Bio safe coomassie stain

Following 2DE, gels were stained with BioSafe Coomassie Stain (Bio-Rad). In preparation, gels were soaked in fixing solution (50% methanol, 5% acetic acid) for 30 min, stained with BioSafe Coomassie Stain for 1 hr, and destained in double distilled water at 4°C overnight. Gels were scanned and individual spots chosen for mass spectrometric analysis were cut from the gel and digested with trypsin as previously described [22 (link)]. Briefly, multiple cycles of dehydration in 50% acetonitrile followed by rehydration in 50 mM NH₄HCO₃ was done to remove the SDS and protein stains from the gel pieces. Following the last wash and dehydration cycle, gel pieces were rehydrated in 50 mM NH₄HCO₃ with 12.5 ng/μl trypsin (sequencing grade) and incubated for 45 min on ice followed by 37°C overnight. Digested peptides were extracted from the gel pieces with 25 mM NH₄HCO₃, acetonitrile, and 10% formic acid and dried by vacuum centrifugation.

Protein concentrations of the pooled protein samples were confirmed, and equal amounts of protein were denatured by boiling in NuPAGE®LDS Sample Buffer (Invitrogen, Carlsbad, CA) for 10 min at 70°C. Denatured lysates were loaded in triplicate (15 μg per lane) and resolved on a 4–12% Bis-Tris Gel (Figure 1B) in MOPS running buffer (Invitrogen, Carlsbad, CA). Gels were fixed with 50% methanol in 10% acetic acid, washed in ultra-pure water, then stained with Bio-Safe Coomassie Stain (Bio-Rad, Hercules, CA) per manufacturer's instructions. Gel lanes were then divided into 5 evenly spaced horizontal regions using protein bands that were common to all samples as guides. The middle half of each sample lane was cut length-wise. Gel sections were digested with trypsin following a previously described protocol (Hogan et al., 2015 (link)). In brief, each gel section was de-stained, reduced with dithiothreitol and alkylated with iodoacetamide. Proteins were digested overnight at 37°C with 140 ng of trypsin dissolved in 25 mM Tris (pH 8.2). Peptides were extracted from the gel piece with 50% acetonitrile (ACN) in 4% trifluoroacetic acid (TFA), followed by two additional extractions with ACN. The combined extracts were evaporated to dryness on a vacuum concentrator and stored at −80°C until further analysis.

Blue shark (Prionace glauca) skins were removed from cold-stored fish body parts in an industrial plant and kindly provided by Vitorino & Filhos, Lda (Peniche, Portugal). Skins were washed with ice-cold deionized water and stored frozen at a temperature of −20°C until use. Acetic acid (99.7%) and citric acid were purchased from Panreac (Germany). Xylitol, commercial Type I collagen from calf skin and ß-mercaptoethanol were supplied by Sigma-Aldrich (United States). The protein ladder Precision Plus Protein™ All Blue Prestained Protein Standard, Laemmli Sample Buffer, Tris/Glycine/SDS Running Buffer, and Bio-Safe™ Coomassie Stain were purchased from Bio-Rad (United States). Minimum essential medium (MEM) with Earle′s balanced salts and 2.0 mM l-glutamine, phosphate-buffered saline (PBS), pH = 7.4, and dimethyl sulfoxide (DMSO) were purchased from Sigma (United States). Non-essential amino acids (NEAA), fetal bovine serum (FBS) and 0.25% (w/v) Trypsin-EDTA were purchased from Gibco (Life Technologies, United States). CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (MTS) reagent assay was obtained from Promega (Madison, United States).