2.4.3. Protein Identification by Mass Spectrometry

Mass spectrometry (MS) analysis was performed by the Proteomics Platform of the Research Center of the *Centre Hospitalier Universitaire* (CHU) of Quebec City (QC, Canada). First, final hydrolysates (120 or 240 min of hydrolysis) generated by Alcalase® and pepsin in vitro digestion for each condition (control, pretreatment, and simultaneous conditions) were desalted on an Oasis HLB column (Waters, Mississauga, ON, Canada) and the peptides were quantified at 205 nm while using a nanodrop (ThermoFisher, Waltham, MA, USA). Peptide samples (1 µg) were analyzed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) on an Ekspert NanoLC425 (Eksigent, Redwood City, CA, USA) coupled to a 5600+ Triple TOF mass spectrometer (Sciex, Framingham, MA, USA) that was equipped with a nanoelectrospray ion source. The peptides were trapped on a pepmap 5 mm × 0.3 mm (ThermoFisher, Waltham, MA, USA) cartridge at 4 µL/min then separated on a self-packed picofrit column (New Objective, Woburn, MA, USA) with Reprosil 3 µL, (120A C18, 15 cm × 0.075 mm internal diameter), (Dr Maisch, Ammerbuch, Germany). The peptides were eluted with a linear gradient from 8–35% solvent B (acetonitrile, 0.1% V/V formic acid) in 30 min, at 300 ηL/min. Mass spectra were acquired using a data dependent acquisition mode and Analyst software version 1.7 (Sciex, Framingham, MA, USA). Each full scan mass spectrum (400 to 1,250 m/z) was followed by collision-induced dissociation of the twenty most intense ions. Dynamic exclusion was set for a period of 20 s and a tolerance of 100 ppm.

Mascot Generic Format (MGF) peak list files were created using Protein Pilot software (version 4.5, Sciex, Concord, ON, Canada). The MGF sample files were then analyzed using Mascot software (version 2.5.1, Matrix Science, London, UK). Uniprot databases were used to detect contaminants and align protein sequences while using the *Tenebrionidae* family (24,496 entries) database, assuming no enzyme. A fragment ion mass tolerance of 0.100 Da and a parent ion tolerance of 0.100 Da were used. The deamidation of asparagine and glutamine, and oxidation of methionine, were specified in Mascot as variable modifications.

Scaffold software (version 4.8.4, Proteome Software Inc., Portland, OR, USA) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at a probability greater than 95.0% by the Scaffold Local False Discovery Rate (FDR) algorithm. Protein identifications were also accepted if they could be established at a probability that is greater than 95.0% and if they contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm [30]. Proteins that contained similar peptides that could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
