Abstract
An extensive mass spectrometry analysis of the human milk peptidome has revealed almost 700 endogenous peptides from 30 different proteins. Two in-house computational tools were created and used to visualize and interpret the data through both alignment of the peptide quasi-molecular ion intensities and estimation of the differential enzyme participation. These results reveal that the endogenous proteolytic activity in the mammary gland is remarkably specific and well conserved. Certain proteins-not necessarily the most abundant ones-are digested by the proteases present in milk, yielding endogenous peptides from selected regions. Our results strongly suggest that factors such as the presence of specific proteases, the position and concentration of cleavage sites, and, more important, the intrinsic disorder of segments of the protein drive this proteolytic specificity in the mammary gland. As a consequence of this selective hydrolysis, proteins that typically need to be cleaved at specific positions in order to exert their activity are properly digested, and bioactive peptides encoded in certain protein sequences are released. Proteins that must remain intact in order to maintain their activity in the mammary gland or in the neonatal gastrointestinal tract are unaffected by the hydrolytic environment present in milk. These results provide insight into the intrinsic structural mechanisms that facilitate the selectivity of the endogenous milk protease activity and might be useful to those studying the peptidomes of other biofluids.
Highlights
From the ‡Department of Chemistry, University of California at Davis, One Shields Avenue, Davis, California 95616; ¶Department of Food Science, University of California at Davis, One Shields Avenue, Davis, California 95616; ʈFoods for Health Institute, University of California at Davis, One Shields Avenue, Davis, California 95616
This proteolytic pattern is explained as a result of the action of endopeptidases cleaving in specific protein regions and the subsequent partial degradation of these initial fragments by exopeptidases [18]
Mechanistic Peptidomics of Human Milk last factor is related to the accessibility of the enzyme to the cleaving site, and it is commonly accepted that proteolysis happens in solvent-exposed, flexible substrate regions [22, 23]
Summary
Chemicals and Sample Set—Acetonitrile, formic acid, and trifluoroacetic acid were obtained from Thermo Fisher Scientific (Waltham, MA), and trichloroacetic acid was obtained from EMD Millipore (Darmstadt, Germany). Mass Spectrometry Analysis—Samples were analyzed in positive mode on an Agilent (Santa Clara, CA) nano-LC-chip-Q-TOF-MS/MS instrument (Chip-Q-TOF) with a chip C18 column at a flow rate of 0.3 l/min. Each sample was analyzed in the MS/MS mode using the iterative exclusion lists method four times. This methodology helps the instrument to fragment peaks of lesser abundance, which in turn allows deeper exploration of the samples. Protein Quantification—An aliquot of 5 l from each human milk sample was used to determine the protein content using the BCA method [41]. The protein content was determined by means of interpolation of the absorbance values with the bovine serum albumin standard curve
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