Label-free liquid chromatography–tandem mass spectrometry analysis with automated phosphopeptide enrichment reveals dynamic human milk protein phosphorylation during lactation
Section snippets
Materials
All water used was generated by Milli-Q filtration and was measured as 18 MΩ water. The phosphopeptide kit, containing the phosphochip and necessary elution and regeneration solutions, was provided by Agilent Technologies (Santa Clara, CA, USA). Sequencing-grade modified trypsin was purchased from Promega (Madison, WI, USA). Formic acid (high-performance liquid chromatography [HPLC] grade), acetic acid (HPLC grade), dithiothreitol, iodoacetamide, and Tris buffer all were purchased from
Validation of the experimental approach
To validate the quantification strategy outlined above, a standard containing equimolar amounts of a BSA standard and FQsEEQQQTEDELQDK phosphopeptide from bovine β-casein was used. First, varying amounts of the standard were injected, and a standard curve of the intensity of the FQsEEQQQTEDELQDK phosphopeptide versus the amount injected was calculated. This plot possessed a high linearity (see Supplementary Fig. 1 in supplementary material). The MS intensity of each peptide identified was
Discussion
Tracking relative changes in protein phosphorylation of milk proteins is especially difficult because the protein amounts are known to vary during lactation. Therefore, a normalization scheme was implemented to correct for changes in protein expression. The total peptide intensity from each protein was used as a proxy for changes in protein expression. The CVs for this method after three replicate analyses were comparable to those in quantitation studies that use isotopically labeled peptides;
Conclusions
In this study, a label-free LC–MS/MS quantification method was applied to the study of human milk phosphorylation from an individual during the first month of lactation. This is the first known report of relative quantitation of human milk proteins during lactation. Because the expression levels of milk proteins are known to vary during the course of lactation, a normalization strategy based on total peptide signal was developed and incorporated into the quantitative method to elucidate changes
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2013, Journal of ProteomicsCitation Excerpt :Characterization of low abundance phosphoproteins requires selective enrichment. This can be achieved by immobilized metal or titanium dioxide affinity chromatography, applied either to intact proteins or to tryptic peptides, followed by protein identification and characterization by mass spectrometry [81]. In human milk this type of modification is very important because it potentially modulates the anti-microbial functions of milk and modifies its probiotic function.