Reprosil pur c18 aq 5 μm resin

Nanoflow LC-MS/MS was performed by coupling an Eksigent nanoLC-Ultra 1D + (Eksigent, Dublin, CA) to a LTQ-Orbitrap XL ETD (Thermo Scientific, Bremen, Germany). Peptides were delivered to a trap column (100 μm × 2 cm, packed in-house with Reprosil-Pur C₁₈-AQ 5 μm resin, Dr. Maisch, Ammerbuch, Germany) at a flow rate of 5 μL/min in 100% solvent A (0.1% formic acid in HPLC grade water). After 10 min of loading and washing, peptides were transferred to an analytical column (75 μm × 40 cm, packed in-house with Reprosil-Pur C₁₈-GOLD, 3 μm resin, Dr. Maisch, Ammerbuch, Germany) and separated using a 210 min gradient from 4% to 32% of solvent B (0.1% formic acid, 5% DMSO in acetonitrile; solvent A: 0.1% formic acid, 5% DMSO in water) at 300 nL/minute flow rate. The LTQ Orbitrap XL was operated in data dependent mode, automatically switching between MS and MS [2 (link)]. Full scan MS spectra were acquired in the Orbitrap at 60,000 (m/z 400) resolution after accumulation to a target value of 1,000,000. Tandem mass spectra were generated for up to eight peptide precursors in the linear ion trap by using collision-induced dissociation at a normalized collision energy of 35% after accumulation to a target value of 5,000 for max 100 ms.

The SGs were dissected from 5^th instar nymphs and adults at 5, 12 and 24 days post blood meal and carefully punctured at 4°C. Following centrifugation (16.000 × g, 15 min, 4°C), the soluble protein fraction from fifteen pairs of SG homogenates was ethanol/acetone precipitated. Resuspended proteins were consecutively alkylated, reduced, digested by trypsin, and subjected to LC-MS/MS analysis as previously described [27 (link)]. Briefly, the tryptic peptides were loaded onto a 2 cm fused silica trap column (150 μm inner diameter) packed in-house with reverse phase capillary column ReproSil-Pur C18-AQ 5 μm resin (Dr. Maisch GmbH, Germany) and separated using a DIONEX 3000 nanoUPLC system coupled to an LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific, Waltham, USA). MS1 spectra were recorded in the Orbitrap mass analyzer with 120,000 resolution. After ion fragmentation, MS/MS spectra of the 15 most intense ions were acquired. Raw files were generated and used for protein identification using Proteome Discoverer v.1.3 (Thermo Scientific, Waltham, USA) with in-house SequestHT algorithm for R. neglectus SG transcriptome and human keratins, BSA and porcin trypsin. The false discovery rate was less than 1%, with peptide rank of 1 and at least 2 peptides per protein.