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A universal peptide matrix interactomics approach to disclose motif dependent protein binding

ABSTRACT: Protein-protein interactions (PPIs) mediated by intrinsically disordered regions (IDRs) are often based on short linear motifs (SLiM). SLiMs are implicated in signal transduction and gene regulation, yet remain technically laborious and notoriously challenging to study. Here, we present an optimized method for a PRotein Interaction Screen on a peptide MAtrix (PRISMA) in combination with quantitative mass spectrometry. The protocol was benchmarked with previously described SLiM based PPIs using peptides derived from EGFR, SOS1, GLUT1 and CEBPB and extended to map binding partners of kinase activation loops. The detailed protocol provides practical considerations for setting up a PRISMA screen and subsequently implementing PRISMA on a liquid handling robotic platform as a cost effective high-throughput method. Optimized PRISMA can be universally applied to systematically study SLiM based interactions and associated post translational modifications (PTMs) or mutations to advance our understanding of the largely uncharacterized interactomes of intrinsically disordered protein regions.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Hela Cell

SUBMITTER: Evelyn Ramberger

LAB HEAD: Philipp Mertins

PROVIDER: PXD027265 | Pride | 2021-08-11

REPOSITORIES: Pride

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Project description:Alterations in glycosylation are seen in many types of cancer, including colorectal cancer (CRC). CRC is known to display glycosylation alterations. Glycans, the sugar moieties of glycoconjugates, are involved in many important functions relevant to cancer, such as cell signaling and adhesion, and may can be of value as biomarkers. In this study, we have used mass spectrometry to analyze the N-glycan profiles of 35 CRC tissue samples from patients with tumors in the right or left colon and 10 healthy tissue samples from non-CRC patients who underwent operations for other reasons. The tumor samples were divided into groups depending on tumor location (right or left colon) and stage (II or III), while the healthy samples were divided into right or left side of the colon. The levels of neutral and acidic N-glycan compositions and glycan classes were analyzed in a total of ten different groups. Surprisingly, there were no significant differences in glycan levels when all right- and left-sided CRC samples were compared, and few differences (such as in the abundance of the neutral N-glycan H3N5) were seen when the samples were divided according to both location and stage. Multiple significant differences were found in the levels of glycans and glycan classes when stage II and III samples were compared, and these glycans could be of value as candidates for new markers of cancer progression. In order to validate our findings, we analyzed healthy tissue samples from the right and left colon and found no significant differences in the levels of any of the glycans analyzed, confirming that our findings when comparing CRC samples from the right and left colon are not due to normal variations in the levels of glycans between the healthy right and left colon. Additionally, the levels of the acidic glycans H4N3F1P1, H5N4F1P1, and S1H5N4F1 were found to change in a cancer-specific but colon location-nonspecific manner, indicating that CRC affects glycan levels in similar ways, regardless of tumor location.

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A universal peptide matrix interactomics approach to disclose motif dependent protein binding

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