Quantification of FAM20A in human milk and identification of calcium metabolism proteins.

Vaksha Patel,Graciana Jaureguiberry,Markus Bleich,Naomi Issler,Enriko Klootwijk,Jasminka Godovac‐Zimmermann,Detlef Bockenhauer,Robert Kleta,Keith Siew,Stephen Walsh,Jun Wheeler,Gail Whiting,Nina Himmerkus

doi:10.14814/phy2.15150

Abstract

BackgroundFAM20A, a recently discovered protein, is thought to have a fundamental role in inhibiting ectopic calcification. Several studies have demonstrated that variants of FAM20A are causative for the rare autosomal recessive disorder, enamel‐renal syndrome (ERS). ERS is characterized by defective mineralization of dental enamel and nephrocalcinosis suggesting that FAM20A is an extracellular matrix protein, dysfunction of which causes calcification of the secretory epithelial tissues. FAM20A is a low‐abundant protein that is difficult to detect in biofluids such as blood, saliva, and urine. Thus, we speculated the abundance of FAM20A to be high in human milk, since the secretory epithelium of lactating mammary tissue is involved in the secretion of highly concentrated calcium. Therefore, the primary aim of this research is to describe the processes/methodology taken to quantify FAM20A in human milk and identify other proteins involved in calcium metabolism.MethodThis study used mass spectrometry‐driven quantitative proteomics: (1) to quantify FAM20A in human milk of three women and (2) to identify proteins associated with calcium regulation by bioinformatic analyses on whole and milk fat globule membrane fractions.ResultsShotgun MS/MS driven proteomics identified FAM20A in whole milk, and subsequent analysis using targeted proteomics also successfully quantified FAM20A in all samples. Combination of sample preparation, fractionation, and LC‐MS/MS proteomics analysis generated 136 proteins previously undiscovered in human milk; 21 of these appear to be associated with calcium metabolism.ConclusionUsing mass spectrometry‐driven proteomics, we successfully quantified FAM20A from transitional to mature milk and obtained a list of proteins involved in calcium metabolism. Furthermore, we show the value of using a combination of both shotgun and targeted driven proteomics for the identification of this low abundant protein in human milk.

Highlights

Human milk is produced by mammary epithelial cells (MEC)
A recent publication has identified FAM20A protein to be present in lactating but absent in non-lactating mammary glands (Cui et al, 2015), this suggested a potentially important role for FAM20A in lactation. This is further supported by data from the gene annotation portal, BioGPS which shows mRNA expression of FAM20A to be the highest in the lactating mammary gland compared to any other tissues in mouse. To investigate this further we aimed to quantify FAM20A in human milk and identify other proteins involved in calcium metabolism
To assess whether an absence of protease inhibitors (PI) in the samples influenced the identification and quantification of FAM20A, each of the three samples was divided into two aliquots

Summary

Introduction

Human milk is produced by mammary epithelial cells (MEC). The milk produced is comprised of three fractions: the MEC, whey, and milk fat globule membrane (MFGM). ERS is characterized by defective mineralization of dental enamel and nephrocalcinosis suggesting that FAM20A is an extracellular matrix protein, dysfunction of which causes calcification of the secretory epithelial tissues. We speculated the abundance of FAM20A to be high in human milk, since the secretory epithelium of lactating mammary tissue is involved in the secretion of highly concentrated calcium. Method: This study used mass spectrometry-driven quantitative proteomics: (1) to quantify FAM20A in human milk of three women and (2) to identify proteins associated with calcium regulation by bioinformatic analyses on whole and milk fat globule membrane fractions. Conclusion: Using mass spectrometry-driven proteomics, we successfully quantified FAM20A from transitional to mature milk and obtained a list of proteins involved in calcium metabolism. We show the value of using a combination of both shotgun and targeted driven proteomics for the identification of this low abundant protein in human milk

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