Abstract
Until now, cheese peptidomics approaches have been criticised for their lower throughput. Namely, analytical gradients that are most commonly used for mass spectrometric detection are usually over 60 or even 120 min. We developed a cheese peptide mapping method using nano ultra-high-performance chromatography data-independent acquisition high-resolution mass spectrometry (nanoUHPLC-DIA-HRMS) with a chromatographic gradient of 40 min. The 40 min gradient did not show any sign of compromise in milk protein coverage compared to 60 and 120 min methods, providing the next step towards achieving higher-throughput analysis. Top 150 most abundant peptides passing selection criteria across all samples were cross-referenced with work from other publications and a good correlation between the results was found. To achieve even faster sample turnaround enhanced DIA methods should be considered for future peptidomics applications.
Highlights
During cheese ripening, caseins undergo a progressive breakdown by enzymatic action, releasing peptides and amino acids, which contributes to the development of cheese flavour and texture [1].The term “peptidomics” has been extensively used in dairy science for comprehensive analysis of peptides released during proteolysis in different cheese varieties [2,3,4] as well as characterisation of bioactive peptides with potential nutritional and health-promoting effects [5,6]
The key analytical tool employed in cheese peptidome research as well as exploring the possibilities of controlling the cheese maturation process, is currently mass spectrometry (MS) coupled with liquid chromatography (LC) [12,13,14]
Across all samples analysed with either Data-independent acquisition (DIA)- or dependent acquisition (DDA)-based approaches, peptides from αs1 -CN
Summary
Caseins undergo a progressive breakdown by enzymatic action, releasing peptides and amino acids, which contributes to the development of cheese flavour and texture [1].The term “peptidomics” has been extensively used in dairy science for comprehensive analysis of peptides released during proteolysis in different cheese varieties [2,3,4] as well as characterisation of bioactive peptides with potential nutritional and health-promoting effects [5,6]. Caseins undergo a progressive breakdown by enzymatic action, releasing peptides and amino acids, which contributes to the development of cheese flavour and texture [1]. Several researchers have been focusing on the identification of phosphorylated peptides [7] and the determination of specific bitter peptides and their contribution to cheese flavour [8,9]. The key analytical tool employed in cheese peptidome research (i.e., increasing the knowledge of proteolytic events occurring during ripening) as well as exploring the possibilities of controlling the cheese maturation process, is currently mass spectrometry (MS) coupled with liquid chromatography (LC) [12,13,14]. The most widely used hyphenation is nano ultra (high) performance liquid chromatography (nanoUHPLC) coupled with high-resolution mass spectrometry (HRMS) based on data-dependent acquisition (DDA) mode [15,16,17]
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