sEV proteins were digested by trypsin52 (link) and the tryptic peptides were analyzed by nano-ultra-high-performance LC (UPLC) (Waters) and tandem mass spectrometry using a Q-Tof Premier (Waters)53 (link),54 (link). Digested peptides were injected into a 2 cm × 180 μm trap column and resolved in a 25 cm × 75 μm nanoACQUITY C18 column (Waters) using the LC system. All samples were analyzed in triplicate. For protein identification, MS raw data were converted into peak lists using MASCOT Distiller version 2.1 (Matrix Science, London, UK) with default settings. All MS/MS raw data were analyzed using MASCOT version 2.2.1 (Matrix Science)53 (link). Mascot was used to search the SwissProt database (release 2018_07) with human taxonomy. Quantification was performed using PEAKS Studio version 10.0 (Bioinformatics Solution Inc., Waterloo, Canada). For Label-free protein quantification, identified peptides were filtered based on False Discovery rate <1%. The abundance of each peptide was determined using ion chromatography extraction and the protein ratio was calculated using the average abundance among the corresponding peptides. Protein ratios were considered acceptable when the proteins contained more than one unique peptide.
Nanoacquity c18 column
Manufactured by Waters Corporation
Sourced in Germany
The NanoACQUITY C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of small molecules. The column features a C18 stationary phase, which is commonly used for the separation of a wide range of organic compounds. The column is suitable for use in nano-scale HPLC applications, where small sample volumes and high sensitivity are required.
Automatically generated - may contain errors
Lab products found in correlation
Q tof premier, by Waters Corporation (4 mentions)
Mascot version 2, by Matrix Science (2 mentions)
Mascot distiller version 2, by Matrix Science (2 mentions)
Ultra performance liquid chromatography (uplc), by Waters Corporation (1 mentions)
Nanoacquity uplc system, by Waters Corporation (1 mentions)
5 protocols using nanoacquity c18 column
1
Proteomic Characterization of Extracellular Vesicles
2
LC-MS Peptide Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LC-MS analysis was performed as described previously [15 (link)]. Resulting peptide was analyzed by nano-UPLC mass spectrometry using Q-Tof Premier (Waters, Manchester, UK). Peptides were injected into the trap column then resolved by nanoACQUITY C18 column (Waters). The peptides were resolved with a gradient of 3% to 45% CAN with 0.1% FA over 160 min at a 300 nL/min flow rate.
3
Proteomic Analysis of Small Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Small-EVs were analyzed by LC–MS/MS as described previously [27 (link)]. One microgram of extracted peptide was analyzed by nano-ultra-high-performance LC (UPLC) (Waters) and tandem mass spectrometry using a Q-Tof Premier (Waters). Peptides were injected into a 2 cm × 180 μm trap column and resolved in a 25 cm × 75 μm nanoACQUITY C18 column (Waters) using the LC system. Mobile phase A was composed of water containing 0.1% FA and mobile B phase was 0.1% FA in ACN. The peptides were resolved using a gradient of 3–45% mobile phase B over 135 min at a flow rate of 300 nL/min. All samples were analyzed in triplicate. The method included a full sequential MS scan (m/z 150–1600, 0.6 s) and five MS/MS scans (m/z 100–1990, 0.6 s/scan) for the five most intense ions present in the full-scan mass spectrum.
4
Quantitative Proteomic Analysis of Exosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All proteomic experimental procedures were performed according to a previous report (Im et al., 2019 ). Exosomal proteins were digested with trypsin (Cho et al., 2017 (link)). The tryptic peptides were analysed using nano‐ultra‐high‐performance LC (UPLC, Waters) and tandem mass spectrometry using a Q‐Tof Premier (Waters) (Cho et al., 2012 ; Moon et al., 2011 (link)). The digested peptides were injected into a 2 cm × 180 μm trap column and resolved using a 25 cm × 75 μm nano ACQUITY C18 column (Waters) on the LC system. All samples were analysed in triplicate. For protein identification, MS raw data were converted into peak lists using MASCOT Distiller version 2.1 (Matrix Science) using the default settings. All MS/MS raw data were analysed using MASCOT version 2.2.1 (Matrix Science). (Cho et al., 2012 ) The MASCOT was used to search the SwissProt database (release 2018_07) with human taxonomy. Quantification was performed using PEAKS Studio version 10.0 (Bioinformatics Solution Inc.). For label‐free protein quantification, the identified peptides were filtered based on a false discovery rate <1%. The abundance of each peptide was determined using ion chromatography extraction and the protein ratio was calculated using the average abundance of the corresponding peptides. Protein ratios were considered acceptable when the proteins contained more than one unique peptide.
5
Nano-LC-MS/MS Peptide Separation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tryptic peptides mixtures were separated using a nano Acquity UPLC system (Waters GmbH, Eschborn, Germany). Peptides were trapped on a nano Acquity C18 column, 180 µm x 20 mm, particle size 5 µm (Waters GmbH, Eschborn, Germany). The liquid chromatography separation was performed on a C18 column (BEH 130 C18 100 µm x 100 mm, particle size 1.7 µm (Waters GmbH, Eschborn, Germany) with a flow rate of 400 nl /min. Chromatography was carried out using a 1h gradient of solvent A (98.9% water, 1% acetonitrile, 0.1% formic acid) and solvent B (99.9% acetonitrile and 0.1% µl formic acid) in the following sequence: from 0 to 4% B in 1 min, from 4 to 40% B in 40 min, from 40 to 60% B in 5 min, from 60 to 85% B in 0.1 min, 6 min at 85% B, from 85 to 0% B in 0.1 min, and 9 min at 0% B. The nanoUPLC system was coupled online to an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Bremen, Germany). The mass spectrometer was operated in the data-dependent mode to automatically measure MS1 and MS2. Following parameters were set: ESI voltage 2400 V; capillary temperature 200 °C, normalized collision energy 35 V. Data were acquired by scan cycles of one FTMS scan with a resolution of 60000 at m/z 400 and a range from 300 to 2000 m/z in parallel with six MS/MS scans in the ion trap of the most abundant precursor ions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!