Gels were fixed for 1 h in 40% methanol/10% acetic acid, followed by staining for 2 h with Coomassie Brilliant Blue (CBB) G-250

Gels were fixed for 1 h in 40% methanol/10% acetic acid, followed by staining for 2 h with Coomassie Brilliant Blue (CBB) G-250. band (proline-rich proteinsPRPs) increased only after yoghurt intake. Walnuts snack was the one resulting in lower changes, probably due to lower amounts eaten. Even so, it resulted in the increase in one PRPs band. In conclusion, changes in saliva composition varies with foods, with variable changes in proteins related to oral food processing and belief. 0.05; Two-way ANOVA; Bonferrronis post-test); 1 Salivary amylase enzymatic activity. 2.2. Salivary Protein Profile SDS PAGE protein separation allowed the observation of 11 bands, consistently present in the several profiles (Physique 2). The tryptic digestion, followed by mass spectrometry, resulted in the identification of 8 different proteins (Table 2). Open in a separate window Astilbin Physique 2 SDS-PAGE profiles representative of saliva collected before and after the ingestion of the four snacks studied. Bands A and G decreased immediately after ingestion (0) of snacks (red arrow); Band B decreased immediately after yoghurt and increased immediately after apple intake (white arrowhead); Bands J + K increased immediately Astilbin after the intake of all foods except walnuts (white arrows). Table 2 Mass spectrometry Astilbin identification of salivary proteins present in the SDS-PAGE bands. 4 C for 20 min, to precipitate insoluble material and recover homogeneous liquid samples. 4.3.1. Salivary Flow Rate, Total Protein Concentration and Alpha Amylase Activity Saliva flow rate was assessed by assuming that saliva density is usually 1.0. The weight of empty tubes was subtracted from the weight of the tubes made up of saliva and the final value was divided by 4 (minutes of collection) to obtain the secretion rate (mL/min). Total protein concentration was determined by the Bradford method, using bovine serum albumin (BSA) as standard, and plates were read at 600 nm in a microplate reader (Glomax, Promega, Madison, WI, USA). For salivary amylase enzymatic activity quantification, a Salimetrics? kit was used according to the manufacturers recommendations, as previously described and using saliva samples diluted 200X [20]. Absorbance values were read at 405 nm in a plate reader spectrophotometer (BioRad, Hercules, CA, USA), at two time points and the enzymatic activity of amylase (U/mL) was calculated. Astilbin 4.3.2. SDS-PAGE Salivary Protein Separation Each saliva sample was run in duplicate. For each sample, a volume corresponding to 6.5 g total protein was mixed with sample buffer and run on each lane of a 14% polyacrylamide mini-gel (Protean xi, Bio-Rad, CA, USA) using a Laemmli buffer system, as described elsewhere [19]. An electrophoretic run was performed at a constant voltage of 140 V until front dye reached the end of the gel. Gels were fixed for 1 PTPRC h in 40% methanol/10% acetic acid, followed by staining for 2 h with Coomassie Brilliant Blue (CBB) G-250. Gel images were acquired using a scanning Molecular Dynamics densitometer with internal calibration and LabScan software (GE Healthcare, Chicago, IL, USA), and images were analyzed using GelAnalyzer software (GelAnalyzer 2010a by Istvan Lazar, www.gelanalyzer.com, assessed on February 2020) for the normalized volume (volume percentage) of each protein band. Molecular masses were determined in accordance with molecular mass standards (Bio-Rad Precision Plus Protein Dual Colour 161C0394) run with protein samples. 4.3.3. Protein Identification by Mass Spectrometry Bands of interest were manually excised from gels and proteins were in-gel digested following a protocol previously described [13]. To identify target proteins, peptide mixtures were analyzed by MALDI- FTICR-MS in a Bruker Apex Ultra, Apollo II combi-source (Bruker Daltonics, Bremen, Germany), with a 7 Tesla magnet (Magnex corpora- tion, Oxford, UK). After samples were desalted and concentrated, using reverse phase Poros R2 (Applied Biosystems, Foster City, CA, USA), they were eluted directly to the MALDI target AnchorChip (BrukerDaltonics, Bremen, Germany) with a-cyano-4-hydroxycinnamic acid (CHCA; Fluka, Buchs, Switzerland) matrix, prepared at a concentration of 10 lg/ll in 50% ACN with 0.1% TFA. Monoisotopic peptide masses were decided using the SNAP 2 algorithm in Astilbin Data Analysis software version 3.4 (BrukerDalton- ics, Bremen, Germany). External calibration was performed using the BSA tryptic digest spectrum, processed and analyzed with Biotools 3.1 (BrukerDaltonics, Bremen, Germany). Monoisotopic peptide masses were used to search for protein identification with.