The proteomic composition of the pooled CM were analyzed using LC-MS/MS as previously described [42 (link)]. Briefly, the total protein concentration was measured using a bicinchoninic acid assay (Pierce BCA Kit, Thermo Fisher), and 10 μg of lyophilized protein was processed to obtain tryptic peptides. About 0.5 µg protein as tryptic peptides dissolved in 2% acetonitrile and 0.5% formic acid was injected into an Ultimate 3000 RSLC system connected online to a Exploris 480 mass spectrometer equipped with EASY-spray nano-electrospray ion source (all from Thermo Scientific, Sunnyvale, CA, USA). Additional details of LC-MS/MS are reported in the Supplementary Materials .
Easy spray nano electrospray ion source
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The EASY-Spray nano-electrospray ion source is a laboratory instrument designed for the generation of ions from liquid samples. It utilizes a spray nozzle to convert the liquid sample into a fine mist, enabling the subsequent ionization and analysis of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Pierce bca kit, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 rslc system, by Thermo Fisher Scientific (2 mentions)
Dionex ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
Q exactive, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 uhplc system, by Thermo Fisher Scientific (2 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
Amicon ultrafiltration unit, by Merck Group (1 mentions)
Acclaim pepmap100, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Promega (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Reprosil pur 120 c18 aq, by Dr. Maisch (1 mentions)
Thermo xcalibur 2, by Thermo Fisher Scientific (1 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
11 protocols using easy spray nano electrospray ion source
1
Proteomic Profiling of Conditioned Media
2
Proteomic Analysis of Osteoarthritic Synoviocytes
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CM collected from untreated (pCTR) and CS- and BC-treated osteoarthritic human synoviocytes were concentrated to a final volume of 500 µL with Amicon ultrafiltration units (MWCO 3kDa, Millipore, Billerca, MA, USA) and subjected to trichloroacetic acid (TCA) precipitation [72 (link)]. Protein pellets were air dried and then resuspended in 50 µL of triethylammonium bicarbonate (TEAB). Protein concentration was determined by Quantum Micro Protein Assay Kit- BCA Total Protein Assay Kit for dilute samples (Euroclone S.p.A., Milan, Italy). Equal amounts of proteins (10 µg) were reduced, alkylated and digested as reported elsewhere [73 (link)]. Following tryptic digestions, samples were centrifuged at 10,000× g for 15 min and supernatants were dried under vacuum in a SpeedVac Vacuum (Savant Instruments, Holbrook, NY, USA). Samples were then resuspended in 25 μL of H2O/TFA 2% and centrifuged at 10,000× g for 15 min. Aliquots of the samples (5 μL) were analysed in triplicate on a Q-Exactive Orbitrap mass spectrometer equipped with an EASY-Spray nano-electrospray ion source (Thermo Fisher Scientific, Bremen, Germany) and coupled to a Dionex UltiMate 3000RSLC nano system (Thermo Fisher Scientific) [74 (link),75 (link)].
3
Mass Spectrometry Analysis of Tryptic Digests
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Mass spectrometry analyses of tryptic digests (2 µg) were performed as previously described25 (link) on a Q-Exactive Orbitrap mass spectrometer equipped with an EASY-Spray nanoelectrospray ion source (Thermo Fisher Scientific, Bremen, Germany) and coupled to a ThermoScientific Dionex UltiMate 3000RSLC nano system (Thermo Fisher Scientific).
4
Proteomic Analysis by Mass Spectrometry
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IP protein samples were separated by SDS-PAGE, and 8–10 fractions were cut from each gel lane. Each fraction of the samples was reduced, alkylated, digested (trypsin, Promega), and desalted on C18 columns, as described before [33 (link)]. MS analysis was performed by a nano Liquid-Chromatography (nLC) (EASY-nLC 1000, Thermo Fisher Scientific, Waltham, MA, USA) coupled to a mass spectrometer (Q Exactive, Thermo Fisher Scientific) through an EASY-Spray nano-electrospray ion source (Thermo Fisher Scientific). A pre-column (Acclaim® PepMap 100, 75 μm × 2 cm, nanoviper fitting, C18, 3 μm, 100 Å, ThermoFisher Scientific) and analytical column (EASY-Spray Column, PepMap, 75 μm × 15 cm, nanoviper fitting, C18, 3 μm, 100 Å, Thermo Scientific) were used to trap and separate peptides, respectively. For nLC separation, buffer A was 0.1% FA and buffer B was 100% ACN/0.1% FA. A 30 min gradient of 5% to 30% buffer B was used for peptide separation. Mass spectrometry constituted of full scans (m/z 300–1800) at a resolution of 70,000 followed by top-10 MS/MS scans at a resolution of 17,500. HCD collision energy was 23–28%. Dynamic exclusion of 30 s as well as rejection of precursor ions with charge state +1 and above +8 were employed.
5
Trypsin-mediated HSP10 Proteolysis
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Purified HSP10-p.wt or HSP-p.Leu73Phe protein at 0.1 mg/ml in 100 mM Tris-HCl (pH 7.8) was incubated at 37°C. Trypsin was added to a final concentration of 0.01 mg/ml (molar ratio HSP10:trypsin 10:1). Samples were taken immediately before and 2, 10, 25, 60, and 120 min after addition of trypsin. Samples were quenched by addition of SDS PAGE sample buffer and incubation at 95°C for 2 min. As control, series without addition of trypsin were performed. Samples were subjected to SDS PAGE and proteins stained with Coomassie.
For analysis of the content of HSP10 bands from SDS PAGE, Coomassie–stained bands were excised and peptides extracted as described (Edhager et al., 2014 (link)). Peptides were analyzed using a nano Liquid-Chromatography system (Ultimate 3000, Dionex) coupled to a mass spectrometer (Q Exactive, Thermo Fisher Scientific) through an EASY-Spray nano-electrospray ion source (Thermo Scientific).
For analysis of the content of HSP10 bands from SDS PAGE, Coomassie–stained bands were excised and peptides extracted as described (Edhager et al., 2014 (link)). Peptides were analyzed using a nano Liquid-Chromatography system (Ultimate 3000, Dionex) coupled to a mass spectrometer (Q Exactive, Thermo Fisher Scientific) through an EASY-Spray nano-electrospray ion source (Thermo Scientific).
6
Label-Free Quantitative Proteomics of BMSC-CM and LPRF-CM
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BMSC-CM and LPRF-CM (n = 3 donors each) were analyzed using LC-MS/MS via label-free quantitation (LFQ), as previously described [81 (link)]. Briefly, total protein concentration of each sample was measured using a bicinchoninic acid assay (Pierce BCA Kit, Thermo Fisher, Waltham, MA, USA) and 10 μg protein was processed to obtain tryptic peptides. About 0.5 μg protein, as tryptic peptides dissolved in 2% acetonitrile and 0.5% formic acid, was injected into an Ultimate 3000 RSLC system connected online to an Exploris 480 mass spectrometer equipped with EASY-spray nano-electrospray ion source (all from Thermo Scientific, Sunnyvale, CA, USA). Additional details of LC-MS/MS are reported in the Supplementary Methods . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (https://www.ebi.ac.uk/pride/ accessed on 18 April 2023) with the dataset identifier PXD041617 and 10.6019/PXD041617.
7
Proteomic Workflow for Liquid Chromatography-Mass Spectrometry
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Desalted samples were separated on an UltiMate 3000 UHPLC system (Thermo Fischer Scientific, UK) and electro-sprayed directly into a Q-Exactive Mass Spectrometer (Thermo Fischer Scientific) through an EASY-Spray nano-electrospray ion source (Thermo Fischer Scientific). The peptides were trapped on a C18 Acclaim™ PepMap™ 100 pre-column (300 μm i.d. Â 5 mm, 100 Å, Thermo Fisher Scientific) using solvent A (0.1% FA in MilliQ) at a pressure of 500 bar. The peptides were separated on an in-house packed analytical column (75 μm i. d. Â 50 cm packed with ReproSil-Pur 120 C18-AQ, 1.9 μm, 120 Å, Dr. Maisch GmbH, Germany) using a linear gradient (length: 65 min, 15%-35% solvent B (0.1% FA in ACN), flow rate: 200 nl min À1 ). Data were acquired in a data-dependent mode. Full scan MS spectra were acquired in the Orbitrap (scan range 350-1500 m/z, resolution 70 000, AGC target 3e6, maximum injection time 50 ms À1 ). The 3 or 20 most intense peaks were selected for HCD fragmentation at 30% of normalised collision energy (resolution 17 500, AGC target 5e4, maximum injection time 120 ms À1 ) with first fixed mass at 180 m/z. A Top 3 method (three MS/MS collected per MS scan) was used for the label-free quantitative comparisons while a separate run was performed using a Top 20 method for qualitative analysis.
8
High-Resolution Proteomic Analysis
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Aliquots of TMT‐labeled peptides (2.5 μg) were analyzed by high‐resolution nanoLC–tandem mass spectrometry using a Q‐Exactive Orbitrap mass spectrometer equipped with an EASY‐Spray nanoelectrospray ion source (Thermo Scientific, Rockford, Illinois, USA), coupled to a Thermo Scientific Dionex UltiMate 3000 RSLCnano system (Thermo Scientific, Rockford, Illinois, USA) as reported elsewhere (Russo et al, 2019 ).
9
LC-MS/MS Analysis of Osteo- and Naïve-EVs
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Protein lysates collected from Osteo- and Naïve-EVs (n = 3 each) were analysed using LC–MS/MS with Label-Free Quantitation [25 (link), 26 ]. In brief, approximately 25 μg of protein was digested into tryptic peptides. About 0.5 µg protein dissolved in 2% acetonitrile and 0.5% formic acid was injected into an Ultimate 3000 RSLC system connected online to an Exploris 480 mass spectrometer equipped with EASY-spray nano-electrospray ion source (all from Thermo Scientific, Sunnyvale, CA, USA) [26 ].
10
Peptide Identification Using Nano-LC-MS/MS
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The peptides were identified using an UltiMate® 3000 RSLCnano system (Thermo Fisher Scientific, USA) coupled with Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA) through EASY-Spray nano-electrospray ion source (Thermo Fisher Scientific, USA). The samples were loaded with 5–7% Acetonitrile (ACN) in 5 min, 7–45% in 60 min, 45–50% in 5 min, and 50–97% in 5 min, followed by washing at 100% at 300 nL/min flow rate for 90 min. Full MS scan was carried with mass ranges of m/z 200–2000. Precursor ions with + 1 and greater than + 8 charge state were excluded. Fragmentation of precursor ions was performed using Higher-energy collisional dissociation and data acquisition was performed by Thermo Xcalibur 2.2 (Thermo Fisher Scientific, USA).
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