Elsevier

Analytical Biochemistry

Volume 408, Issue 1, 1 January 2011, Pages 136-146
Analytical Biochemistry

Label-free liquid chromatography–tandem mass spectrometry analysis with automated phosphopeptide enrichment reveals dynamic human milk protein phosphorylation during lactation

https://doi.org/10.1016/j.ab.2010.08.031Get rights and content

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.

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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