PXD042569

PXD042569 is an original dataset announced via ProteomeXchange.

Title	N-terminal modification of eGFP by LC-MSMS
Description	We used an in-depth analysis of the characteristics of eGFP as a proof-of-principle analyte to establish a robust assay for the determination of degradation rates on the single-cell level using microinjection and live-cell microscopy. We aimed to determine the influence of the exposed n-terminal aminoacid on the proteins degradation. The high sensitivity of our single-cell analysis in combination with the controlled starting conditions of microinjection provides an unique advantage to examine differences in rates with an improved resolution and to compare the influence of different n-terminal aminoacids on eGFP-degradation without accounting for differences during translation. We produced XS-eGFP, with X being substituted by different proteinogenic amino acids, from E.coli and extensively purified it by a three column process. We could show pronounced differences in degradation rates for the constructs with differing N-terminal aminoacids. We also chemically acetylated the N-terminal α amino groups of methionine, alanine, threonine, valine and serine that would normally occur co translationally, in vitro before, microinjection to analyze the influence of acetylation on target degradation. Acetylation at the N-terminus was confirmed by LC-MS-MS. Acetylation reduced degradation of the respective construct. Finally we purified eGFP by anion-exchange from eGFP expressing HEK293 cells to elucidate modifications of eGFP after eukaryotic expression. We could show that eGFP is likely cleaved during HEK293 expression and n-terminally acetylated, which leads to a starkly reduced degradation rate. The degradation rate was similarly reduced for a N-terminally shortened eGFP produced in E.coli and acetylated in-vitro.
HostingRepository	PRIDE
AnnounceDate	2025-05-06
AnnouncementXML	Submission_2025-05-06_09:37:37.383.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dorothea Winkelvoss
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	acetylated residue
Instrument	Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2023-05-31 02:34:39	ID requested
⏵ 1	2025-05-06 09:37:38	announced

Vukovic D, Winkelvo, ß D, Kapp JN, H, ä, nny AC, B, ü, rgisser H, Riermeier L, Udovcic A, Tiefenboeck P, Pl, ü, ckthun A, Protein degradation kinetics measured by microinjection and live-cell fluorescence microscopy. Sci Rep, 14(1):27153(2024) [pubmed]

10.1038/s41598-024-76224-0;

submitter keyword: LC-MSMS,N-degron, PTM, eGFP, Acetylation

Andreas Plückthun
contact affiliation	Department of biochemistry, University of Zürich, Zürich, Switzerland
contact email	plueckthun@bioc.uzh.ch
lab head
Dorothea Winkelvoss
contact affiliation	University of Zürich
contact email	d.winkelvoss@bioc.uzh.ch
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/05/PXD042569

PRIDE project URI

• PRIDE
  1. PXD042569
     1. Label: PRIDE project
     2. Name: N-terminal modification of eGFP by LC-MSMS