Easy nlc liquid chromatography system

Whole glycoproteins digested with trypsin were also examined using a Q Exactive Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, United States) coupled with an Easy nLC liquid chromatography system (Thermo Fisher Scientific). LC was operated by a C18 reverse phase trap column (Scientific Acclaim PepMap100, 100 μm × 2 cm, nanoViper, Thermo Fisher Scientific) connected to a C18 reversed-phase analytical column Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin, Thermo Fisher Scientific) in 0.1% formic acid and separated with a linear gradient of buffer (acetonitrile and 0.1% formic acid). The flow rate was at 300 ml/min and the system was controlled using IntelliFlow technology. The MS system was operated in positive ion mode. The optimal source/gas parameters were as follows: automatic gain control target, 3e6; maximum inject time, 10 ms. The dynamic exclusion duration was 40.0 s and the normalized collision energy was 30 eV. The survey scan for higher-energy collisional dissociation (HCD) fragmentation was set at 300–1800 m/z, with HCD spectra set to 17,500 at m/z 200, and an isolation width was of 2 m/z. The instrument was run with an enabled mode of peptide recognition.

Three HucMSC-EVs samples from independent experiments were collected and prepared as protein samples. Briefly, the protein concentration was measured by Qubit 4.0. A total of 200 µg of protein solution was used and digested with sequencing-grade trypsin (Promega, Madison, USA). Then, a C18 Cartridge was used to desalt the digested peptide. Liquid chromatography-tandem MS (LC‒MS/MS) analysis was subsequently conducted on a Q-Exactive mass spectrometer (Thermo Fisher, USA) coupled with an Easy-nLC liquid chromatography system (Thermo Fisher, USA) for 120 min in Applied Protein Technology (APTBIO, Shanghai, China) according to a previously published protocol [25 ]. Maxquant software (version 1.5.3.17) and the SwissProt Human proteome database containing 20,395 proteins (SwissProt_Homo_sapiens_20395_20210106.fasta) were utilized to analyse the raw data. The proteins identified by the database search met the set filter parameter of a false discovery rate (FDR) ≤ 0.01. The identified proteins were compared with available extracellular vesicle data from the ExoCarta database (http://www.exocarta.org). Both Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) term analyses were performed using the DAVID online tool (https://david.ncifcrf.gov).

Peptide samples were dried in the Speed-Vac, resuspended in 0.1% formic acid (FA) and analyzed using an EASY-nLC liquid chromatography system (Thermo Scientific) coupled to LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). The peptides were loaded on a Reprosil-Pur C18-AQ (3 μm) column and separated in an organic solvent gradient from 100% phase A (0.1% FA) to 30% phase B (0.1% FA, 95% ACN) during 80 min for a total gradient of 105 min at a constant flow rate of 300 nL/min. The LTQ-Orbitrap Velos was operated in positive ion mode with data-dependent acquisition. The full scan was acquired in the Orbitrap with an automatic gain control (AGC) target value of 10e6 ions and a maximum fill time of 500 ms. Peptides were fragmented by collision-induced dissociation. Ions selected for tandem mass spectrometry (MS/MS) were dynamically excluded for a duration of 30 s. Each MS scan was acquired at a resolution of 60,000 FWHM followed by 20 MS/MS scans of the most intense ions. All raw data were accessed with the Xcalibur software (Thermo Scientific).