Recognition of transmembrane helices by the endoplasmic reticulum translocon

Tsybin

et al. 2009

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...

Section: Ecd Pia Analysismentioning

confidence: 99%

Section: Ecd Pia Analysismentioning

confidence: 99%

See 1 more Smart Citation

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

Tsybin

et al. 2009

“…Indeed, both radical and prime product ions in decoupled ETD and CRCID demonstrate distinct differences. ETD and CRCID results in Figures 1 and 2 show a drastic product ion amplitude change between the different regimes of ion activation/dissociation, particularly for Substance P. We include amino acid hydrophobicity distribution along peptide sequence (Figures 1 and 2), to indicate a possible correlation between intermediate lifetime and amino acid properties [41]. Hydrophobic amino acids are characterized by a lower probability of hydrogen bond formation and may allow facile separation of products after backbone cleavage.…”

Section: Complementarity Of Etd/crcid To Dr Ecdmentioning

confidence: 99%

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

Chiappe

Hartmer

et al. 2008

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...

“…In total, a set of 17 amino acid property scales out of the 60 probed [31] returned a correlation factor R higher than 0.5 for all amino acids but Arg, Lys, His, Gly, Pro, Cys, and Leu. Among others, the following scales were considered: hydrophobicity (Hessa [32], Kyte and Doolittle [33], Black [34]), polarity (Grantham [27], and Zimmerman et al [35]), ␣-helix tendency [36], ␤-turn tendency [36], and transmembrane tendency [37]. In addition, we correlated other physicochemical properties of amino acids with the ECD PIA, including [31]: dipole moment [38], molecular weight, bulkiness [35], average flexibility index [39], pKa, substituent effect scale [40], and diverse electron properties of the amino acid side chains [41].…”

Section: Correlation Of Ecd Pia With Amino Acid Propertiesmentioning

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

Tsybin

2009

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 ...