Proteomics

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Non-covalently associated peptides are observed during liquid chromatography-mass spectrometry and affect cross-link analyses


ABSTRACT: Cross-Linking/mass spectrometry draws structural information out of covalently linked peptide pairs. When these links do not match to previous structural models they may indicate changes of protein conformation. Unfortunately, such links can also be the result of experimental errors or artefacts. Here we describe the observation of non-covalently associated peptides during liquid chromatography-mass spectrometry analysis which can easily be misidentified as cross-linked. Strikingly, they often mismatch to the protein structure or may falsely suggest protein homo-dimerization. Non-covalently associated peptides presumably form during ionization and can be distinguished from cross-linked peptides by observing co-elution of the corresponding linear peptides in MS1, as well as the presence of the individual peptide fragments in mixed MS2 spectra. Interestingly, non-covalently associated peptides seem sensitive to ionization source design as we see them more prevalently on a Q Exactive than on an Orbitrap Velos mass spectrometer. Finally we show how more disruptive ionization settings such as moderate in-source fragmentation suppresses their presence.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive HF, Q Exactive

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine) Gallus Gallus (chicken)

SUBMITTER: Sven Giese  

LAB HEAD: Juri Rappsilber

PROVIDER: PXD010895 | Pride | 2019-01-25

REPOSITORIES: Pride

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Publications

Noncovalently Associated Peptides Observed during Liquid Chromatography-Mass Spectrometry and Their Effect on Cross-Link Analyses.

Giese Sven H SH   Belsom Adam A   Sinn Ludwig L   Fischer Lutz L   Rappsilber Juri J  

Analytical chemistry 20190201 4


Cross-linking mass spectrometry draws structural information from covalently linked peptide pairs. When these links do not match to previous structural models, they may indicate changes in protein conformation. Unfortunately, such links can also be the result of experimental error or artifacts. Here, we describe the observation of noncovalently associated peptides during liquid chromatography-mass spectrometry analysis, which can easily be misidentified as cross-linked. Strikingly, they often mi  ...[more]

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