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

Abstract

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 mismatch to the protein structure. Noncovalently associated peptides presumably form during ionization and can be distinguished from cross-linked peptides by observing coelution of the corresponding linear peptides in MS1 spectra, as well as the presence of the individual (intact) peptide fragments in MS2 spectra. To suppress noncovalent peptide formations, increasingly disruptive ionization settings can be used, such as in-source fragmentation.

Retention Time Prediction Using Neural Networks Increases Identifications in Crosslinking Mass Spectrometry

“…Thus, the model implicitly learned that the RT of a crosslinked peptide should differ from the RT of the individual peptides. This might become useful especially for distinguishing consecutive 45 from crosslinked peptides or when dealing with gas-phase associated peptides 36 .…”

“…This analysis revealed a similar magnitude for all 15 SHAP values implying that a single feature alone is insufficient to recognize false matches. Notably, the top crosslinked peptides or when dealing with gas-phase associated peptides 36 .…”

“…Another consequence of coverage gaps is the misidentification of noncovalently associated peptides as crosslinks. 36 The severity of this coverage issue depends on the applied acquisition strategy 37 , crosslinker chemistry 38 , and the details of the implemented scoring in the search engine. Nevertheless, assuming RT predominantly depends on both peptides of a crosslink, it could complement mass spectrometric information and thus improve existing scoring routines and lead to more crosslinks at the same confidence (i.e.…”

Liposomes as Carriers of Membrane‐Associated Proteins and Peptides for Mass Spectrometric Analysis

“…Note that, contrary to expectations,noncovalent association of coeluting peptides was recently described for this specific instrument. [30] (ii)W ea lso employed aQ -ToF Ultima mass spectrometer modified for transmission of high-mass complexes,t hereby preserving non-covalent interactions (Figure 1, step v). [31] This type of mass spectrometer is commonly applied in native MS and allows the analysis of intact protein complexes and their interactions with ligands including lipids.…”

Liposomen als Überträger membranassoziierter Proteine und Peptide für die massenspektrometrische Analyse

“…Man beachte,dass entgegen den Erwartungen eine nicht-kovalente Assoziation von koeluierenden Peptiden kürzlich auch fürd ieses Massenspektrometer beschrieben wurde. [30] 2) Wirv erwendeten ein "Q-ToF Ultima"-Massenspektrometer,w elches fürd en Tr ansfer großer Komplexe in die Gasphase modifiziert wurde,u mn icht-kovalente Wechselwirkungen zu erhalten (Abbildung 1, Schritt v). [31] Diese Art von Massenspektrometer wird üblicherweise in der nativen MS eingesetzt und ermçglicht die Analyse intakter Proteinkomplexe und deren Wechselwirkungen mit Liganden, einschließlich Lipiden.…”