Computational protocol: Dataset of milk whey proteins of three indigenous Greek sheep breeds

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Protocol publication

[…] Protein extraction and peptide generation, was performed as described by our group elsewhere, with few modifications , . In brief, 200 ng whey fractions were treated with urea and triethyl ammonium bicarbonate (TEAB) under mild sonication in a water-bath for 30 min and reduction and alkylation steps of proteins were carried out using dithiothreitol and iodoacetamide solutions, The final step of processing included overnight digestion of extracted proteins of all samples by trypsin (Roche Diagnostics, Basel, Swiss) at a final concentration of 500 ng/μl.Digested samples were analyzed using a LTQ Orbitrap Elite coupled to a Dionex 3000 nanoHPLC system (Thermo Scientific, Rockford, IL, USA). LC separation of peptides took place on two Thermo Scientific columns (PepMap® RSLC, C18, 100 Å, 3 μm-bead-packed 15 cm column and 2 μm-bead-packed 50 cm column) at a flow rate of 3 nL/min. The mobile phases A and B were 0.1% formic acid in water and 99% ACN in water, respectively. The gradient elution profile was as follows: 2.0% B (98.0% A) for 10 min, 2.0–35.0% B (98.0–65.0% A) for 325 min, 80.0% B (20.0% A) for 10 min, 2.0% B (98.0% A) for 10 min. Data were collected in the data-dependent MS/MS mode using a standard top-20 method. Full-scan data were acquired at a resolving power of 60,000 with a maximum integration time of 250 ms. Scan range was fixed at 250–1250 m/z and peptide fragmentation was performed in a higher-energy collision dissociation (HCD) mode with a normalized collision energy of 36%. MS/MS spectra were acquired with 15,000 resolving power and a maximum integration time of 120 ms. Measurements were performed using m/z 445.120025 as lock mass. Dynamic exclusion settings were set to repeat count 1, repeat duration 30 s, exclusion duration 120 s, and exclusion mass width 0.6 m/z (low) and 1.6 m/z (high).The *.raw data files were analyzed using the Proteome Discoverer software (Thermo Scientific), using the Sequest search engine applying the Ovis aries for milk of sheep *.fasta databases. MS/MS searches were performed using a 10 ppm parent ion mass tolerance and a 0.05 fragment mass tolerance. Trypsin was selected as the cleavage enzyme with up to 2 missed cleavage points. Cysteine methylthio modification was selected as a fixed modification and oxidation of methionine were selected as a variable. Peptide identifications were considered valid at 1% False Discovery Rate (q-value<0.01) (percolator maximum Delta Cn was 0.05). The minimum length of acceptable identified peptides was set as 6 amino acids. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet, Percolator
Application MS-based untargeted proteomics
Organisms Ovis aries, Capra hircus