Digestpro msi

Peptide analysis of the culture media samples from BM-MSC under TNFα and C1 conditions (3 samples each condition) was carried out by quadrupole-orbitrap nano-HPLC-ESI-MS/MS. Culture media was lyophilized and dissolved in water. Gel-assisted proteolysis was performed for the proteins entrapped in a polyacrylamide gel matrix. Protein digestion and sample preparation were carried out using DigestPro MSI (INTAVIS Bioanalytical Instruments AG, Cologne, Germany). Peptides were purified and concentrated using C18 ZipTip (Merck Millipore) according to the manufacturer’s instructions. Samples were injected into an Easy nLC 1200 UHPLC system coupled to a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFisher). Data was acquired using Tune 2.9 and Xcalibur 4.1.31.9 (ThermoFisher). Swiss-Prot database was used to identify Mus musculus proteins. These proteins were analyzed using Proteome Discoverer 2.2 (Thermo Fisher) and the tandem mass spectrometry data analysis program SEQUEST. The false discovery rate (FDR) was calculated using Percolator. Minora feature detector in Proteome Discoverer 2.2 (Thermo Fisher) was used for the label-free quantification.

One quarter (25 μL) of the ProteoMiner enriched sheep acid whey protein fraction obtained after 2D Clean Up Kit desalting was subjected to 1D-PAGE on a 12 well Novex BOLT 4–12% bis-Tris electrophoresis gel run in a Novex BOLT mini-gel electrophoresis system (Life Technologies, Auckland, NZ). The protein sample was added to Novex BOLT LDS sample buffer (4X) and Novex BOLT sample reducing agent (10X) according to supplier's recommendations, prior to electrophoresis. Novex Sharp Pre-Stained Protein Standard (Life Technologies, Auckland, NZ) was run in one lane for calibration. After electrophoresis the gels were washed in MQ-water 3 x 5 min and then stained with Simply Blue SafeStain (Invitrogen, Auckland, NZ) according to supplier's instructions. The whole gel lane containing the ProteoMiner enriched sheep acid whey fraction was excised from the gel and then divided into 6 segments prior to in-gel tryptic digestion. Each gel lane segment was subjected to in-gel digestion with trypsin using a robotic workstation for automated protein digestion (DigestPro Msi, Intavis AG, Cologne, Germany). The protocol for automated in-gel digestion was based on the method of Shevchenko, Jensen [24 (link)]. Eluted peptides were dried using a Savant Speed Vac (Savant, France) centrifugal concentrator, prior to LCMS/MS.

All proteomic analyses were performed at the Proteomics Core Facility of the University of Málaga. The bands identified on the western blot were isolated from the gel, destained, reduced with DTT, alkylated with iodoacetamide and digested in-gel with trypsin (Promega, Southampton, UK) automatically in a DigestPro MSI (INTAVIS Bioanalytical Instruments AG, Köln, Germany). Bands corresponding to extractions from atrial, sinusal, ventricular and conal myocardium were used.

Based on initial 1D gels, equal volumes of ovarian fluid from each of 20 females were combined and mixed (we omitted 5 females which showed unusually strong protein bands indicating contamination with albumin and other plasma proteins). From this mixture, a 3 µl aliquot was run on SDS-PAGE and stained with homemade colloidal Coomassie [38] (link). Five regions (up to 10 kDa, 10–18 kDa, 20–55 kDa, 55–90 kDa, and 90–260 kDa) were excised from the gel (Fig. 1B) and subjected to in-gel digestion with trypsin [39] using a robotic workstation for automated protein digestion (DigestPro Msi, Intavis AG, Cologne, Germany). Two prominent bands (major bands at 10 kDa and 18–20 kDa) were also excised to avoid interference of high abundant proteins. Eluted peptides were dried using a centrifugal concentrator (Savant Speed Vac SC 100; Savant, France).