Separation and identification of structural isomers by quadrupole collision-induced dissociation-hydrogen/deuterium exchange-infrared multiphoton dissociation (QCID-HDX-IRMPD)

Abstract

A new approach that uses a hybrid Q-FTICR instrument and combines quadrupole collisioninduced dissociation, hydrogen-deuterium exchange, and infrared multiphoton dissociation (QCID-HDX-IRMPD) has been shown to effectively separate and differentiate isomeric fragment ion structures present at the same m/z. This method was used to study protonated YAGFL-OH (free acid), YAGFL-NH 2 (amide), cyclic YAGFL, and YAGFL-OCH 3 (methyl ester). QCID-HDX of m/z 552.28 (C 29 T andem mass spectrometry is widely used for the identification of peptides and proteins [1,2]. Generally, protonated peptides at a given m/z are selected and fragmented via collision-induced dissociation (CID), generating 'b' and 'y' fragment ions, which allows the peptide sequence to be determined.In the course of a common proteomics experiment, several hundred to several thousand spectra are generated for a single sample, making manual interpretation of the peptide fragmentation spectra impractical. To speed up the assignment of MS/MS spectra, protein identification algorithms are often used [3,4]. These algorithms generate theoretical peptide spectra or m/z lists based loosely on prevalent fragmentation models, including the mobile proton model [5] and the pathways-in-competition model [6], and compare them with experimental spectra to determine peptide sequences. While these models can help to predict many trends in peptide fragmentation, actual dissociation chemistry is much more complex, and the complete dissociation chemistry in an MS/MS spectrum cannot always be accurately predicted by using existing theoretical models. As a result, false and missed identifications frequently occur, with only a small percentage of the spectra being correctly assigned [7][8][9][10]. Incorporation of additional chemical information and fragmentation models into sequencing algorithms could depict fragmentation processes more completely, potentially allowing more accurate kinetic modeling of spectra to be possible, and the success of identification algorithms would likely improve.One potential cause for missed or false identifications of peptide fragmentation spectra is the presence of multiple isomeric ion structures at a single m/z. If multiple isomers are present at one m/z, the MS/MS spectrum of that m/z could contain fragments from each of the structures present. For example, the b n ions from peptides of length n and the corresponding [MH Ϫ H 2 O] ϩ ions are isomeric. While water loss at the peptide C-terminus results in b 5 ion formation for pentapeptides, water can also be lost involving oxygen from an Address reprint requests to Dr.

Conformation-Specific Spectroscopy of Peptide Fragment Ions in a Low-Temperature Ion Trap

“…Different tools, sometimes in combination, have been applied for obtaining information on the structures of peptide fragment ions, including theoretical calculations [9,10], multi-stage mass spectrometry (MS/MS, or MS n ) [3,4,[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28], ion mobility spectrometry (IMS) [29,30], hydrogen-deuterium exchange (HDX) [22,31,32], and infrared multiple photon dissociation (IRMPD) spectroscopy [5,22,[31][32][33][34][35][36][37][38][39][40][41][42]. While spectroscopy is a particularly powerful way to obtain detailed information about the structures of gas phase molecules, approaches based on IRMPD feature certain drawbacks.…”

“…With increasing complexity of the fragment ion structures, support from other sources of information becomes necessary for unraveling complicated IRMPD spectra. This can be achieved by combining spectroscopy with other techniques [22,31,32] or by comparison of the spectra with chemically modified species in which cyclization is either induced or inhibited [36,38,39,42,44].…”

Diagnosing the Protonation Site of b2 Peptide Fragment Ions using IRMPD in the X–H (X = O, N, and C) Stretching Region

“…Most of the attention concerning peptide fragments has been devoted to b n ions [12,[17][18][19][20][21][22][23][24][25]. Nonetheless, a n CID fragments [26][27][28] as well as c n fragment ions [29] generated via electron capture dissociation have also been investigated by IR spectroscopy.…”

Disfavoring Macrocycle b Fragments by Constraining Torsional Freedom: The “Twisted” Case of QWFGLM b6

“…In a complementary IRMPD spectroscopy and HDX study on oligoglycine b fragments, Chen et al demonstrated a size effect on the propensity to form macrocycle structures: the smaller b 2 and b 3 exclusively adopt oxazolone structures, whereas mid-sized fragments (b 4 -b 7 ) exhibit a mixture of oxazolone and macrocycle structures; for the largest b fragment, b 8 , only macrocycle structures were confirmed [18]. Similar trends were observed for b 2 -b 4 for Leuenkephalin [21], as well as b 5 from YAGFL-NH 2 [22]. Computational results by Bleiholder et al confirmed that for permuted b 5 fragment ions, the macrocycle structures were energetically favored over the linear oxazolones [30].…”

“…"Fast-" and "slow"-exchanging b fragment structures have been distinguished in this way by a number of groups [18][19][20][21][22][23]. Quantification of "fast" versus "slow" structures can be performed by an analysis of the kinetic rates.…”