EVs (10 μg of total protein, quantified by BCA protein assay kit; Thermo Fisher) were obtained from MC38 cells, as described above. The sample was processed as previously described29 (link). Peptides were desalted using StageTips and dried in a vacuum concentrator. For LC-MS/MS analysis, the peptides were separated by reverse phase chromatography on a Dionex Ultimate 3000 RSLC nano UPLC system connected in-line with an Orbitrap Elite (Thermo Fisher Scientific). Database search was performed using Mascot 2.5 (Matrix Science) and SEQUEST in Proteome Discoverer v.1.4. against a murine Uniprot protein database. Data were further processed and inspected in ScaffoldTM 4.8.4 (Proteome Software). Two independent analyses were performed on one EV preparation.
Dionex ultimate 3000 rslcnano
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom
The Dionex Ultimate 3000 RSLCnano is a high-performance liquid chromatography (HPLC) system designed for nano-scale separations. It features a compact design and is capable of handling flow rates in the nanoliter-per-minute range.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive, by Thermo Fisher Scientific (4 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (4 mentions)
Orbitrap fusion, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Proteome discoverer 2, by Thermo Fisher Scientific (4 mentions)
Mascot 2, by Matrix Science (3 mentions)
Scaffoldtm 4, by Proteome Software (3 mentions)
Orbitrap elite, by Thermo Fisher Scientific (3 mentions)
Q exactive plus hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Xcalibur software, by Thermo Fisher Scientific (3 mentions)
Proteome discoverer v1, by Matrix Science (2 mentions)
15 cm c18 zorbax column, by Agilent Technologies (2 mentions)
Orbitrap fusion lumos tribrid mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Orbitrap exploris 480 mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Irt synthetic peptide mixture, by Biognosys (2 mentions)
Skyline daily, by Thermo Fisher Scientific (2 mentions)
Pierce retention time calibration mixture, by Thermo Fisher Scientific (2 mentions)
Acclaim pepmap rslc column, by Thermo Fisher Scientific (2 mentions)
Fused silica column, by New Objective (2 mentions)
Fusion mass spectrometer, by Thermo Fisher Scientific (2 mentions)
48 protocols using dionex ultimate 3000 rslcnano
1
Proteomic Analysis of Extracellular Vesicles
2
Doxorubicin Quantification in Serum
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To measure the DOX concentration in the serum, blank serum samples (190 μL) were spiked with DOX standard solution (10 μL) to make the standard curve in serum, which ranged from 5 ng/mL to 600 ng/mL.
Daunorubicin (Meilune Biological Technology Co., Ltd., Dalian, China) in methyl alcohol at a concentration of 2 μg/mL and dose of 10 μL, as the internal standard solution, were added to all of the standards and samples. The precipitation of protein was done according to the established protocols for LC–MS/MS (Hronek & Reed, 2015 ; Tao et al., 2015 (link)).
High-performance liquid chromatography (HPLC) was carried out using a Dionex UltiMate 3000 RSLC Nano (Thermo Fisher Scientific Inc., Waltham, MA). A XBridge C18 column (2.5 μm, 2.1 × 50 mm column; Waters Corporation, Dublin, Ireland) was used, with the flow rate 0.4 mL/min. The mobile phase consisted of A (acetonitrile [AcCN]) and B (0.1% formic acid, 99.9% water) at a ratio of 79:21. The injection volume of the standards or samples was 20 μL.
Mass spectrometry was carried out on an API 4000 QTRAP LC-MS/MS System (Applied Biosystems/MDS Sciex, Concord, Ontario, Canada). The lower limit of quantity (LOQ) of LC–MS/MS for DOX was 5 ng/mL.
Daunorubicin (Meilune Biological Technology Co., Ltd., Dalian, China) in methyl alcohol at a concentration of 2 μg/mL and dose of 10 μL, as the internal standard solution, were added to all of the standards and samples. The precipitation of protein was done according to the established protocols for LC–MS/MS (Hronek & Reed, 2015 ; Tao et al., 2015 (link)).
High-performance liquid chromatography (HPLC) was carried out using a Dionex UltiMate 3000 RSLC Nano (Thermo Fisher Scientific Inc., Waltham, MA). A XBridge C18 column (2.5 μm, 2.1 × 50 mm column; Waters Corporation, Dublin, Ireland) was used, with the flow rate 0.4 mL/min. The mobile phase consisted of A (acetonitrile [AcCN]) and B (0.1% formic acid, 99.9% water) at a ratio of 79:21. The injection volume of the standards or samples was 20 μL.
Mass spectrometry was carried out on an API 4000 QTRAP LC-MS/MS System (Applied Biosystems/MDS Sciex, Concord, Ontario, Canada). The lower limit of quantity (LOQ) of LC–MS/MS for DOX was 5 ng/mL.
3
Serum-free Conditioned Media Proteomic Analysis
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To produce serum-free conditioned media, cells were grown to 70% confluency on three 100-mm cell culture dishes per sample. The cells were then washed twice with 1× PBS and then cultured in 8 ml of serum-free RPMI 1640 for 48 hours. The collected conditioned media were centrifuged at 300g at 4°C for 15 min to remove dead cells from the media. To purify proteins secreted by the cells, 15 ml of conditioned media was transferred into an Amicon Ultra-15 Centrifugal Filter Unit with a filter size of 3 kDa and centrifuged at 4000g at 4°C for 60 min in a swinging bucket rotor. Fifteen milliliters of RPMI 1640 without serum was also concentrated using the same method. LC-MS/MS data acquisition was performed, as previously described (59 (link)), using a Dionex Ultimate 3000 RSLCNano and monitored on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Analysis of MS data was performed, as previously described. Peptides were identified by matching the spectra with Proteome Discoverer 2.0 (Thermo Fisher Scientific) against the human Uniprot database (90,300 target sequences).
4
LC-MS Analysis of Erythrocyte Frataxin
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The LC-MS analysis of peptides was performed as previously described22 (link). Briefly, MS was conducted using a Q Exactive™ HF coupled to Dionex Ultimate 3000 RSLCnano with capillary flowmeter chromatographic systems (Thermo Fisher Scientific, San Jose, CA, USA). The analytical column was a C18 AQ capillary column with a 10 µm pulled tip (75 µm × 25 cm, 3 µm particle size; Columntip, New Haven, CT). A data dependent scan was used for initial identification of the erythrocyte frataxin N-terminal peptide (Acetyl-MNLRKSGTLGHPGSL). For peptide identification and quantification, the scheduled PRM was used instead.
5
Proteome Analysis of Multiple Myeloma Samples
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Proteome analysis was performed for 35 MM patient samples as described previously (Tierney et al., 2021 (link)). Briefly, CD138 + cells were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, United States). The whole cell lysates were then digested and loaded onto a Q Exactive (Thermo Fisher Scientific, Hemel Hempstead, United Kingdom) high-resolution accurate mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) chromatography system (Thermo Fisher Scientific, Hemel Hempstead, United Kingdom). Protein identification and quantification was performed using MaxQuant v1.5.2.83 . Perseus v.1.5.6.04 was used for data analysis, processing, and visualization. Normalized label-free quantification intensity values were used as the quantitative measurement of protein abundance for subsequent analysis. The dataset is available on the OSF platform (see text footnote 1).
6
Quantitative Proteomic Analysis Pipeline
7
Orbitrap-Based Proteomic Workflow
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The analysis was conducted on a Dionex Ultimate 3000 RSLCnano coupled to a LTQ Orbitrap XL (ThermoFisher Scientific). A total of 10 μl of each sample was injected onto the column. Mobile phase A was 100% H2O, 0.1% formic acid, and mobile phase B was 80% ACN, 0.08% formic acid. The flow rate was set at 300 nl/min. The column oven was set at 40°C. A gradient of 5–45% mobile phase B was run over 105 min followed by a wash cycle and equilibration of the column. Total run time on the HPLC was 138 min. An EASY-Spray column (2-μm particle size, 25 cm × 75 μm ID, PepMap C18) was used to separate the peptides, and an EASY-Spray ionization source was used for ionization. Data-dependent acquisition was conducted with the mass spectrometer. The first scan was recorded with the Orbitrap followed by 10 subsequent ion trap scans (FT-IT detection) on the top 10 most abundant ions. Collision-induced dissociation (CID) was used as the activation source with normalized collision energy at 35. Charge state rejection was enabled for +1 charged ions, and dynamic exclusion was enabled for a list size of 500 over 30 s. The data were analyzed using PEAKS 8.5 software.
8
Identifying IRAK1-Interacting Proteins in Glioma
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To investigate IRAK1-binding proteins in glioma cells, U251 cells were pretreated with MG132 (10 μM) for 4 h and then harvested using lysis buffer. The immunoprecipitates isolated by anti-IRAK1 and IgG control antibodies were analyzed using Dionex Ultimate 3000 RSLCnano (Buffer A: 0.1% formic acid solution; Buffer B: 0.1% formic acid + 80% acetonitrile solution; Thermo Scientific, USA). The peptides were then passed through a chromatographic column (Acclaim PepMap 75um X 150 mm,160321; Thermo) at a flow rate of 300 nL/min of Buffer A. After chromatographic separation, mass spectrometry analysis was performed based on a Q Exactive mass spectrometer (Thermo Scientific, USA) and MASCOT (http://www.matrixscience.com/ ).
9
Phytochemical Profiling of P. emblica Nanoherbal
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Determination of the phytochemical constituents of the P. emblica fruit nanoherbal was carried out with UHPLC Thermo Scientific Dionex Ultimate 3000 RSLCnano—HRMS Thermo Scientific Q Exactive (LSIH, Brawijaya University) with mobile phase A (0.1% formic acid in water) and phase B (0.1% formic acid in acetonitrile) following the gradient method. A 40 μL/min flow rate was used for the Hypersil GOLD aQ column, 50 × 1 mm × 1.9 m. The column oven temperature was 30 °C, and the time for analysis was 30 min. The results were analyzed using Compound Discoverer software with mzCloud MS/MS Library 2.1 [46 (link)].
10
Neorhodomela munita Metabolite Analysis
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The extract of Neorhodomela munita prepared using 100 ppm in methanol (HPLC grade, Sigma-Aldrich, Saint Louis, MO, USA) was analyzed using a Thermo Q-Exactive Orbitrap plus mass spectrometer connected to a Dionex Ultimate 3000 RSLC nano HPLC using an acquity UPLC BEH C18 (1.7 μm, 2.1 × 100 mm) column (Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase consisted of (A) 0.1% formic acid in HPLC-grade H2O and (B) 0.1% formic acid in HPLC-grade acetonitrile at a flow rate of 400 μL/min, with a column temperature of 45 °C. The Orbitrap MS scan was performed in positive ion mode with a 70,000 resolution and a scan range of 80–1000 m/z. The data-dependent MS/MS was performed with a resolution of 17,500, an isolation window of 2.0 m/z, and a loop count of 5. The estimation of the elemental composition of ions was performed based on the registered Chemspider (http://www.chemspider.com by Royal Society of Chemistry, London, UK) database and mz Cloud MS database (http://www.mzcloud.org by HighChem LLC, Bratislava, Slovakia).
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