Highlights • Single-pot workflow for manual or automated enrichment of N-terminal peptides. • Sensitive enrichment of protein N termini from 10,000 cells or 2 g crude proteome. • Data independent acquisition improves precision of peptide level quantification. • First degradomic analyses of sorted immune cells, single seedlings, and mitochondria from patient cells.

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We previously reported that the expression of KIAA1199 in human colorectal tumors (benign and malignant) is markedly higher than that in the normal colonic mucosa. In this study, we investigated the functions of the protein encoded by this gene, which are thus far unknown. Immunostaining studies were used to reveal its subcellular localization, and proteomic and gene expression experiments were conducted to identify proteins that might interact with KIAA1199 and molecular pathways in which it might play roles. Using colon cancer cell lines, we showed that both endogenous and ectopically expressed KIAA1199 is secreted into the extracellular environment. In the cells, it was found mainly in the perinuclear space (probably the ER) and cell membrane. Both cellular compartments were also over-represented in lists of proteins identified by mass spectrometry as putative KIAA1199 interactors and/or proteins encoded by genes whose transcription was significantly changed by KIAA1199 expression. These proteomic and transcriptomic datasets concordantly link KIAA1199 to several genes/proteins and molecular pathways, including ER processes like protein binding, transport, and folding; and Ca2+, G-protein, ephrin, and Wnt signaling. Immunoprecipitation experiments confirmed KIAA1199’s interaction with the cell-membrane receptor ephrin A2 and with the ER receptor ITPR3, a key player in Ca2+ signaling. By modulating Ca2+ signaling, KIAA1199 could affect different branches of the Wnt network. Our findings suggest it may negatively regulate the Wnt/CTNNB1 signaling, and its expression is associated with decreased cell proliferation and invasiveness.

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