Easy nlc 2 system

As previously described, all digested samples were analyzed by nLC-MS/MS [16 (link)]. Briefly, peptides were chromatographically separated using the EASY-nLC II system (Thermo Fisher Scientific, Waltham, MA, USA) using A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile as the mobile phase. Samples were chromatographically resolved using a 5–70–95% B gradient for 80 min at a flow rate of 300 nL/min. Peptides were analyzed using LTQ Orbitrap XL (Thermo Fisher Scientific Inc., Bremen, Germany) in a data-dependent mode with the 10 most intense precursors subjected to fragmentation by collision-induced dissociation.

The peptide samples were chromatographically separated on an EASY-nLC II system (Thermo Fisher Scientific Inc., Bremen, Germany) with two columns set up: a trap column C18-A1, 2 cm (SC001, Thermo Fisher Scientific, Waltham, MA, USA) and an analytical column PepMap C18, 15 cm × 75 µm, 3 µm particles, and 100 Å pore size (ES800, Thermo Fisher Scientific, Waltham, MA, USA). Samples (4–8 μL) were analyzed with an LTQ Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific Inc., Bremen, Germany). MS data were acquired in the data-dependent MS² mode. Data acquisition was controlled by XCalibur 2.1 software (Thermo Fisher Scientific Inc., Bremen, Germany).

The dried samples were dissolved in 0.1% formic acid to be analyzed in an Easy-nLC II system coupled to an ion trap LTQ-Orbitrap-Velos-Pro hybrid mass spectrometer (Thermo Scientific) [17 (link)].
The detection was performed with the following experimental conditions: survey scans from 400 to 1600 amu (1 μscan), twenty data dependent MS/MS scans, isolation width of 2u (in mass-to-charge ratio units), normalized collision energy of 35%, and dynamic exclusion applied for 30 s periods. Peptide identification from raw data was carried out using the SEQUEST algorithm (Proteome Discoverer 1.4, Thermo Scientific). A database search was performed against uniprot-Homo.fasta, uniprot-Bos.fasta and uniprot-Homo-Bos.fasta.

All excised and digested bands were analyzed by LC⿿MS/MS. Peptides were chromatographically separated using the EASY-nLC II system (Thermo, Germany) with a 2 column set up: trap column C18-A1, 2 cm (Thermo, Germany) and analytical column PepMap C18, 15 cm ÿ 75 μm, 3 μm particles, 100 ÿ pore size (Thermo, Germany). Mobile phases used were A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile. All solvents used were MS grade (Sigma, Germany). Total of 2 μL of each sample was loaded and separated by a gradient over the course of 80 min with a flow rate of 300 nL/min. The flow gradient was (i) 0⿿5 min at 5% B, (ii) 5⿿55 min, 5⿿70% B, (iii) 55⿿60 min 70⿿95% B, (iv) 60⿿70 min 95% B, (v) 70⿿75 min 95⿿5% B, (vi) 75⿿80 min 5% B.
Peptides were analyzed by LTQ Orbitrap XL mass spectrometer in data dependent mode with nano-ESI spray voltage of 1.9 kV, capillary temperature of 275 °C and tube lens value set at 110 V. All spectra were acquired in positive mode with high-resolution full scan in the mass range m/z 300⿿2000 and Orbitrap resolution of 30,000. The 5 most intense precursors were subjected to collision induced dissociation (CID) with normalized collision energy of 35 and activation time of 30 ms. Dynamic exclusion with 1 repeat count over 10 s and exclusion for 10 s was applied.

Analysis of the SCX-LC fractions was performed as previously described [32 (link)]. Rapidly, selected samples are loaded on the pre-column (NS-MP-10, Nano-separation,) of the Easy nLC II system (Thermo Scientific) followed by the separation on a C18 reverse phase column (NikkyoTechnos capillary column,) at a flow of 300 nL/min on 40 min gradient. The nano-LC was coupled to an Orbitrap™ Velos (Thermo Scientific). The survey scan was acquired by Fourier-Transform MS scanning 400–2000 Da at 30 000 resolution using internal calibration (lock mass) using the Top-20 acquisition method with 20 s exclusion time. Raw data files were extracted and exported with Proteome Discoverer (Thermo Scientific, Ver. 1.4) for ion signal higher than 1 counts and S/N higher than 1.5.