Each fraction of redissolved iTRAQ-labeled peptides was sequentially injected in triplicate and separated in a home-packed nanobore C18 column with a picofrit nanospray tip (75 μm ID × 15 cm, 5 μm particles) (New Objectives, Woburn, MA, USA) on a Tempo nano-MDLC system coupled with a QSTAR Elite Hybrid MS (Applied Biosystems/MDS-SCIEX). Each fraction was independently analyzed by the LC–MS/MS over a gradient of 90 min with the constant flow rate of 300 nl/min. Data acquisition in QSTAR Elite was set to positive ion mode using Analyst QS 2.0 software (Applied Biosystems). The precursors with a mass range of 300–1600 m/z and calculated charge from + 2 to + 5 were selected for fragmentation. For each MS spectrum, 5 most abundant peptides at most above a 5-count threshold were selected for MS/MS, and the selected precursor was dynamically excluded for 20s with a mass tolerance of 0.1 Da. Smart information-dependent acquisition was activated with automatic collision energy and automatic MS/MS accumulation. The fragment intensity multiplier was set to 20 and maximum accumulation time was 2 s.
Analyst qs 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Analyst QS 2.0 is a compact and robust liquid chromatography (LC) system designed for routine analysis in laboratories. It features a modular design and supports a range of LC techniques, including high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC). The Analyst QS 2.0 provides reliable and consistent performance for a variety of analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Qstar elite, by Thermo Fisher Scientific (3 mentions)
Qstar elite hybrid ms, by Thermo Fisher Scientific (2 mentions)
Proteinpilot software 3, by Thermo Fisher Scientific (2 mentions)
Reprosil pur c18 aq, by Dr. Maisch (1 mentions)
Zorbax 300sb c18 capillary column, by Agilent Technologies (1 mentions)
Zorbax 300sb c18 trap column, by Agilent Technologies (1 mentions)
Fused silica emitter tip, by New Objective (1 mentions)
Qstar elite mass spectrometer, by Thermo Fisher Scientific (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
Qtof ms 6545 mass spectrometer, by Agilent Technologies (1 mentions)
9 protocols using analyst qs 2
1
Tandem Mass Spectrometry Peptide Sequencing
2
Glycopeptide Analysis by LC-MS/MS
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LC–MS/MS of glycopeptides:
Analyst QS 2.0 software (Applied Biosystems, Foster City, CA) was
used to process MS data sets, and glycoproteins were identified by
Mascot search engine. Protein sequences were downloaded from UniProt
database. MS/MS spectra were converted to mzXML format using msconvert
from the ProteoWizard project. GlycoPeptideSearch (GPS) engine was
used for finding of glycopeptides forms for each glycoprotein.24 (link),25 (link) Identified glycopeptides were further confirmed by manual data inspection.
We adopt the N-glycan nomenclature from the NIBRT GlycoBase databases.26 (link) Peak area counts from extracted ion chromatogram
(XIC) of the precursor ion of each identified glycoform were used
for calculation of percentage distribution of glycopeptides and also
for calculation of ratio between fucosylated and nonfucosylated glycoforms.
All samples were analyzed in triplicate.
Analyst QS 2.0 software (Applied Biosystems, Foster City, CA) was
used to process MS data sets, and glycoproteins were identified by
Mascot search engine. Protein sequences were downloaded from UniProt
database. MS/MS spectra were converted to mzXML format using msconvert
from the ProteoWizard project. GlycoPeptideSearch (GPS) engine was
used for finding of glycopeptides forms for each glycoprotein.24 (link),25 (link) Identified glycopeptides were further confirmed by manual data inspection.
We adopt the N-glycan nomenclature from the NIBRT GlycoBase databases.26 (link) Peak area counts from extracted ion chromatogram
(XIC) of the precursor ion of each identified glycoform were used
for calculation of percentage distribution of glycopeptides and also
for calculation of ratio between fucosylated and nonfucosylated glycoforms.
All samples were analyzed in triplicate.
3
Sequential iTRAQ Peptide Analysis
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Each fraction of redissolved iTRAQ-labeled peptides was sequentially injected in triplicate and separated in a home-packed nanobore C18 column (75 μm ID × 15 cm, 2.4 μm particles, Reprosil-Pur C18-AQ, Dr. Maisch GmbH, Ammerbuch, Germany) with a picofrit nanospray tip (New Objectives, Woburn, MA, USA) on a Tempo nano-MDLC system coupled with a QSTAR Elite Hybrid MS (Applied Biosystems/MDS-SCIEX). Each fraction was independently analyzed by the LC-MS/MS over a gradient of 90 min with the constant flow rate of 300 nl/min [29 (link), 30 (link)]. Data acquisition in QSTAR Elite was set to positive ion mode using Analyst QS 2.0 software (Applied Biosystems, Foster City, CA, USA). The precursors with a mass range of 300–1600 m/z and calculated charge from +2 to +5 were selected for fragmentation. For each MS spectrum, 5 most abundant peptides at most above a 10-count threshold were selected for MS/MS, and the selected precursor was dynamically excluded for 15 s with a mass tolerance of 50 mDa. Smart information-dependent acquisition was activated with automatic collision energy and automatic MS/MS accumulation. The fragment intensity multiplier was set to 20 and maximum accumulation time was 2 s.
4
LC/MS/MS Quantification of Analytes
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LC/MS/MS experiments were carried out as previously method by Chang et al. (2013c) (link). This analysis was performed at the National Instrumentation Center for Environmental Management (NICEM) of Seoul National University in Korea using an integrated system consisting of an auto switching nano pump, autosampler (TempoTM nano LC system, MDS SCIEX, Canada), and a hybrid Quadrupole-TOF MS/MS spectrometer (QStar Elite, Applied Biosystems, USA) equipped with a nano-electrospray ionization source and fitted with a fused silica emitter tip (New Objective, USA). The precise method was as previously described by Chang et al. (2013a (link), 2013c) (link). The injection volume was 2 μL into an LC-MS/MS on a Zorbax 300 SB-C18 trap column (300 μm i.d × 5 mm, 5 μm, 100; Agilent Technologies, USA; part number 5065-9913), at a flow rate of 5 μL/min, and the sample was separated on a Zorbax 300SB-C18 capillary column (75 μm i.d × 150 mm, 3.5 μm, 100; part number 5065-9911) at a flow rate of 300 nL/min. The gradient was carried out as follows: 2% to 35% solvent B over 30 min, then from 35% to 90% over 10 min, followed by 90% solvent B for 5 min, and 5% solvent B for 15 min. Resulting peptides were electrosprayed and mass data were acquired automatically using Analyst QS 2.0 software (Applied Biosystems) with the 200-2000 range of m/z.
5
SCX Fractionation and Retinal Proteome
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For the strong cation exchange (SCX) fractionation, the mixed peptides were resuspended in buffer A containing 5 mM KH2PO4 and 20% acetonitrile, pH 2.7. The SCX was performed on a polysulfoethyl column (2.1×50 mm, 5 µm×200 Å) and used a KCl gradient from 0 to 0.5 M in 50 min to fractionate the peptides. Twelve fractions were collected and desalted using C18 ZipTips (Millipore, Billerica, MA) before the MS analysis.
A QSTAR ELITE mass spectrometer (Applied Biosystems, Foster City, CA) coupled with a nanoflow HPLC system (Tempo™, Applied Biosystems) was used for the relative quantitation of the retinal proteome before and after the I/R treatment. The LC-MS/MS method was performed as previously described [23] . Briefly, a 130-min liquid chromatography (LC) gradient was used to separate the peptide mixture with mobile phase A (2% ACN, 0.1% formic acid) and mobile phase B (98% ACN, 0.1% formic acid). The IDA (information dependent acquisition) mode was used to generate the MS/MS data. For the MS scan, the m/z range was set from 400 to 1800 with a charge state from 2 to 5, and each MS scan was followed by 5 MS/MS events. The raw MS data were generated by Analyst QS 2.0 (Applied Biosystems).
A QSTAR ELITE mass spectrometer (Applied Biosystems, Foster City, CA) coupled with a nanoflow HPLC system (Tempo™, Applied Biosystems) was used for the relative quantitation of the retinal proteome before and after the I/R treatment. The LC-MS/MS method was performed as previously described [23] . Briefly, a 130-min liquid chromatography (LC) gradient was used to separate the peptide mixture with mobile phase A (2% ACN, 0.1% formic acid) and mobile phase B (98% ACN, 0.1% formic acid). The IDA (information dependent acquisition) mode was used to generate the MS/MS data. For the MS scan, the m/z range was set from 400 to 1800 with a charge state from 2 to 5, and each MS scan was followed by 5 MS/MS events. The raw MS data were generated by Analyst QS 2.0 (Applied Biosystems).
6
Quantitative Proteome Analysis Protocol
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The Analyst QS 2.0 software (Applied Biosystems) was used for spectral data acquisition. ProteinPilot Software 3.0, Revision Number: 114,732 (Applied Biosystems) was used for the peak list generation, protein identification, and quantification against the concatenated target-decoy Uniprot human database (191,242 sequences, downloaded on 10 March 2012). The protein ratios were calculated from the peptide-level iTRAQ ratios of confidently detected unique peptides after averaging them. The false discovery rates (FDR) of peptide and protein identification were set to be less than 1% (FDR = 2.0 × decoy_hits/total_hits) [19 (link)]. Details of the analysis strategy have been described in SI Methods section.
7
Label-Free Proteomic Quantification
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The Analyst QS 2.0 software (Applied Biosystems) was used for the spectral data acquisition. ProteinPilot Software 3.0, Revision Number: 114 732 (Applied Biosystems) was used for the peak list generation, protein identification and quantification against the concatenated target-decoy Uniprot human database (191242 sequences). The false discovery rate (FDR) of peptide identification was set to be less than 1% (FDR = 2.0×decoy_hits/total_hits). Details of the analysis strategy have been described previously [23] .
8
Mass Spectrometry Analysis with QTOF/MS-6545
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Mass spectrometry was achieved on a QTOF/MS-6545 mass spectrometer (Aglient, CA, USA) with an electrospray ionization (ESI) interface. The scan range was from m/z 100 to 1000. Nitrogen (N2) and helium (He) were respectively used as the sheath and auxiliary gas and collision gas and. The acquisition parameters were as follows: ion spray voltage floating,+2500/− 1500 V; nebulizer gas pressure, 40 V; Fragmetor potential, 135 V; gas temperature, 325 ℃; sheath temperature, 325 ℃; gas flow, 8.0 L/min; Analyst QS 2.0 (Applied Biosystems/MDS Sciex) was applied for data acquisition and processing.
9
Mass Spectrometry Protein Analysis
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Mass-spectrometry measurements were performed on a hybrid quadrupole-Time-of-Flight (Q-TOF) instrument (QSTAR ELITE, Applied Biosystems, Foster City, CA, United States), equipped with a nano-ESI sample source. Metal-coated borosilicate capillaries (Proxeon, Odense, DK), with medium-length emitter tip of 1-mm internal diameter, were used to infuse the sample. The instrument was calibrated using the renine-inhibitor (1757.9 Da) (Applied Biosystems, Foster City, CA, United States) and its fragment (109.07 Da) as standards. Spectra were acquired in the 1500–3000 m/z range, with accumulation time of 1 s, ion-spray voltage of 1200–1500 V, declustering potential of 80 V, and instrument interface of 50°C. Spectra were averaged over a time period of at least 3 min. Data analysis was performed by the program Analyst QS 2.0 (Applied Biosystems, Foster City, CA, United States). The samples were prepared in 5 mM ammonium acetate pH 6.5.
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