Online comprehensive hydrophilic interaction chromatography × reversed phase liquid chromatography coupled to mass spectrometry for in depth peptidomic profile of microalgae gastro-intestinal digests

In this study, a comprehensive hydrophilic interaction chromatography x reversed phase coupled to high resolution mass spectrometry was developed for the peptide profile of microalgae formulations subjected to gastro-intestinal digestion. A BEH Amide column was employed in the first dimension, while a BIOshell ES-C18 Peptide in the second. As modulation interface, two trapping columns, in house packed with 1.9 mu m fully porous monodisperse C18 particles characterized by high retention and efficiency, were tested and compared with SecurityGuard C18 cartridges, together with a dilution flow, to reduce first dimension mobile phase strength. The platform was coupled to both diode array detector and Orbitrap mass spectrometry. The developed setup provided high peak capacity (n(c): 957) in only 60 min and a good orthogonality (A(0): 0.70). The employment of the custom made C18 traps resulted in improved sensitivity (signal enhancement-4) and a higher number of peptides detected (+58) especially of short lenght (<= 6 aminoacids), with respect to the setup based on the security guard C18 traps. 184 phycocyanin-derived peptides were detected in Klamath and Spirulina gastro-intestinal digests, whose sequence and protein origin has been elucidated in detail by mass spectrometry. The results show the potential of the developed HILIC x RP-MS platform for in depth peptide mapping of microalgae and its possible application to highlight the products of gastro-intestinal digestion of other microalgae species. (C) 2019 Elsevier B.V. All rights reserved.