Nano easy spray source

The desalted protein digest was analyzed by RP-LC-ESI-MS/MS in an EASYnLC1000 System, coupled to the Q-Exactive-HF mass spectrometer through the Nano-Easy spray source (Thermo Scientific, Waltham, MA, USA). Peptide identifications were carried out using the Mascotv.2 search engine through the Protein Discoverer Software. A database search was performed against SwissProt. Mascot Scores were adjusted by a percolator algorithm. The acceptance criteria for protein identification were a false discovery rate (FDR) <1% and at least one peptide identified with high confidence (CI > 95%). To determine the abundances of the identified peptides and proteins, a processing free label workflow was initiated in the first step. Finally, the results were normalized to the total amount of the peptides, equaling the total abundance among the different samples. The proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE [27 (link),28 (link)].

Samples were suspended in a 20 μL loading buffer and analysed on an Ultimate 3000 nano UPLC system online coupled to either an Orbitrap Fusion^Tm Tribrid^Tm Mass Spectrometer or a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific). Peptides were separated on a 75 μm × 50 cm PepMap C18 column using a 1 or 2 h linear gradient from 2–5% buffer A to 35% buffer B at a flow rate of 250 nL/min (approx. 600 bar at 40 °C). Peptides were introduced into the mass spectrometer using a nano Easy Spray source (Thermo Scientific) at 2000 V. The ion transfer tube temperature was set to either 305 °C (Fusion Lumos), or 250 °C (HF-X). Subsequent isolation and higher-energy C-trap dissociation (HCD) were induced on the 20 most abundant ions per full MS scan with an accumulation time of 128 ms and an isolation width of 1.2 Da (Fusion Lumos) or 1.6 Da (HF-X). All fragmented precursor ions were actively excluded from repeated selection for 8 s (Fusion) or 15 s (HF-X). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [31 (link)] with the dataset identifiers PXD008151 and PXD024053.

Fresh CM samples were concentrated from 20 mL to approximately 2 mL by using Vivaspin^® 20 Centrifugal Concentrators 10K (Sartorius Stedim Lab Ltd., Stonehouse, UK). Proteinase K 500 μg/mL in PBS was added to break the exosomal membranes. A total of 6 samples corresponding to three biological replicates of each group (control and treated samples) were analysed. The proteomic analysis was performed at the Proteomics Unit of the Complutense University of Madrid (Spain) as reported elsewhere [93 (link)]. Briefly, 50 μg of each protein extract was concentrated in a stacking gel. The bands of proteins were cut from the gel, reduced, alkylated and trypsin digested o/n. Then the peptides from the digested proteins were desalted and concentrated with C18 reverse phase chromatography, eluted, freeze-dried in speed-vac and resuspended in acetonitrile/formic acid. The desalted peptides were analysed by a reverse phase liquid chromatography electrospray ionisation tandem mass spectrometry (RP-LC-ESI-MS/MS) in an EASY-nLC 1000 System coupled to the Q-Exactive HF mass spectrometer (an ultra-high-field mass orbitrap analyser) through the Nano-Easy spray source (all from Thermo Scientific, Bremen, Germany). All data were acquired using data-dependent acquisition (DDA) and in positive mode with the Xcalibur 4.0 software (Thermo Fisher Scientific).

Protein profile of EVs and cell-extracts was analysed by 10% SDS-PAGE using 20 μg protein from each extract of the three replicates. Several bands were excised and identified by peptide fingerprint and fragmentation following comparison with the NCBIprot database (Inbiotec, Leon, Spain). In addition, PBS-extracts obtained in three independent biological replicates were mixed and analysed by liquid chromatography and mass spectrometry (Proteomic Unit, CAI Técnicas Biológicas, U.C.M. Madrid). In short, desalted protein digests were analysed by RP-LC–ESI–MS/MS in an EASY-nLC 1000 System coupled to the Q-Exactive HF mass spectrometer through the Nano-Easy spray source (all from Thermo Scientific, Mississagua, ON, Canada). Database search was performed against UniProt database (SwissProt and TrEMBL) with taxonomic restriction to L. plantarum. Detailed information of these procedures is described in the supplementary material.

Samples were suspended in 20 μl of loading buffer (1% acetonitrile, 0.1% TFA in water) and analyzed on an Ultimate 3000 nano UPLC system online coupled to a Fusion Lumos mass spectrometer (Thermo Scientific). Peptides were separated on a 75 μm × 50 cm PepMap C18 column using a 1h linear gradient from 5 to 25% buffer B in buffer A at a flow rate of 250 nL/min (∼600 bar). Peptides were introduced into the mass spectrometer using a nano Easy Spray source (Thermo Scientific) at 2000V and ion transfer tube temperature of 305 °C. Subsequent isolation and higher energy C-trap dissociation (HCD) was induced on the most abundant ions per full MS scan at 2 s cycle time. Ions with a charge of 2–4 were measured at an accumulation time of 120 ms, AGC target of 200,000, quadrupole isolation width of 1.2 Da, and energy level 28. All fragmented precursor ions were actively excluded from repeated selection for 60 s.