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Human milk and gastric peptidomics
It is unclear to what degree protein degradation occurs in the infant stomach and whether peptides previously annotated for bioactivity are released. This study combined nanospray liquid chromatography separation with time of flight mass spectrometry, comprehensive structural libraries and informatics to interrogate milk of three human mothers and the gastric aspirates from their 4–12 day post-partum infants. Milk from the mothers contained almost two hundred distinct peptides, demonstrating enzymatic degradation of milk proteins beginning either during lactation or between milk collection and feeding. In the gastric samples, 649 milk peptides were identified, demonstrating that digestion continues in the infant stomach. The majority of peptides in both the intact milk and gastric samples were derived from β-casein. The numbers of peptides from β-casein, lactoferrin, α-lactalbumin, lactadherin, κ-casein, serum albumin, bile-salt associated lipase and xanthine dehydrogenase/oxidase were significantly higher in the gastric samples than the milk samples (p<0.05). Six hundred three peptides were significantly different in abundance between milk and gastric samples (p<0.05). Most of the identified peptides have previously identified biological activity. Gastric proteolysis occurs in the term infant in the first two weeks of life releasing biologically active milk peptides with immunomodulatory, antibacterial, and calcium-binding activity of clinical relevance to the proximal intestinal tract.
Sample Processing Protocol
Materials. Acetonitrile (ACN)9, formic acid (FA) and trifluoroacetic acid (TFA) were obtained from Thermo Fisher Scientific (Waltham, MA) and trichloroacetic acid (TCA) from EMD Millipore (Darmstadt, Germany). All water used was nanopure (18.2 Ohm). Subjects and samples. This study was approved by the Institutional Review Board of the University of California Davis. Following informed consent, intact milk samples were obtained from three healthy mothers who delivered at term (see Table 1 for infant metadata). The breast was cleansed with water on a wash cloth (no soap or alcohol) and then the milk samples were collected by an electric pump into a sterile plastic container. Samples from both breasts were combined and then frozen in a home freezer and transported on ice to the Neonatal Intensive Care Unit at UC Davis Children’s Hospital and kept frozen at -20°C. Time of day of pumping was not specified or recorded. The three term infants were hospitalized due to health problems unrelated to the gastrointestinal tract (Table 1). Two of the infants received intravenous antibiotics prior to sample collection which may alter the gastric microbiota. None of the babies received oral antibiotics or acid-suppressing agents. The infants’ conditions precluded normal feeding, therefore, a naso-gastric tube was placed for each. The milk samples were thawed in warm (not hot) water and stored in a refrigerator at 4°C until feeding. Just prior to feeding, an aliquot of milk was re-frozen at -40°C. The unfortified milk was fed via the naso-gastric tube over 30 minutes. Two hours after the initiation of the feeding, a fraction of the gastric contents was collected back through the tube via suction for each infant and stored at –40°C. Milk and gastric acid samples were transported to the UC Davis laboratory on dry ice and stored at –80°C until analysis. Sample preparation. Samples were removed from the freezer and thawed on ice. Then, they were vortexed for 1 min each. Peptides were extracted, with the following modifications. A 100 μL sample of mother’s expressed breast milk and 100 μL of infant’s gastric fluid were used. To each sample, 100 μL of nanopure water were added, and the samples were again vortexed. The samples were then incubated at 100°C to prevent any further enzyme-driven proteolysis. Briefly, samples were delipidated by centrifugation and proteins in the infranate samples were precipitated with 1:1 (sample to solution) 200 g/L TCA, followed by plate vortexing, centrifugation and removal of the supernatant. Supernatants were applied to 96-well C18 solid-phase extraction plate to purify peptides. Eluted peptides were dried by vacuum centrifugation and rehydrated in 60 μL nanopure water for quadrupole time-of-flight (Q-TOF) analysis. Two microliters of 0.1 μg/μL peptide standards containing equal parts Leu-enkephalin, gonadoliberin, angiotensin I and neurotensin (ProteoChem, Loves Park, IL, USA) were added to map retention time reproducibility of the samples.
Data Processing Protocol
Peptide analysis. The analysis was performed with the Agilent nano-liquid chromatography chip-cube 6520 quadrupole time-of-flight tandem mass spectrometer (Santa Clara, CA, USA). The chip employed contained an enrichment and analytical column packed with a C18 stationary phase. . Briefly, the gradient elution solvents were (A) 3% ACN/0.1% FA and (B) 90% ACN/0.1% FA. The gradient employed was ramped from 0–8% B from 0–5 min, 8–26.5% B from 5–24 min, 26.5–100% B from 24–48 min, followed by 100% B for 2 min and 100% A for 10 min (to re-equilibrate the column). Each sample was run in triplicate on the Q-TOF in MS-only mode. Creation of peptide library. To create a peptide library, these gastric and intact samples for term-delivered infants were run using the same method. Data were exported to Mascot Generic Format (.mgf) and analyzed via the downloadable version of X!Tandem (thegpm.org) against a library of human milk proteins derived from previous human milk proteomes. Briefly, no complete modifications were required, but possible phosphorylations of serine, threonine or tyrosine, deamidation of glutamine or asparagine, and oxidation of methionine or tryptophan were allowed. Error tolerances employed were 20 ppm for precursor masses and 40 ppm for fragment masses. The results from X!Tandem for all samples were compiled for milk samples and for gastric samples, separately. Peptides with e-values ≤ 0.01 were removed (a 99% confidence level threshold). To isolate only unique peptide sequences, all duplicates of sequence, protein and modifications combined were eliminated with the “remove duplicates” function in Excel. Peptides representing identical amino acid sequences and modifications but modified in different positions were also removed as duplicates. The compiled peptide libraries were then used to make an exclusion list for recursive analysis. Recursive analysis was repeated until the number of new peptides returned for each sample was ≤10. Three rounds of recursive analysis were completed for the milk samples (for a total of four MS/MS runs for each). Five rounds were completed for two of the gastric samples (for a total of six MS/MS runs for each). One gastric sample with retention times repeatedly different than the other two gastric samples was examined alone—and required ten rounds of recursive analysis before the number of new peptides returned was ≤10. Peptides were identified via the above procedure and the results from the recursive analysis were added to the original peptide results. Peptide identification. Compounds were identified from all samples by the “Batch Targeted Feature Extraction” function in Agilent MassHunter Profinder B.06.00. The data were searched against the peptide library based on the molecular formula. One library generated from milk peptide library was used to extract milk sample peptide peak areas and two different libraries were used to extract the gastric peptides (as one sample had consistently different retention times). The milk peptide library could not be applied to the gastric samples because they showed retention time shifts in comparison with the milk peptides, likely due to molecular interactions within the sample matrices. For library searching, the following parameters were employed. The maximum number of matches per formula was 1. Peaks were matched on both mass (within 20 ppm error) and retention time (within 1 min error). A height threshold of 500 ion counts was employed. The charge carrier was protons, and the allowed charge states were 1-7. The isotope model was “peptides.” Extracted ion chromatogram peaks were smoothed with the Gaussian function and the resulting peaks were integrated via the “agile” algorithm. After compounds were extracted, each peak was manually inspected for peak integration and any incorrect assignments were corrected within the Profinder program.
Dallas DC, Guerrero A, Khaldi N, Borghese R, Bhandari A, Underwood MA, Lebrilla CB, German JB, Barile D. A Peptidomic Analysis of Human Milk Digestion in the Infant Stomach Reveals Protein-Specific Degradation Patterns. J Nutr. 2014 Apr 3 PubMed(s) : 24699806
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||34656||milk vs. gastric peptidomics||13||194||107||956||174||
|2||34657||milk vs. gastric peptidomics||15||192||115||997||178||
|3||34654||milk vs. gastric peptidomics||5||49||21||967||29||
|4||34655||milk vs. gastric peptidomics||5||59||32||1062||49||
|5||34652||milk vs. gastric peptidomics||4||63||22||499||55||
|6||34653||milk vs. gastric peptidomics||6||67||33||367||64||
|7||34650||milk vs. gastric peptidomics||18||149||83||904||118||
|8||34651||milk vs. gastric peptidomics||20||276||140||949||236||
|9||34675||milk vs. gastric peptidomics||17||196||107||921||178||
|10||34674||milk vs. gastric peptidomics||8||63||41||973||62||