Toward an efficient workflow for the analysis of the human milk peptidome

Kelly A Dingess,Henk W P Van Den Toorn,Albert J R Heck,Bernd Stahl,Marko Mank

doi:10.1007/s00216-018-01566-4

Abstract

There is a growing interest for investigating endogenous peptides from human biofluids which may provide yet unknown functional benefits or provide an early indication of disease states as potential biomarkers. A major technical bottleneck in the investigation of endogenous peptides from body fluids, e.g., serum, urine, saliva, and milk, is that each of these fluids seems to require unique workflows for peptide extraction and analysis. Thus, protocols optimized for serum cannot be directly translated to milk. One biofluid that is readily available, but which has not been extensively explored, is human milk, whose analysis could contribute to our understanding of the immune development of the newborn infant. Due to the occurrence of highly abundant lipids, proteins, and saccharides, milk peptidomics requires dedicated sample preparation steps. The aim of this study was to develop a time and cost-efficient workflow for the analysis of the human milk peptidome, for which we compared peptide extraction methodologies and peptide fragmentation methods. A method using strong acid protein precipitation and analysis by collision-induced dissociation fragmentation was found to be superior to all other test methods, allowing us qualitative and quantitative detection of about 4000 endogenous human milk peptides in a total analysis time of just 18 h.

Highlights

The study of endogenous biological peptides from tissue or biofluids was originally termed peptidomics by SchulzKnappe [1]
We argued that an optimized workflow would be the one that enables the identification of a high number of total and unique peptides across a large dynamic range in a relatively short amount of time
Peptidomics builds upon technologies being introduced for proteomics, the nature and origin of the endogenous peptides analyzed by petpidomics require several different and more specialized approaches

Summary

Introduction

The study of endogenous biological peptides from tissue or biofluids was originally termed peptidomics by SchulzKnappe [1]. Peptides are mostly defined as low molecular weight compounds (< 10 kDa) [3, 4] These peptides are associated with the proteome of the biofluids from which they originate. These peptides can exhibit distinctly different functions compared to their precursor proteins. They are mostly derived by specific proteases and can be further modified by posttranslational modifications (PTMs). Because of their size, endogenous peptides are found throughout the body, in both the intercellular and extracellular spaces, and potentially have unrestricted vascular and interstitial access [3]. Samples for the analysis of peptidomes from human biofluids, like plasma, saliva, and urine, are readily available and require less invasive sample collections, especially when compared to tissue biopsies

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