The case study was approved by the Institutional Review Board (IRB) of the National Cheng Kung University Hospital (NCKUH) (A-ER-103–395, B-ER-110–342, and B-ER-110–418), and the healthy control study was approved by the IRB of NCKUH (B-ER-110–442). The study was conducted in accordance with the Declaration of Helsinki. We used LC‒MS for amino acid and related amine analysis and nuclear magnetic resonance (NMR) for nonamine metabolite analysis. Plasma was collected from a patient with MSI cancer and healthy control subjects. Protein precipitation using methanol was carried out as described by Gowda [30 (link)]. NMR experiments were conducted at 298 K on a Bruker Avance III 600 MHz spectrometer (Billerica, MA, USA) equipped with a triple-inverse probe and a Z-gradient. CPMG (Carr − Purcell − Meiboom − Gill) pulse sequences and presaturation for water suppression were used for 1H 1D NMR experiments. For the LC‒MS-based metabolomics study, the amino acid derivatives were prepared according to the methods described in the Kairos™ amino acid kit manual of Waters™ (Milford, MA, USA). The precipitated samples were derivatized using the AccQ Tag™ Derivatization kit (Waters Corporation). The LC‒MS system consisted of an ACQUITY® UPLC® H-Class Plus System (Waters Corporation) and an ACQUITY® QDa® Mass Detector (mass spectrometry detector; Waters Corporation) equipped with an electrospray ionization interface. Ultra-performance liquid chromatography‒mass spectrometry (UPLC-QDa, UPLC‒MS) was used for analysis. A CORTECS® UPLC® C18 column (2.1 mm × 150 mm, 1.6 μm particle size) was used for compound separation. Information regarding the identified metabolites was confirmed in our preliminary results by matching the LC‒MS or NMR information with the analysis of various metabolites of the internal standard.
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