A fragmentation geometry based upon axial acceleration of m/z-selected protein ions into a linear octopole ion trap allowed simultaneous production and external accumulation of fragment ions prior to m/z measurement in a FT mass spectrometer. Improved dynamic range resulting from this octopole collisionally activated dissociation resulted in a 2.5؋ increase in experimental throughput and a 2؋ increase in fragment ion matches to gene products identified and characterized in the top down fashion. The acceleration voltage for optimal fragmentation has a m/z and mass dependence, knowledge of which facilitated an automated platform for top down MS/MS on a quadrupole FT hybrid mass spectrometer. Controlled by improved software for data acquisition (e.g. using dynamic exclusion of previously identified species), automated octopole collisionally activated dissociation of samples fractionated using chromatofocusing and reversed-phase liquid chromatography achieved a significant increase in protein identification rate versus previous benchmarks. Also a batch analysis version of ProSight PTM facilitated probability-based identification of intact proteins obtained in a higher throughput fashion. In total, 101 unique proteins (5-59 kDa) were identified from whole cell lysates of Methanosarcina acetivorans grown anaerobically, including the characterization of several mispredicted start sites and biologically relevant mass discrepancies. Molecular & Cellular Proteomics 5:14 -25, 2006.Over the last 3 years a platform consisting of two-dimensional fractionation (1), an automated (2, 3) quadrupole (Q) 1 FT mass spectrometer (4), and new informatic tools (5-7) has emerged for "top down" analysis (8 -11) of intact proteins with DNA-predicted sequences. Application of the nascent platform to Methanococcus jannaschii and Saccharomyces cerevisiae led to the identification of ϳ230 expressed gene products (Ͻ39 kDa) with 100% sequence coverage and simultaneous characterization of ϳ50 post-translational modifications (PTMs) (12, 13). Although yielding promising results, throughput has been limited compared with bottom up platforms based on tryptic peptides (14 -19).Poor dynamic range associated with ESI (20) of intact proteins in complex mixtures, with up to 15 observed proteins per fraction, is minimized by preselection of pseudomolecular ions with quadrupole mass filter-FT or quadrupole ion trap-FT hybrid instruments (4,(21)(22)(23)(24)(25). Given limitations in ion transmission of the quadrupole mass filter at 1-Da resolution, isolation windows in excess of ⌬m/z ϭ 5 were commonly used in prior studies for multiplexed fragmentation of several precursor proteins at once and detection of hundreds of fragment ions (4). This challenges the specificity of protein identification because the search depends largely upon the mass accuracy and the ratio n/f of the number of matching fragment ions for a protein in a database (n) relative to the number observed (f) (5).Controlled fragmentation with infrared multiphoton dissociation (26) facilitate...
Virtual Router Redundancy Protocol Status of this Memo This document specifies an Internet standards track protocol for the Internet community, and requests discussion and suggestions for improvements. Please refer to the current edition of the "Internet Official Protocol Standards" (STD 1) for the standardization state and status of this protocol. Distribution of this memo is unlimited.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.