title: Label-free liquid chromatography-tandem mass spectrometry analysis with automated phosphopeptide enrichment reveals dynamic human milk protein phosphorylation during lactation.
abstract: Protein phosphorylation is a critical posttranslational modification that affects cell-cell signaling and protein function. However, quantifying the relative site-specific changes of phosphorylation occupancies remains a major issue. An online enrichment of phosphopeptides using titanium dioxide incorporated in a microchip liquid chromatography device was used to analyze trypsin-digested human milk proteins with mass spectrometry. The method was validated with standards and used to determine the dynamic behavior of protein phosphorylation in human milk from the first month of lactation. α-Casein, β-casein, osteopontin, and chordin-like protein 2 phosphoproteins were shown to vary during this lactation time in an independent manner. In addition, changes in specific regions of these phosphoproteins were found to vary independently. Novel phosphorylation sites were discovered for chordin-like protein 2, α-lactalbumin, β-1,4-galactosyl transferase, and poly-Ig (immunoglobulin) receptor. Coefficients of variation for the quantitation were comparable to those in other contemporary approaches using isotopically labeled peptides, with a median value of 11% for all phosphopeptide occupancies quantified.
authors.affiliation: Department of Chemistry, University of California, Davis, 95616, USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Froehlich
authors.forename: Caroline S
authors.initials: CS
authors.lastname: Chu
authors.forename: Ning
authors.initials: N
authors.lastname: Tang
authors.forename: Keith
authors.initials: K
authors.lastname: Waddell
authors.forename: Rudolf
authors.initials: R
authors.lastname: Grimm
authors.forename: Carlito B
authors.initials: CB
authors.lastname: Lebrilla
mesh_terms: D001331:Automation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry; D007774:Lactation; D008894:Milk Proteins / Q000737:chemistry*; D008895:Milk, Human / Q000737:chemistry*; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014025:Titanium / Q000737:chemistry
chemical_list: D008894:Milk Proteins; D010748:Phosphopeptides; C009495:titanium dioxide; D007768:Lactalbumin; D014025:Titanium
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