Computational protocol: Do hypoxia/normoxia culturing conditions change the neuroregulatory profile of Wharton Jelly mesenchymal stem cell secretome?

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[…] Samples were analyzed on a Triple TOF™ 5600 System (ABSciex, Framingham, MA, USA) in two phases: information-dependent acquisition (IDA) was followed by SWATH (Sequential Windowed data independent Acquisition of the Total High-resolution Mass Spectra) acquisition on the same sample []. Peptides were resolved by liquid chromatography (nanoLC Ultra 2D, Eksigent, California, USA) on a ChromXP™ C18AR reverse phase column (300 μm ID × 15 cm length, 3 μm particles, 120 Å pore size; Eksigent) at 5 μL/min. Peptides were eluted into the mass spectrometer with an ACN gradient in 0.1 % FA (2 % to 35 % ACN, in a linear gradient for 25 min), using an electrospray ionization source (DuoSpray™ Source, ABSciex).For IDA, the mass spectrometer was set to scanning full spectra (350–1250 m/z) for 250 ms, followed by up to 30 MS/MS scans (100–1500 m/z for 75 ms each). Candidate ions with a charge state between +2 and +5 and counts above a minimum threshold of 70 counts/s were isolated for fragmentation and one MS/MS spectra was collected for 75 ms before adding those ions to the exclusion list for 15 s (mass spectrometer operated by Analyst® TF 1.6, ABSciex). Rolling collision was used with a collision energy spread of 5. Peptide identification was performed with Protein Pilot software (v4.5, ABSciex). Search parameters used were the following: SwissProt database, against a database composed of human and bovine species from SwissProt database (release at February 2014), GFP and iRT peptides, and using MMTS alkylated cysteines as fixed modification. An independent false discovery rate (FDR) analysis using the target-decoy approach provided with the Protein Pilot software was used to assess the quality of the identifications, and positive identifications were considered when identified proteins and peptides reached a 5 % local FDR [, ].The SWATH setup was essentially that used by Gillet et al. [], with the same chromatographic conditions used as in the IDA run described above. For SWATH-MS based experiments, the mass spectrometer was operated in a looped product ion mode. The instrument was specifically tuned to allow a quadruple resolution of 25 m/z mass selection. Using an isolation width of 26 m/z (containing 1 m/z for the window overlap), a set of 30 overlapping windows was constructed covering the precursor mass range of 350–1100 m/z. A 250 ms survey scan (350–1500 m/z) was acquired at the beginning of each cycle for instrument calibration and SWATH MS/MS spectra were collected from 100–1500 m/z for 90 ms resulting in a cycle time of 3 s from the precursors ranging from 350 to 1100 m/z. The collision energy for each window was determined according to the calculation for a charge +2 ion centered upon the window with a collision energy spread of 15.A specific library of precursor masses and fragment ions was created by combining all files from the IDA experiments, and used for subsequent SWATH processing. Libraries were obtained using Protein Pilot™ software (v4.5, ABSciex) with the same parameters as described above. Data processing was performed using SWATH™ processing plug-in for PeakView™ (v2.0.01, ABSciex); briefly peptides were selected automatically from the library using the following criteria: (1) the unique peptides for a specific targeted protein were ranked by the intensity of the precursor ion from the IDA analysis as estimated by the ProteinPilot™ software; and (2) peptides that contained biological modifications and/or were shared between different protein entries/isoforms were excluded from selection. Up to 15 peptides were chosen per protein, and SWATH™ quantitation was attempted for all proteins in the library file that were identified below 5 % local FDR from ProteinPilot™ searches. In SWATH™ Acquisition data, peptides are confirmed by finding and scoring peak groups, which are a set of fragment ions for the peptide. Target fragment ions, up to 5, were automatically selected and peak groups were scored following the criteria described in Lambert et al. []. Peak group confidence threshold was determined based on a FDR analysis using the target-decoy approach and 1 % extraction FDR threshold was used for all the analyses. Peptides that met the 1 % FDR threshold in one of the samples were retained, and the peak areas of the target fragment ions of those peptides were extracted across the experiments using an extracted-ion chromatogram (XIC) window of 3.0 min. The levels of the human proteins were estimated by summing all the transitions from all the peptides for a given protein (an adaptation of []) and normalized to the more stable internal standard. […]

Pipeline specifications

Software tools Analyst TF, ProteinPilot
Application MS-based untargeted proteomics
Organisms Homo sapiens
Chemicals Oxygen, Superoxides