Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

Belov, Arseniy M.

doi:10.17760/d20259955

Cited by 1 publication

(3 citation statements)

References 307 publications

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“…The subsequent inject flatapole device was converted into a linear quadrupole trap and was operated in a “trap and release” mode of analysis as developed in ref . Following efficient desolvation in the dual-funnel interface, protein complexes were introduced and activated in the linear ion trap operating at a pressure of 10 –2 –10 –1 mbar.…”

Section: Methodssupporting

confidence: 54%

“…The trapping capability was enabled at the front section of the instrument, as described in detail in the Experimental Section. Specifically, to enable the first step of the activation approach, an inject flatapole device was converted to a linear quadrupole trap and was operated in a “trap and release” mode of analysis (Figure ). Trapping of ions for extended intervals (∼10 ms) ensured a sufficient number of higher-energy collisions, highly efficient fragmentation, collisional relaxation of the ejected subunits, and then purging the fragment ions (or excited precursors) out of the linear ion trap, to be referred to as a release event.…”

Section: Resultsmentioning

confidence: 72%

“…(A) Modifications were introduced in the front section of the instrument (highlighted in colors). Specifically, a pressure-regulated dual-funnel interface was used for improving desolvation and enhancing sensitivity (blue) in the manner described in ref . In addition, a trap-and-release mode of analysis was enabled in the inject flatapole by a front-end trap, to allow pseudo-MS 2 measurements (yellow).…”

Section: Methodsmentioning

confidence: 99%

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Triple-Stage Mass Spectrometry Unravels the Heterogeneity of an Endogenous Protein Complex

et al. 2017

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Protein complexes often represent an ensemble of different assemblies with distinct functions and regulation. This increased complexity is enabled by the variety of protein diversification mechanisms that exist at every step of the protein biosynthesis pathway, such as alternative splicing and post transcriptional and translational modifications. The resulting variation in subunits can generate compositionally distinct protein assemblies. These different forms of a single protein complex may comprise functional variances that enable response and adaptation to varying cellular conditions. Despite the biological importance of this layer of complexity, relatively little is known about the compositional heterogeneity of protein complexes, mostly due to technical barriers of studying such closely related species. Here, we show that native mass spectrometry (MS) offers a way to unravel this inherent heterogeneity of protein assemblies. Our approach relies on the advanced Orbitrap mass spectrometer capable of multistage MS analysis across all levels of protein organization. Specifically, we have implemented a two-step fragmentation process in the inject flatapole device, which was converted to a linear ion trap, and can now probe the intact protein complex assembly, through its constituent subunits, to the primary sequence of each protein. We demonstrate our approach on the yeast homotetrameric FBP1 complex, the rate-limiting enzyme in gluconeogenesis. We show that the complex responds differently to changes in growth conditions by tuning phosphorylation dynamics. Our methodology deciphers, on a single instrument and in a single measurement, the stoichiometry, kinetics, and exact position of modifications, contributing to the exposure of the multilevel diversity of protein complexes.