Novel Linear Quadrupole Ion Trap/FT Mass Spectrometer:  Performance Characterization and Use in the Comparative Analysis of Histone H3 Post-translational Modifications

Syka, John E. P.; Marto, Jarrod A.; Bai, Dina L.; Horning, Stevan; Senko, Michael W.; Schwartz, Jae C.; Ueberheide, Beatrix; García, Benjamin A.; Busby, Scott A.; Muratore, Tara L.; Shabanowitz, Jeffrey; Hunt, Donald F.

doi:10.1021/pr0499794

Cited by 348 publications

(356 citation statements)

References 11 publications

(27 reference statements)

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“…The highresolution, high mass accuracy mass spectrometer should be operated in positive mode using a data-dependent MS/MS mode setting as described previously (see refs. [13][14][15][16][17].…”

Section: Reagent Setupmentioning

confidence: 99%

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Chemical derivatization of histones for facilitated analysis by mass spectrometry

et al. 2007

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Histone post-translational modifications have been recently intensely studied owing to their role in regulating gene expression. Here, we describe protocols for the characterization of histone modifications in both qualitative and semiquantitative manners using chemical derivatization and tandem mass spectrometry. In these procedures, extracted histones are first derivatized using propionic anhydride to neutralize charge and block lysine residues, and are subsequently digested using trypsin, which, under these conditions, cleaves only the arginine residues. The generated peptides can be easily analyzed using online LC-electrospray ionization-tandem mass spectrometry to identify the modification site. In addition, a stable isotope-labeling step can be included to modify carboxylic acid groups allowing for relative quantification of histone modifications. This methodology has the advantage of producing a small number of predicted peptides from highly modified proteins. The protocol should take approximately 15-19 h to complete, including all chemical reactions, enzymatic digestion and mass spectrometry experiments.

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Section: Reagent Setupmentioning

confidence: 99%

“…The described protocols have been applied for the successful identification and quantification of histone PTMs in cells from various organisms and under various external stimuli [13][14][15][16][17] , but can clearly be extended to histones extracted from any cellular source.…”

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Chemical derivatization of histones for facilitated analysis by mass spectrometry

et al. 2007

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“…Application of ECD to triply charged model amphipathic peptides, AA9 and LL9, is shown in Figure 3, and for a series of naturally occurring antimicrobial peptides of amphipathic nature, piscidins, in Figure 4 and Supplementary Figure S1, which can be found in the electronic version of this [54,55]. The periodic character (e.g.…”

Section: Ecd Of Triply Charged Amphipathic Peptidesmentioning

confidence: 99%

Periodic sequence distribution of product ion abundances in electron capture dissociation of amphipathic peptides and proteins

Hamidane

Tsybin

et al. 2009

J. Am. Soc. Mass Spectrom.

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The rules for product ion formation in electron capture dissociation (ECD) mass spectrometry of peptides and proteins remain unclear. Random backbone cleavage probability and the nonspecific nature of ECD toward amino acid sequence have been reported, contrary to preferential channels of fragmentation in slow heating-based tandem mass spectrometry. Here we demonstrate that for amphipathic peptides and proteins, modulation of ECD product ion abundance (PIA) along the sequence is pronounced. Moreover, because of the specific primary (and presumably secondary) structure of amphipathic peptides, PIA in ECD demonstrates a clear and reproducible periodic sequence distribution. On the one hand, the period of ECD PIA corresponds to periodic distribution of spatially separated hydrophobic and hydrophilic domains within the peptide primary sequence. On the other hand, the same period correlates with secondary structure units, such as ␣-helical turns, known for solution-phase structure. Based on a number of examples, we formulate a set of characteristic features for ECD of amphipathic peptides and proteins: (1) periodic distribution of PIA is observed and is reproducible in a wide range of ECD parameters and on different experimental platforms; (2) local maxima of PIA are not necessarily located near the charged site; (3) ion activation before ECD not only extends product ion sequence coverage but also preserves ion yield modulation; (4) the most efficient cleavage (e.g. global maximum of ECD PIA distribution) can be remote from the charged site; (5) the number and location of PIA maxima correlate with amino acid hydrophobicity maxima generally to within a single amino acid displacement; and (6) preferential cleavage sites follow a selected hydrogen spine in an ␣-helical peptide segment. Presently proposed novel insights into ECD behavior are important for advancing understanding of the ECD mechanism, particularly the role of peptide sequence on PIA. An improved ECD model could facilitate protein sequencing and improve identification of unknown proteins in proteomics technologies. In structural biology, the periodic/preferential product ion yield in ECD of ␣-helical structures potentially opens the way toward de novo site-specific secondary structure determination of peptides and proteins in the gas phase and its correlation with solution-phase structure. (J Am Soc Mass Spectrom 2009, 20, 1182-1192) © 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry B iomolecular fragmentation in the gas phase by electron capture dissociation (ECD) [1-3] and electron-transfer dissociation (ETD) [4,5] is particularly attractive for mass spectrometry (MS)-based peptide and protein structural analysis. In ECD/ETD, interaction of a multiply charged peptide or protein ion with an electron/anion results in cleavage of NOC ␣ bonds along the peptide backbone, including chargesite-remote backbone cleavages [2]. ECD is useful for peptide sequence tag generation on a chromatographic timescale for protein identificat...

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“…nozzles) from Advion (Ithaca, NY, USA) at a flow rate of about 200 nL/min. Desired charge states of peptide cations were isolated in LTQ (isolation window of 4 m/z) and transferred to the ICR ion trap for subsequent MS/MS following standard procedures [36]. ECD was performed with low-energy electrons for about 70 ms. A CO 2 IR laser was used for AI-ECD at a low power level (Ͻ10 W) and for short periods of time (Ͻ100 ms).…”

Section: Ecd-based Tandem Mass Spectrometrymentioning

confidence: 99%

Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

Hamidane

Chiappe

Hartmer

et al. 2008

J. Am. Soc. Mass Spectrom.

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We decoupled electron-transfer dissociation (ETD) and collision-induced dissociation of charge-reduced species (CRCID) events to probe the lifetimes of intermediate radical species in ETD-based ion trap tandem mass spectrometry of peptides. Short-lived intermediates formed upon electron transfer require less energy for product ion formation and appear in regular ETD mass spectra, whereas long-lived intermediates require additional vibrational energy and yield product ions as a function of CRCID amplitude. The observed dependencies complement the results obtained by double-resonance electron-capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ECD in a cryogenic ICR trap. Compared with ECD FT-ICR MS, ion trap MS offers lower precursor ion internal energy conditions, leading to more abundant charge-reduced radical intermediates and larger variation of product ion abundance as a function of vibrational post-activation amplitude. In many cases decoupled CRCID after ETD exhibits abundant radical c-type and even-electron z-type ions, in striking contrast to predominantly even-electron c-type and [7]. In addition to mainly product ion mass-based MS/ MS, product ion abundance (PIA) in ECD is increasingly considered as a new source of information to improve peptide and protein sequencing [8,9], quantitative modification analysis [10, 11], higher-order structure characterization [8,[12][13][14][15], providing new insights into ECD mechanism [16,17], suggesting charge location in peptides and proteins [18,19], and indicating routes toward developing a quantitative model of ECD/ETD [15]. Double-resonance (DR) ECD, with and without ion preactivation, is used to estimate the radical intermediate lifetimes and differentiate between short-lived and long-lived intermediates by monitoring PIA variation attributed to radical intermediates ejection from the ICR trap immediately upon formation [20,21]. An alternative approach to DR-ECD is to compare ECD fragmentation patterns obtained at room-temperature (300 K) and cold (86 K) ICR ion trap conditions [22]. In cold ICR trap long-lived radical intermediates remain inside of the trap but do not have sufficient internal energy to initiate product ion separation and thus do not contribute to the product ion mass spectrum [9,22]. In general, long-lived radical intermediates exhibit a higher yield of radical N-terminal product ions, c· ions, and even-electron or prime C-terminal product ions, z= ions than that of short-lived intermediates, presumably as the result of increased probability of hydrogen atom transfer between ECD products [9,23]. Ion internal energy variation in activated ion (AI)-ECD [24] was shown to influence hydrogen atom rearrangement between ECD products and determine the ratio of radical to prime product ions [9,21]. Consideration of hydrogen atom loss/gain is important for correct product ion assignment and error-free peptide sequencing in proteomics [23,25].Implementation of electron-transfer dissociation (ETD) in io...

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Novel Linear Quadrupole Ion Trap/FT Mass Spectrometer: Performance Characterization and Use in the Comparative Analysis of Histone H3 Post-translational Modifications

Cited by 348 publications

References 11 publications

Chemical derivatization of histones for facilitated analysis by mass spectrometry

Chemical derivatization of histones for facilitated analysis by mass spectrometry

Periodic sequence distribution of product ion abundances in electron capture dissociation of amphipathic peptides and proteins

Electron capture and transfer dissociation: Peptide structure analysis at different ion internal energy levels

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