Label-free quantitative proteomics

Protein extraction and quantification

Total hemocyte proteins are extracted from pelleted shrimp hemocytes with 0.25x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂PO₄, 2 mM KH₂PO₄). After centrifugtion at 13000 ×g for 5 min, the supernatant is collected, quantitated by Protein assay dye (Bio-Rad) and checked for quality using SDS-PAGE electrophoresis. After quantification, each sample of protein lysate (60 μg) is lyophilized and further prepared as described below.

Pre-MS sample preparation

Prior to in-solution digestion, BSA protein is added to the frozen protein lysates as the internal standard for normalizing XIC area readings of endogenous peptides. The protein mixtures are then resolubilized in an 8 m urea/ 25 mM ammonium bicarbonate buffer, reduced for 1 hr at 37°C with 2 mm dithioerythreitol and then alkylated for 1 hr by 2.5 mm iodoacetamide at room temperature. Next, the samples are further diluted with 25 mM ammonium bicarbonate to bring the urea concentration to 1 m. A modified protease that cleaves C-terminally to arginine and lysine residues is added (1:50 w/w) and the mixture is incubated overnight for protein digestion. Protease activity is quenched by acidification of the reaction mixtures with formic acid solution to pH 1~2. The peptide mixture is aliquoted, desalted, concentrated on a C₁₈-StageTip (Proxeon Biosystems), and eluted with 50 % acetonitrile in 0.1% formic acid.

Label-free quantitative proteomic using Nanoflow HPLC-MS/MS

The peptide mixtures are analyzed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) on a nanoAcquity system (Waters, Milford, MA) connected to an LTQ Orbitrap Velos hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a PicoView nanospray interface (New Objective, Woburn, MA). Peptide mixtures are loaded onto a 75 μm ID, 25 cm length C18 BEH column (Waters, Milford, MA) packed with 1.7 μm particles with a pore width of 130 Å and separated using a segmented gradient of 5% to 40% solvent B (acetonitrile with 0.1% formic acid) over 90 min at a flow rate of 300 nl/min and a column temperature of 35°C. Solvent A is 0.1% formic acid in water. The effluent from the HPLC column is directly electrosprayed into the mass spectrometer. The LTQ Orbitrap Velos instrument is operated in data-dependent mode to automatically switch between full scan MS and MS/MS acquisition. The instrument is controled using Tune 2.6.0 and Xcalibur 2.1.

For the CID-MS/MS top20 method, full scan MS spectra (from m/z 350–1600) are acquired in the Orbitrap analyzer after accumulation to a target value of 1×10^-6 in the linear ion trap. Resolution in the Orbitrap system is set to r = 60,000 (all Orbitrap system resolution values are given at m/z 400). Typically, the 20 most intense peptide ions with charge states ≥2 are sequentially isolated to a target value of 5,000 and fragmented in the high-pressure linear ion trap by low-energy CID with normalized collision energy of 35%. The resulting fragment ions are scanned out in the low-pressure ion trap at the normal scan rate and recorded with the secondary electron multipliers. Ion selection threshold is 500 counts for MS/MS, and the maximum allowed ion accumulation times were 500 ms for full scans and 100 ms for CID-MS/MS measurements in the LTQ. An activation q of 0.25 and an activation time of 10 ms are used.

Standard mass spectrometric conditions are set as follow: spray voltage, 1.8 kV; no sheath or auxiliary gas flow; heated capillary temperature, 200 °C; predictive automatic gain control (AGC) enabled, and an S-lens RF level of 50%. For all full scan measurements with the Orbitrap detector a lock-mass ion from ambient air (m/z 391.284286) is used as an internal calibrant. Selected ion monitoring injection of the lock mass is deactivated in order to save time. However, when present the lock mass is still used for real time correction of the mass scale.

Comparison of the changes in metabolic pathways in control and experimental shrimp

MetaCore (GeneGO) software uses the protein profiles of all the samples to generate correlation networks of the host protein. Protein expression ratios (log2 transformed) between the control and experimental groups are calculated and pathways of interest are highlighted.