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

tIn this study, a comprehensive hydrophilic interaction chromatography × reversed phase coupled to highresolution mass spectrometry was developed for the peptide profile of microalgae formulations sub-jected to gastro-intestinal digestion. A BEH Amide column was employed in the first dimension, while aBIOshell ES-C18 Peptide in the second. As modulation interface, two trapping columns, in house packedwith 1.9 m fully porous monodisperse C18 particles characterized by high retention and efficiency, weretested and compared with SecurityGuard C18 cartridges, together with a dilution flow, to reduce firstdimension mobile phase strength. The platform was coupled to both diode array detector and Orbitrapmass spectrometry. The developed setup provided high peak capacity (nc: 957) in only 60 min and a goodorthogonality (A0: 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 (≤ 6aminoacids), with respect to the setup based on the security guard C18 traps. 184 phycocyanin-derivedpeptides were detected in Klamath and Spirulina gastro-intestinal digests, whose sequence and proteinorigin has been elucidated in detail by mass spectrometry. The results show the potential of the devel-oped HILIC × RP-MS platform for in depth peptide mapping of microalgae and its possible application tohighlight the products of gastro-intestinal digestion of other microalgae species.