Dried peptides were reconstituted in 15 μL of 0.1% formic acid in water. An Agilent 1100 series autosampler/HPLC was used to draw 0.5 μL of sample and inject it onto a C18 reverse phase column (GRACE; 150 × 0.500 mm) where an acetonitrile concentration gradient (5–30% in water with 0.1% formic acid) was used to elute peptides for online ESI-MS/MS by a QStar XL mass spectrometer (Applied Biosystems). Pertinent instrument scan settings were as follows: 4800 scans were taken during 140 min of elution for ions between 300 to 1800 m/z with charge states of 2 to 5 exceeding 10 counts. Former target ions were excluded for 300 s. Column cleaning was performed automatically with 2 cycles of a 5–85% acetonitrile gradient lasting 15 min each between runs.
Qstar xl mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QSTAR XL mass spectrometer is a high-performance hybrid quadrupole time-of-flight (QTOF) mass spectrometer designed for advanced analytical applications. It combines the high sensitivity and resolution of a quadrupole with the accurate mass measurement capabilities of a time-of-flight analyzer.
Automatically generated - may contain errors
Lab products found in correlation
1100 series autosampler hplc, by Agilent Technologies (2 mentions)
1100 hplc system, by Thermo Fisher Scientific (1 mentions)
Atr tensor 37 spectrophotometer, by Bruker (1 mentions)
Dip 360, by Jasco (1 mentions)
Itraq kit, by Thermo Fisher Scientific (1 mentions)
Np 40, by Nacalai Tesque (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Simplyblue, by Thermo Fisher Scientific (1 mentions)
Ultracel 10 membrane, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Pronase, by Roche (1 mentions)
Ncs 3500rs nano lc system, by Thermo Fisher Scientific (1 mentions)
Micro bio spin 6 column, by Bio-Rad (1 mentions)
Agilent 1100 series system, by Agilent Technologies (1 mentions)
Analyst software, by Thermo Fisher Scientific (1 mentions)
Hp 5 column, by Agilent Technologies (1 mentions)
Hp 6890 gc system, by Agilent Technologies (1 mentions)
5975c mass spectrometer, by Agilent Technologies (1 mentions)
Ultraflex 3 tof tof, by Bruker (1 mentions)
16 protocols using qstar xl mass spectrometer
1
Peptide Identification by LC-MS/MS
2
Quantitative Proteome Analysis of HGTs vs. PBTs
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The differential proteome between HGTs and PBTs were identified by LC-MS/MS, which was described in detail in our papers [20 (link)–24 (link)]. Generally, 30 μg proteins from Leu-d3-labeling cells were respectively mixed with 30 μg HGT or PBT proteins to separate on 12% SDS-PAGE. Gels were excised to in-gel digest and extract peptides, peptides were identified by LC-nanospray-tandem MS (MS/MS) on a QSTAR XL mass spectrometer (Applied Biosystems, USA). The parameters for database searching were mainly followed as our previous approaches [22 (link), 23 (link), 25 (link)]. The relative tissue protein expression level (SILAC ratio) was quantified by tracking pairs of labeling and unlabeling peptides from MS spectra. The differential expression protein was defined with its change ratio above 2 or below 0.5 times as a significantly up-regulated or down-regulated one between HGTs and PBTs, which was performed following bioinformatics analysis [23 (link), 24 (link)].
3
Quantitative Proteomics of LAMP2A Knockdown
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SN4741 cells transfected with siRNA targeting Lamp2a (siLamp2a-215) or non-specific control (NC) siRNAs for 48 h were harvested and lysed in RIPA lysis buffer. The samples were centrifuged at 13,000 g for 15 min and the supernatants were collected to determine concentration of protein via BCA assay. iTRAQ labels 113 and 114 were used to label the NC group and siLamp2a group, respectively. Mass spectrometric analysis was performed using a QStarXL mass spectrometer (Applied Biosystems, CA, USA) as previously described [62 (link)]. Protein identification was accomplished by searching the spectrums against Uniprot database and ratio of 114:113 was calculated to analyze the relative expression levels of proteins upon LAMP2A knockdown.
4
Quantitative Analysis of Ethanolamine Phospholipids in Serum
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Example 3
The present invention provides a chromatographic method combined with a mass spectrometric detector for the quantitative and qualitative characterization of ethanolamine phospholipids in serum.
For MS/MS applications and experiments involving chromatography, an Agilent 1100 HPLC system was used in combination with an Applied Biosystems QSTAR XL mass spectrometer. An Agilent Zorbax RX-SIL (4.6×150 mm, 5 μm) column was used for normal phase chromatography. Conditions included an isocratic mobile phase (55:40:5 isopropanol:hexane:H2O) at a flow rate of 1.0 mL/min for a total run time of 15 min. The column was heated to 35° C. The sample injection volume was 10 μL. Organic solvent extracts (ethyl acetate) of samples were evaporated to dryness under nitrogen gas and the residue was reconstituted in 100 μL of 55:40:5 isopropanol:hexane:H2O solution prior to injection.
The QSTAR XL instrument was equipped with an APCI (Heated Nebulizer) source operating in negative mode. Values of major instrument parameters were DP, −60; FP, −265; DP2, −15; GS1, 75; GS2, 15; CUR, 30; NC, −3; TEM, 400° C.; Scan range, 50-1500 amu; Accumulation time, 1 sec.
5
Characterization of Organic Compounds
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The measurement of optical rotations was performed on a polarimeter (JASCO DIP 360). The IR spectra were obtained through ATR-Tensor 37 spectrophotometer by Bruker. To record the ESI mass spectra the QSTAR XL mass spectrometer by Applied Biosystems was used with a capillary voltage set from 5–5.5 kV. The NMR spectra (1H and 13C) were recorded on Bruker spectrometer operating at 600 MHz (150 MHz for 13C). The δ values on the chemical shift scale are reported in ppm, while the J values of the coupling constants are given in Hz. Purification of minor compounds was performed by recycling HPLC by using a 7:3 EtOAc/n-hexane solvent system in a silica gel column with a flow rate of 4 mL/min. Pre-coated aluminium silica gel sheets (60 F-254, Merck) were used for TLC (thin layer chromatography), which were visualized under UV light at 254 and 366 nm. Ceric sulphate and ninhydrin reagent were used for spraying the TLC followed by heating.
6
Proteomic Analysis Workflow
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The system in the MARS.14 chromatographic column was obtained from Aglient Co.; the iTRAQ kit, QSTAR XL mass spectrometer, and Protein Pilot4.2 software were obtained from Applied Biosystems (USA); the liquid chromatograph (20AD) was obtained from Shimadzu Co. (Japan); Strong Cation Exchange Chromatography (2.1×100 mm, 5 um, 300 A) was obtained for the Nest Group Inc. System (USA); and the ZORBAX 300SB-C18 column (5 um, 300A, 0.1×150 mm) was obtained from Microm Co. (USA).
7
Protein-Protein Association Analysis
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To investigate protein–protein associations, 293T cells transfected with U14 expressing plasmid were lysed in TNE buffer (10 mM Tris-HCl [pH 7.8], 0.15 M NaCl, 1 mM EDTA, and 1% NP-40 [Nacalai Tesque]). For immunoprecipitations, antibodies were bound to protein G–Sepharose (GE Healthcare) and then cross-linked with protein G using dimethyl pimelimidate (DMP; Thermo Scientific) [39 (link)]. Whole-cell extracts were then incubated with the appropriate protein G–Sepharose-bound antibody. Bound proteins were eluted with 0.1 M glycin (PH 2.5) and neutralized by using 1 M Tris-HCl (PH 9.0). The eluates were further prepared for LC/MS analysis as described elsewhere [40 (link),41 (link)]. LC/MS analysis was performed using a Qstar-XL mass spectrometer (Applied Biosystems).
8
Nano-LC-MS/MS Proteomic Analysis
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The mass spectrometric analysis was performed using a nano-LC column coupled online to a QStarXL mass spectrometer (Applied Biosystems). Peptides were loaded onto a 75 cm × 10 cm, 3-mm fused silica C18 capillary column, and mobile phase elution was performed using buffer A (0.1% formic acid in 2% acetonitrile/98% Milli-Q water) and buffer B (0.1% formic acid in 98% acetonitrile/2% Milli-Q water). The peptides were eluted using a gradient from 2% buffer B to 100% buffer B over 90 min at a flow rate of 300 nl/min. The LC eluent was directed to an ESI source for Q-TOF-MS analysis. The mass spectrometer was set to perform information-dependent acquisition (IDA) in the positive ion mode for a selected mass range of 300–2,000 m/z. Peptides with +2 to +4 charge states were selected for tandem mass spectrometry, and the time of summation of MS/MS events was set to 3 s. The two most abundantly charged peptides above a 10-count threshold were selected for MS/MS and were dynamically excluded for 60 s with a ±50-mmu mass tolerance.
9
Identifying PKCδ Phosphorylation Sites in LCN2
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To identify the PKCδ phosphorylation sites, LCN2 protein was phosphorylated in vitro by PKCδ using 0.5 mM non-radiolabeled ATP for 4 h as described above. The reaction mixtures were fractionated by SDS-PAGE and stained with SimplyBlue (Invitrogen). Gel slices containing the phosphorylated LCN2 were treated with trypsin, and the resulting peptide mixtures were analyzed by nano-liquid chromatography-mass spectrometry/mass spectrometry (nano-LC-MS/MS) serviced at the Protein Chemistry Center, UT Southwestern Medical Center. Samples from the digests were analyzed by nano-LC-MS/MS using a LC-Packings HPLC (Dionex) coupled to a QStar XL mass spectrometer (Applied Biosystems). Data were searched against a home-built database that includes the LCN2 sequence. Four modifications were included in the database search: carbamidomethyl (C), oxidation (M), phospho (ST), and phospho (Y).
10
Nano-LC QTOF Mass Spectrometry
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Mass spectrometric analysis was performed using a nano-LC coupled online to QStarXL mass spectrometer (Applied Biosystems). The mass spectrometer was set to perform information-dependent acquisition (IDA) in the positive ion mode at a mass range of 300-1800 m/z. Peptides with +2 to +4charge states were selected for tandem mass spectrometry, and the time of summation of MS/MS events was set to 3s. The two most abundantly charged peptides above a 10 count threshold were selected for MS/MS and dynamically excluded for 60s with ±50 mDa mass tolerance.
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