Milk Peptidomics to Identify Functional Peptides and for Quality Control of Dairy Products

Abstract

Human milk and dairy products are important parts of human nutrition. In addition to supplying ­nutrients, milk proteins contain fragments—peptides—with important biological functions that are released during processing or digestion. Besides their potential functional relevance, peptides released during processing can be used as markers of ripening stage or product deterioration. Hence, identification and quantification of peptides in milk can be used to assay potential health benefits or product quality. This chapter describes how to extract, identify, and analyze peptides within breast milk, dairy products, and dairy digestive ­samples. We describe how to analyze extracted peptides with liquid chromatography-mass spectrometry, to use software to identify peptides based on database searching, and to extract peak areas for relative quantification of each peptide. We describe methods for data analysis, including predicting which enzymes are responsible for protein cleavage, identifying the site specificity of protein breakdown, mapping ­identified peptides to known bioactive peptides, and applying models to predict novel functional peptides.

Comprehensive Profiling of the Native and Modified Peptidomes of Raw Bovine Milk and Processed Milk Products

“…Therefore, we applied this procedure to extract endogenous peptides from RM, UHT milk, and IF. Confident peptide identification was aimed for by utilizing two fragmentation techniques (i.e., CID and ETD) in DDA mode, processing the data with two software tools (i.e., Proteome Discoverer 2.2 and PEAKS Studio 10.5), and validation with a spectral library generated within Skyline as previously described by Dallas and Nielsen [ 33 ], which allowed confirming the presence of proposed peptides among all analyzed samples ( Figure S1 ). To identify more modified peptides detected with low intensities, previously identified unmodified peptides were excluded from the second analysis of each milk sample.…”

“…Peptides identified in at least two of the three replicates by both software tools were considered for further processing ( Figure S1 ). Their presence in all measured samples was confirmed with Skyline (20.1.0.155, MacCoss Lab, Department of Genome Sciences, University of Washington) by generating a spectral library [ 33 ] and adjusting the parameters for the instrument used. Peptides were considered present if the precursor was detected at the same retention time and the isotope dot-product value (idotp) was above 0.95 in at least two individual replicates.…”

Release of functional peptides from mother's milk and fortifier proteins in the premature infant stomach

“…Spectra were analyzed by database searching in Thermo Proteome Discoverer (v2.1.0.81) using an in-house human and bovine milk protein sequence database as previously described in detail [35]. Potential modifications allowed included phosphorylation of serine and threonine and oxidation of methionine.…”

Hidden “Digestome”: Current Analytical Approaches Provide Incomplete Peptide Inventories of Food Digests

“…These findings highlight the need of standardized peptidomic protocols for the identification of functional peptides, which are currently under development …”

“…These findings highlight the need of standardized peptidomic protocols for the identification of functional peptides, which are currently under development. 30 Identification of Disulfide-Linked Cross-Linked Peptides. Major native bovine whey proteins (including α-La, β-Lg, and BSA) contain disulfide-linked cysteines, which, upon digestion, are likely to generate cross-linked peptide heterodimers or even concatenated multimers in the case of α-La.…”