot software (version 5.0, AB SCIEX) and searched against two complementary Sus scrofa databases: a Swiss-Prot database (2015_10 release, 1422 entries) and a TrEMBL database (2015_10 release, 47465 entries). Quantitation was conducted with or without auto bias correction, an available option implemented in the ProteinPilot software to normalize uneven protein across the multiplex samples, improving further quantitation. Mass tolerances and identification parameters were automatically set and optimized for the ABSciex 5600+ TripleTOF™-generated MS/MS data (MS/MS Fragment mass tolerance was set at 0.1 Da. Precursor mass tolerance was set at 0.05 Da).
The *.group results files (1 per experiment) were exported as Peptide Summaries.
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It was kindly performed by specialist personel (Helinho) in the AB-Sciex Laboratory located in Sao Paulo, SP, Brazil. Plasma samples from 59 breast cancer patients were compared to control group composed of 93 healthy menopaused volunteers (Supplementary Note). Samples were injected onto a Shimadzu Prominence LC system coupled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument with an acquisition scan rate of 100 spectra/sec and stable mass accuracy of ~2 ppm.
Flow Injection Analysis (FIA) was performed using isocratic elution with Methanol/Water (90/10) with 5.0 mM of ammonium formate. Flow rate and injection volumes were 0.025 mL/min and 50 μL respectively.
No ion source or declustering potential (50 V and −40 V) optimization was performed. The following ionization parameters were applied: CUR = 20 psi, GS1 = 20 psi, GS2 = 15 psi, Temp = 250oC, IS = 5000 V (–4000V). MS scan ranging from m/z 100 to 1200 with accumulation time of 0.25 s and product ion scan from m/z 100 to 1200 and accumulation time of 0.03 s were the adopted parameters during survey and dependent scans respectively.
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Trypsin digestion and LC-MS/MS analysis were carried out at the SCSIE_University of Valencia Proteomics Unit, a member of ISCIII ProteoRed Proteomics Platform, following a standardized protocol, described previously [28 ].
Briefly, gel slices were reduced with DTT, alkylated with iodoacetamide and digested with 20 ng/μl of Trypsin (Promega, Madison, USA) overnight at 37 °C. Digestion was stopped with 10% trifluoroacetic acid (TFA) to a final concentration of 0.1%, and the supernatants were filtered through a 0.22 μm filter and dried by centrifugation in a vacuum. Pellets were re-suspended in 6 μl of 5% acetonitrile, 0.1% TFA, and 5 μl of every sample was loaded onto a trap column (Nano LC Column, 3 μ C18-CL, 350 μm × 0.5 mm, Eksigent) and desalted with 0.1% TFA at a flow rate of 3 μl/min for 5 min. The peptides were then loaded onto an analytical column (LC Column, 3 μ C18-CL, 75 μm × 25 cm, Eksigent) and equilibrated in 5% acetonitrile (ACN) and 0.1% formic acid (FA). Elution was carried out with a linear gradient of 5–40% B (B: ACN, 0.1% FA) in A (A: 0.1% FA) for 30 min at a flow rate of 300 nl/min. Eluted peptides were analysed in a nanoESI qQTOF mass spectrometer (5600TripleTOF, ABSCIEX, Ontario, Canada) in IDA mode performing 0.25-s TOF MS scans from 350 to 1250 m/z, followed by 0.05-s product ion scans from 100 to 1500 m/z on the 50 most intense 2–5 charged ions.
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