Project PXD001826

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Kefir peptide profiling


This study examines the proteolytic activity of the kefir grains (a combination of bacteria and yeast) on bovine milk proteins. SDS-PAGE analysis reveals substantial digestion of milk proteins by the kefir grains in comparison with control samples. Mass spectrometric analysis reveals that the kefir microorganisms released 609 new peptide fragments and significantly altered the abundance of around 1,500 peptides compared to the controls. These kefir-digested peptides derived from 55 milk proteins. We show that kefir contains 25 previously identified functional peptides with actions including antihypertensive, antimicrobial, opioid and anti-oxidative .

Sample Processing Protocol

Sample preparation After thawing and gently mixing the raw milk, twelve 1 mL aliquots of milk were collected. Three aliquots were frozen at -20 oC to serve as the untreated standard (Raw Milk = RM). The nine remaining aliquots were pasteurized at 93 oC for 7 min using a thermomixer (Thermo Mixer C, Eppendorf, Hamburg, Germany). The samples were cooled in an ice bath for 20 min, and then brought to room temperature. To have a concentration of 4.5% of kefir grains, 0.0415 g of kefir grains were added in three of the nine samples (Kefir = K). The nine samples were incubated on a thermomixer at 23 oC for 24 h at 800 rpm and then matured at 4 oC for 24 h. During the incubation and maturation steps, the sample vials with kefir were kept open to match the aerobic conditions typical for kefir production. To control for any environmental contamination by air-borne microorganisms, three of the six tubes of pasteurized milk without kefir were closed (Pasteurized milk with closed tubes = PMc), whereas the remaining three were open (Pasteurized milk with open tubes = PMo). After defrosting the three samples of raw milk, the 12 samples were centrifuged at 16,000 xg at 4.5 oC for 10 min in order to separate the cream, solids and skim milk. 500 µL of skim milk was collected. TCA protein precipitation Proteins were precipitated from the skim milk samples by adding 200 g/L trichloroacetic acid (TCA) (EMD Millipore, Darmstadt, Germany) in a 1:1 volume ratio. Then, samples were centrifuged at 4,000 xg, at 20 oC for 10 min. Precipitates were removed and 850 µL of the supernatants from each sample were collected. Extraction of peptides with C18 microplate To remove sugars, salt and TCA from the supernatants, a C18 solid-phase extraction microplate procedure was employed as described previously [19].To remove sugars, salt and TCA from the supernatants, a C18 solid-phase extraction procedure was employed as described previously [19]. A solution of 99.9% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) (Thermo Fisher Scientific, Waltham, MA), followed by a solvent of 1% ACN, 0.1% TFA were added to rehydrate and equilibrate the column. The supernatants were then added. Six column volumes of 1% ACN, 0.1% TFA were added to wash off the sugars, salts and TCA. Three columns of 80% ACN, 0.1% TFA were added to elute the peptide fractions. All the steps of the microplate procedure were centrifuged at 208 xg for 30 s at 23 oC. Peptide fractions were dried by centrifugal evaporation (miVac Quattro, Genevac, Ipswich, UK) at 44 oC, and then preserved at -20 oC.

Data Processing Protocol

Spectral analysis Spectra were analyzed by database searching according to a published procedure [19, 20]. Briefly, data were exported in .mgf format, imported into the offline search engine X!Tandem [21] and searched against a bovine milk library compiled from previous bovine milk proteome literature [22]. The bovine milk protein library was imported in .fasta file format to X!Tandem. [21] and searched against a bovine milk library compiled from previous bovine milk proteome literature [22]. The bovine milk protein library was imported in FASTA file format to X!Tandem. Identified peptides were accepted if e-values were ≤0.01 corresponding to a confidence level of 99%. The peptide mass tolerance was set at 20 ppm for both the intact peptide mass and the fragment masses. No complete (required) modifications or potential modifications were allowed. A non-specific cleavage ([X]|[X]) (where ‘X’ is any amino acid) was used to search against the protein sequences. Because the instrument did not always select the monoisotopic ion for tandem fragmentation, isotope errors were allowed (allowing up to one C13). The data have been deposited to the ProteomeXchange [23] with identifier PXD001079. Peptide identification Our in-house curated bovine milk protein library was imported in .fasta file format into Skyline [24]. A library of identified peptides were uploaded from the .xml outputs of the X!Tandem program for each sample to create the spectral library. After applying all settings, the spectral library was searched against the raw data files (.raw) to extract the peaks for each peptide in each sample. The settings for this extraction are as follows. Unique peptides between 5 and 40 amino acids in length were retained. Precursor mass was calculated based on the monoisotopic ion. Precursor charges were 1–6. MS/MS ion charges were set at 1. Ion types were set as precursor only. The product ions were scanned from “ion 1” to “last ion”. The ion match tolerance was set to 0.5 m/z. “From filtered ion charges and types” was selected. The instrument acquisition window was set between 300 and 1,600 m/z with a tolerance of 0.055 m/z. For MS1 filtering, isotope peaks included by count were employed. The precursor mass analyzer was set to “Orbitrap”. The resolving power was set to 60,000 at 400 m/z. The precursor isotopic import filter was set to a count of 3 (M, M+1, M +2). MS/MS filtering was set to none. Retention time filtering was set to 1 min of the MS/MS IDs. After data import, all peaks were manually inspected for proper peak picking of the MS1-filtered peptides. Only peaks with ≤ 3 ppm mass error and an idotp score of ≥ 80 were only retained. Peaks were examined to make sure that the ratios of M to M+1 to M+2 were as expected based on the molecular weight of the molecule. Peaks were selected based on mass error and retention time proximity to the identified peptide retention time. Peaks that did not match these criteria were deleted. Peaks too close to the noise level to be visually discernable were excluded. After manual inspection, the data were exported to a .csv file. In order to reassign the protein name to peptide sequence and collapse multiple charge states into a single compound, the file was processed through an in-house script.


David Dallas, University of California, Davis
David Dallas, Department of Food Science and Technology University of California, Davis ( lab head )

Submission Date


Publication Date



    Dallas DC, Citerne F, Tian T, Silva VL, Kalanetra KM, Frese SA, Robinson RC, Mills DA, Barile D. Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins. Food Chem. 2016 Apr 15;197(Pt A):273-84 PubMed: 26616950


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# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 46521 kefir experiment 68 5819 1588 28945 3992
2 46532 kefir experiment 64 5409 1511 28855 3712
3 46530 kefir experiment 67 5991 1650 29961 3959
4 46531 kefir experiment 62 5645 1626 29803 3711
5 46528 kefir experiment 60 5487 1547 29954 3589
6 46527 kefir experiment 32 5511 1488 29322 4162
7 46529 kefir experiment 40 5385 1553 29256 4106
8 46524 kefir experiment 74 5461 1575 29388 3770
9 46523 kefir experiment 67 6402 1649 28957 4283
10 46526 kefir experiment 64 6251 1671 29591 4196