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Mapping the cytosolic proteins of HEK293T cells

ABSTRACT: N-terminal proteoforms stem from the same gene but differ at their N-terminus, and most of these are found to be truncated, though some are N-terminally extended caused by ribosomes starting translation from codons in the annotated 5’UTR, and/or carry modified N-termini different from those of the canonical protein. Biological functions of N-terminal proteoforms are emerging, however, it remains unknown to what extend N-terminal proteoforms further expand the functional complexity. To address this in a more global manner, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. For this, we first generated an in-depth catalogue of N-terminal proteoforms in the cytosol of HEK293T cells. In order to aid the selection of pairs of N-terminal proteoforms we generated a cytosolic map to verify their cytosolic interactions. This localization is crucial as Virotrap will be used for interactome analysis and this method works best for cytosolic proteins.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Annelies Bogaert

LAB HEAD: Kris Gevaert

PROVIDER: PXD039127 | Pride | 2023-06-08

REPOSITORIES: Pride

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			Action	DRS
	E21109_u_1p_CMB_492_1.raw	Raw
	E21111_u_1p_CMB_492_2.raw	Raw
	E21113_u_1p_CMB_492_3.raw	Raw
	E21115_u_1p_CMB_492_4.raw	Raw
	E21117_u_1p_CMB_492_5.raw	Raw

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Positional proteomics reveals differences in N-terminal proteoform stability

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Project description:N-terminal proteoforms stem from the same gene but differ at their N-terminus, and most of these are found to be truncated, though some are N-terminally extended caused by ribosomes starting translation from codons in the annotated 5’UTR, and/or carry modified N-termini different from those of the canonical protein. Biological functions of N-terminal proteoforms are emerging, however, it remains unknown to what extend N-terminal proteoforms further expand the functional complexity. To address this in a more global manner, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. For this, we first generated an in-depth catalogue of N-terminal proteoforms in the cytosol of HEK293T cells. From which 20 pairs of canonical protein and N-terminal proteoform(s) were selected. Protein-protein interaction profiling was done with Virotrap. Virotrap was selected as this methods avoids lysis before the isolation of the complexes and allows the detection of weak and cytosolic proteins as well. Our analysis of these pairs revealed that the overlap of the interactomes for both proteoforms is in general high, showing their functional relation. However, for all pairs tested we do report differences as well. We show that N-terminal proteoforms can be engaged in new/different interactions and as well can lose several interactions compared to the canonical protein. We used AP-MS to validate differences in the interaction networks reported for three of these 20 pairs of N-terminal proteoform and canonical counterpart.

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