Electron capture dissociation implementation progress in fourier transform ion cyclotron resonance mass spectrometry

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We report the use of unimolecular dissociation by infrared radiation for gaseous multiphoton energy transfer to determine relative activation energy (E a,laser ) for dissociation of peptide sequence ions. The sequence ions of interest are mass-isolated; the entire ion cloud is then irradiated with a continuous wave CO 2 laser, and the first order rate constant, k d , is determined for each of a series of laser powers. Provided these conditions are met, a plot of the natural logarithm of k d versus the natural logarithm of laser power yields a straight line, whose slope provides a measure of E a,laser . This method reproduces the E a values from blackbody radiative dissociation (BIRD) for the comparatively large, singly and doubly protonated bradykinin ions (nominally y 9 and y 9 2+ ). The comparatively small sequence ion systems produce E a,laser values that are systematic underestimates of theoretical barriers calculated with density functional theory (DFT). However, the relative E a,laser values are in qualitative agreement with the mobile proton model and available theory. Additionally, novel protonated cyclic-dipeptide (diketopiperazine) fragmentation reactions are analyzed with DFT. FT-ICR MS provides access to sequence ions generated by electron capture dissociation, infrared multiphoton dissociation, and collisional activation methods (i.e., b n , y m , c n , z m• ions).

Section: Sample Preparationmentioning

confidence: 99%

Relative Stability of Peptide Sequence Ions Generated by Tandem Mass Spectrometry

Bythell

Hendrickson

Marshall

2012

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“…Gated ion trapping was used without cooling gas. For ECD experiments, a variable delay period (typically 50 ms at 10 V trapping voltage) was used to optimize ion magnetron motion phase [41]; a 3-mm-diameter electron beam (1-100 ms) was then injected into the ICR trap, followed by an electron clean-up event (100 ms) [40,42]. The cathode potential during electron injection was Ϫ5 V and kept at ϩ10 V otherwise.…”

Section: Tandem Mass Spectrometrymentioning

confidence: 99%

“…Accelerating grid voltage was at ϩ5 V during electron injection and at Ϫ200 V otherwise. A multiple-pass electron injection regime was used (transfer octopole DC offset Ϫ60 V) [40,42]. For AI-ECD experiments, precursor ions were activated by CO 2 IR laser irradiation (50 -100 ms at ϳ25 W continuous-wave laser output power; the laser was equipped with a 2.5ϫ beam expander to improve overlap with the ion cloud) before electron injection [39].…”

Section: Tandem Mass Spectrometrymentioning

confidence: 99%

“…ECD FT-ICR MS of six doubly protonated horse myoglobin tryptic fragments ( [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16], [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], [32][33][34][35][36][37][38][39][40][41][42] Figure S2) and with vibrational activation ( Figure 5) confirm preferential and sometimes periodic product ion formation from peptides with mainly ␣-helical or ␤-turn secondary structure in solution (before protein digestion). Predominantly z-ions are observed because of preferential charge retention at the C-terminal Lys or Arg basic residue upon electron capture, as expected for doubly charged tryptic peptides.…”

Section: Ecd Of Doubly Charged Amphipathic Peptidesmentioning

confidence: 99%

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

Hamidane

Tsybin

et al. 2009

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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...

“…Doubly protonated peptides were isolated in the LTQ (isolation window of 4 Th) and transferred to the ICR ion trap for subsequent tandem mass spectrometry following standard procedures [20]. ECD was performed with low-energy electrons for ϳ70 ms using a pencil electron beam from a dispenser cathode located in the homogenous region of the magnetic field [21]. The variable delay before electron injection was optimized to account for ion magnetron motion [22].…”

Section: Ecd-based Tandem Mass Spectrometrymentioning

confidence: 99%

Electron capture dissociation product ion abundances at the X amino acid in RAAAA-X-AAAAK peptides correlate with amino acid polarity and radical stability

Vorobyev

Hamidane

Tsybin

2009

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We present mechanistic studies aimed at improving the understanding of the product ion formation rules in electron capture dissociation (ECD) of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry. In particular, we attempted to quantify the recently reported general correlation of ECD product ion abundance (PIA) with amino acid hydrophobicity. The results obtained on a series of model H-RAAAAXAAAAK-OH peptides confirm a direct correlation of ECD PIA with X amino acid hydrophobicity and polarity. The correlation factor (R) exceeds 0.9 for 12 amino acids (Ile, Val, His, Asn, Asp, Glu, Gln, Ser, Thr, Gly, Cys, and Ala). The deviation of ECD PIA for seven outliers (Pro is not taken into consideration) is explained by their specific radical stabilization properties (Phe, Trp, Tyr, Met, and Leu) and amino acid basicity (Lys, Arg). Phosphorylation of Ser, Thr, and Tyr decreases the efficiency of ECD around phosphorylated residues, as expected. R evealing peptide and protein structure-activity and structure-function relationships is an important and challenging step in the drug discovery process. Further insights into the influence of specific amino acids on molecular conformation and physicochemical properties are needed to increase the throughput and accuracy of the methods and techniques used to investigate these critical relationships [1,2]. Electron capture dissociation (ECD) [3] and electron-transfer dissociation (ETD) [4,5] are generally employed to determine peptide and protein primary structures and to characterize labile modifications in tandem mass spectrometry (MS/MS)-based proteomics. In addition, recent findings demonstrate that the probability of a given N-C ␣ bond in a peptide backbone being cleaved by ECD/ETD, which can be monitored by product ion abundance (PIA), is governed by peptide or protein conformation [6 -9]. A number of experiments ranging from targeted peptide analysis to statistical interpretation of fragmentation have been performed for several thousand tryptic peptides [6, 7, 10 -13] to examine the dependence of ECD PIA on peptide sequence. The experimental results have confirmed the complementarity of ECD and vibrationally induced dissociation, e.g., collision-induced dissociation (CID), but have failed to provide a general quantitative model for ECD. Enhancing models to include intramolecular hydrogen bonding has provided the best quantitative description of ECD PIA to date, as shown by Zubarev and coworkers [14]. However, their reported correlation is built upon extensive molecular dynamics simulations performed for a specific case of a selected part of a small (20-amino acid) protein, Trp cage. Although general application of the model would be time-consuming, it demonstrates the importance of hydrogen bonds when attempting to quantitatively describe ECD. Recent molecular dynamics modeling complemented with quantum chemistry calculations on penta-and hexapeptides supports the hypothesis that ECD/ETD possess strong conformational selectivity [15]. The ...