Towards understanding the tandem mass spectra of protonated oligopeptides. 1: Mechanism of amide bond cleavage

Collision-activated dissociation (CAD) of tryptic peptides is a cornerstone of mass spectrometrybased proteomics research. Principal component analysis of a database containing 15,000 high-resolution CAD mass spectra of gas-phase tryptic peptide dications revealed that they fall into two classes with a good separation between the classes. The main factor determining the class identity is the relative abundance of the peptide bond cleavage after the first two N-terminal residues. A possible scenario explaining this bifurcation involves trans-to cis-isomerization of the N-terminal peptide bond, which facilitates solvation of the N-terminal charge on the second backbone amide and formation of stable b 2 ions in the form of protonated diketopiperazines. Evidence supporting this scenario is derived from statistical analysis of the high-resolution CAD MS/MS database. It includes the observation of the strong deficit of a 3 ions and anomalous amino acid preferences for b 2 ion formation. do not predict relative abundances of fragment ions and are mostly concerned with matching ionic masses of certain fragment types. At the same time, fragment ion abundances in CAD MS/MS spectra can be relatively reproducible and are far from homogeneous-and to use the patterns of these abundances for sequence verification has for a long time been a tempting idea. Two alternative approaches were pursued in implementation of this idea. One approach was to collect a massive database of tandem mass spectra of all possible peptides and to make direct comparison between the standard MS/MS datasets and the experimental ones [3]. Although this approach has many merits, it faces a huge challenge in the form of an astronomical number of peptide sequences existing in nature. The alternative approach is to predict fragment abundances for a given sequence. The abundancepredicting algorithm by Zhang has more than 200 adjustable parameters but still exhibits only 70% accuracy [4,5]. The global model of CAD fragmentation developed by us for tryptic peptide dications uses only 20 parameters, one for each amino acid [6]. In preliminary assessment of the prediction accuracy of that model we have found a bifurcating behavior. It appeared that, although a large group of mass spectra behaved in good agreement with the model's predictions, another large group showed significantly deviating features. Thus the question arose as to whether all tryptic peptide dications constitute one group in the statistical sense. This question is critical for every abundance-predicting algorithm because different models of fragmentation should be applied to describe the behavior of each group. The obvious hypothesis that one group represented peptides ending with Lys and another one with Arg was quickly tested and rejected. Another suggestion was that the division line was the presence or absence of a particular amino acid, e.g., proline, histidine, or aspartic acid, which are known for their effect on bond fragmentation [7,8]. This hypothesis was also tested and rejected. An...

Section: Special Casesmentioning

confidence: 99%

Bifurcating fragmentation behavior of gas-phase tryptic peptide dications in collisional activation

Savitski

Fälth

Fung

et al. 2008

“…Our conformational search engine [29 -34] scans on amide nitrogen protonated species, oxazolone terminated structures, and b n -y m type [31,36] transition structures (TS). During the MD simulations, structures were regularly saved for further refinement by full geometry-optimization using the same force field.…”

Section: Calculationsmentioning

confidence: 99%

“…Two major forms were considered here, the trans-trans and cis-trans isomerization states of the backbone amide bonds (Structure A in Figure S1, and Structure A in Figure S2, Supplementary Material, which can be found in the electronic version of this article). The trans-trans structure is responsible for formation of oxazolone type b ions on the b n -y m pathways (Scheme 3, for more details, see below) [13,31,36,42,43]. On the other hand, the cis-trans isomer is necessary to form diketopiperazine isomers of b ions on the cyclic-peptide pathways (Scheme 4, for more details, see below) [13,31,44].…”

Section: Structure Of [Proϫhis-gly ϩ 2h] 2ϩmentioning

confidence: 99%

Fragmentation of doubly-protonated Pro-His-Xaa tripeptides: Formation of b₂²⁺ ions

Knapp-Mohammady

Young

Paizs

et al. 2009

When ionized by electrospray from acidic solutions, the tripeptides Pro-His-Xaa (Xaa ϭ Gly, Ala, Leu) form abundant doubly-protonated ions, [M ϩ 2H] 2ϩ . Collision-induced dissociation (CID) of these doubly-protonated species results, in part, in formation of b 2 2ϩ ions, which fragment further by loss of CO to form a 2 2ϩ ions; the latter fragment by loss of CO to form the Pro and His iminium [immonium is commonly used in peptide MS work] ions. Although larger doubly-charged b ions are known, this represents the first detailed study of b 2 2ϩ ions in CID of small doubly protonated peptides. The most abundant CID products of the studied doubly-protonated peptides arise mainly in charge separation involving two primary fragmentation channels, formation of the b 2 /y 1 pair and formation of the a 1 /y 2 pair. Combined molecular dynamics and density functional theory calculations are used to gain insight into the structures and fragmentation pathways of doubly-protonated Pro-His-Gly including the energetics of potential protonation sites, backbone cleavages, post-cleavage charge-separation reactions and the isomeric structures of b 2 2ϩ ions. Three possible structures are considered for the b 2 2ϩ ions: the oxazolone, diketopiperazine, and fused ring isomers. The last is formed by cleavage of the His-Gly amide bond on a pathway that is initiated by nucleophilic attack of one of the His side-chain imidazole nitrogens. Our calculations indicate the b 2 2ϩ ion population is dominated by the oxazolone and/or fused ring isomers. (J Am Soc Mass Spectrom 2009, 20, 2135-2143

“…As evident in Figure S from NicGGOMe. The first would involve proton transfer and nucleophilic attack steps characteristic of the conventional b n /y n oxazolone pathway [9,10,26], as shown in Scheme 1. In this process, NicGGOMe is converted from a ring-protonated structure to a "charge-solvated" structure by transfer of a proton from the nicotinic acid ring to the proximate carbonyl.…”

Section: Resultsmentioning

confidence: 99%

“…E ffective application of tandem mass spectrometry, collision induced dissociation (CID), and bioinformatics for protein identification requires a clear understanding of peptide ion fragmentation mechanisms. Low-energy CID of protonated peptides promotes rearrangement reactions in which the added proton presumably migrates to the amide bond that is ultimately cleaved [1][2][3][4][5][6][7][8][9][10][11][12], as treated in the "mobileproton" (MP) [13][14][15][16][17][18][19][20][21][22][23][24][25], and "pathways in competition" (PIC) models of peptide dissociation [26]. Experimental studies have established that the C-terminus-containing y n ϩ fragments are truncated peptides [6,27,28], while the N-terminus-containing b n ϩ and a n ϩ species have substituted oxazolone ring and imine structures, respectively [4,5,11,12,29,30].…”

mentioning

confidence: 99%

Spectroscopic evidence for mobilization of amide position protons during CID of model peptide ions

Molesworth

Leavitt

Groenewold

et al. 2009

Infrared multiple photon dissociation (IRMPD) spectroscopy was used to study formation of b 2 ϩ from nicotinyl-glycine-glycine-methyl ester (NicGGOMe). IRMPD shows that NicGGOMe is protonated at the pyridine ring of the nicotinyl group, and more importantly, that b 2 ϩ from NicGGOMe is not protonated at the oxazolone ring, as would be expected if the species were generated on the conventional b n ϩ /y n ϩ oxazolone pathway, but at the pyridine ring instead. IRMPD data support a hypothesis that formation of b 2 ϩ from NicGGOMe involves mobilization and transfer of an amide position proton during the fragmentation reaction. E ffective application of tandem mass spectrometry, collision induced dissociation (CID), and bioinformatics for protein identification requires a clear understanding of peptide ion fragmentation mechanisms. Low-energy CID of protonated peptides promotes rearrangement reactions in which the added proton presumably migrates to the amide bond that is ultimately cleaved [1][2][3][4][5][6][7][8][9][10][11][12], as treated in the "mobileproton" (MP) [13][14][15][16][17][18][19][20][21][22][23][24][25], and "pathways in competition" (PIC) models of peptide dissociation [26]. Experimental studies have established that the C-terminus-containing y n ϩ fragments are truncated peptides [6,27,28], while the N-terminus-containing b n ϩ and a n ϩ species have substituted oxazolone ring and imine structures, respectively [4,5,11,12,29,30].Wavelength-selective infrared multiple photon dissociation (IRMPD) spectroscopy has recently been used to probe and confirm proposed structures of peptides and peptide dissociation products, with studies of the latter focusing primarily on b n ϩ and a n ϩ ions [11,12,29,30]. Our group has designed model peptides and approaches to probe intramolecular migration of protons during peptide dissociation reactions [31,32]. Versions of these model peptides are the subject of the present study, in which IRMPD was used to determine the structure of protonated nicotinic acid-glycine-glycinemethyl ester (NicGGOMe), and the b 2 ϩ fragment ions from NicGGOMe and benzoic acid-glycine-glycinemethyl ester (BzGGOMe). The pyridine ring of the nicotinic acid residue is used to sequester the "mobile" proton added to the peptide to produce (M ϩ H) ϩ , and inhibit migration to the site of intramolecular nucleophilic attack during fragmentation reactions. IRMPD spectroscopy provides strong evidence for the mobilization and migration of amide-position protons during dissociation reactions of model peptides. Experimental Mass Spectrometry and IRMPD SpectroscopyCID experiments were performed using a ThermoFinnigan (San Jose, CA, USA) LCQ-Deca quadrupole ion trap (QIT) mass spectrometer. IRMPD spectra were collected using the FT-ICR mass spectrometer coupled to the beamline of the free electron-laser user facility (FELIX) infrared free electron laser [33][34][35]. Ions produced by electrospray ionization (ESI) were accumulated in a hexapole ion trap, and isolated and irradiated with FELIX for 2 s at a...