Easy nano liquid chromatography system

To identify the proteins of the differentially abundant spots, a 2D-DIGE pick gel was prepared and separated. After Coomassie blue staining, spots of interest were excised with the Screen Picker (Proteomics Consult), destained overnight with 25 mM ammonium bicarbonate in 50% acetonitrile, dehydrated with 100% acetonitrile, dried, and digested overnight with trypsin (200 ng/spot; T6567 Sigma-Aldrich) in 40 mM ammonium bicarbonate, 9% acetonitrile at 37 °C. The trypsin digest was extracted with 1% trifluoroacetic acid (Sigma-Aldrich), lyophilized and stored at − 80 °C until shipping.
Samples were analyzed at the Proteomics Facility of CEINGE-Biotecnologie Avanzate (Naples, Italy). LC–MS/MS was done on a Proxeon EASY nano liquid chromatography system coupled with an LTQ Orbitrap XL mass spectrometer with ETD (Thermo Fisher Scientific, Massachusetts, USA). CEINGE provided raw MS data in.mgf format.
Mascot Server v2.3 (Matrix Science, Boston, USA) was used to search for matches between the MS data and proteins in the NCBI nr and Swiss-Prot databases, selected for human taxonomy.

The gel pieces were processed with standard mass spectrometry protocols. The tryptic digests were analyzed in an Easy-nano liquid chromatography system (Thermo Scientific) equipped with an Easy-Spray 15-cm column (C18, 2 μm, 75 μm × 15 cm; Thermo Scientific) coupled to an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific) as described previously [64 (link)].
High-resolution mass spectra of the peptide mixture were deconvoluted using Xtract software (Thermo Scientific). The analysis of the mass spectrometric RAW data was carried out using Proteome Discoverer 2.1. The coding products from predicted ORFs of ADRV (KC865735.1) and RGV (JQ654586.1) and the products of the unigenes from transcriptomes of Chinese giant salamander (SRP115981) were used as the search database [6 (link), 40 (link), 55 (link)]. Three independent iPOND experiments containing click and nonclick controls were performed for each virus. The positive identification of a protein followed the following criteria: the protein was present in the click sample but absent in the nonclick control, or the abundance of the protein in the click sample was at least twofold greater than that in the nonclick control.

Each component was further analyzed using a Q Exactive mass spectrometer (Thermo Finnigan) connected to the Easy nano Liquid Chromatography system. Briefly, dried fraction was resuspended in buffer A (0.1% formic acid) and packed with C18-reversed phase column (2 cm × 100 μm, 5 μm, Thermo Scientific). Peptides were eluted onto an analytical C18 column (75 um × 100 mm, 3 μm, Thermo Scientific) with a gradients of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nl/min over 60 min. The mass spectrometer was performed at a data-dependent mode. In a full MS scan range of m/z 300–1800, the ten most abundant precursor ions were choosed for high-energy collisional dissociation (HCD) fragmentation. Survey scans were acquired at a resolution of 70,000 at m/z 200 and resolution for HCD spectra was set to 17,500 at m/z 200.