New aspects in fragmentation of peptide nucleic acids: comparison of positive and negative ions by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The use of peptide nucleic acids (PNAs) is steadily increasing in biochemistry and diagnostics. So far, PNAs have mostly been investigated using cationic conditions in mass spectrometry. Furthermore, the use of fragmentation techniques developed for peptides and proteins like infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) has barely been examined. However, especially the fragmentation behavior of PNA oligomers in negative ion mode is of high importance, due to the ability to interact with nucleic acids which are almost exclusively analyzed in the negatively charged state. In the current study PNA fragmentations under cationic and anionic conditions were investigated and different fragmentation techniques like collision‐induced dissociation (CID), IRMPD and ECD were applied. Especially when using CID and IRMPD, amide bonds were broken, whereas ECD resulted in the elimination of nucleobases. Differences were also observed between positive and negative ionization, while the sequence coverage for the negative ions was superior to positive ions. The fragmentation behavior using IRMPD led to almost complete sequence coverage. Additionally, in anions the interesting effect of multiple eliminations of HNCO was found. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of higher energy collisional dissociation (HCD) to the fragmentation of new DOTA‐based labels and N‐termini DOTA‐labeled peptides

1,4,7,10‐Tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) derivatives are applied in quantitative proteomics owing to their ability to react with different functional groups, to harbor lanthanoides and hence their compatibility with molecular and elemental mass spectrometry. The new DOTA derivatives, namely Ln‐MeCAT‐Click and Ln‐DOTA‐Dimedone, allow efficient thiol labeling and targeting sulfenation as an important post‐translational modification, respectively. Quantitative applications require the investigation of fragmentation behavior of these reagents. Therefore, the fragmentation behavior of Ln‐MeCAT‐Click and Ln‐DOTA‐Dimedone was studied using collision‐induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and higher‐energy collision dissociation (HCD) using different energy levels, and the efficiency of reporter ion production was estimated. The efficiency of characteristic fragment formation was in the order IRMPD > HCD (normal energy level) > CID. On the other hand, the application of HCD at high energy levels (HCD@HE; NCE > 250%) resulted in a significant increase in reporter ion production (33–54%). This new strategy was successfully applied to generate label‐specific reporter ions for DOTA amino labeling at the N‐termini and in a quantitative fashion for the estimation of amino:thiol ratio in peptides. Copyright © 2017 John Wiley & Sons, Ltd.     

MeCAT peptide labeling for the absolute quantification of proteins by 2D‐LC‐ICP‐MS

Metal‐Coded Affinity Tags (MeCAT) reagents were devised for the absolute quantification of labeled proteins and peptides using inductively coupled plasma mass spectrometry (ICP‐MS). After the recent publication of quantification approaches for digested proteins, this work presents a multidimensional strategy for the application of MeCAT to samples which require higher chromatographic resolution. Two‐dimensional separations based on strong cation exchange (SCX) and reversed‐phase (RP) chromatography, were used for the quantification of lysozyme, bovine serum albumin and transferrin after tryptic digestion. The elution protocols were optimized to improve the resolution of the MeCAT‐labeled peptides which led to faster elutions in SCX and longer retention times in RP compared with unlabeled peptides. The optimized method provided enough resolution for the samples analyzed. Peptides losses during the whole procedure were studied. Although recoveries of greater than 90% were found in the RP dimension, important global losses in the two‐dimensional offline approach forced us to use specific internal standards, in this case MeCAT‐labeled standard peptides. External calibration and label‐specific isotope dilution analysis (IDA) were tested and compared as possible quantification techniques. While both techniques showed accurate and precise determinations, the label‐specific IDA technique resulted in more straightforward measurements and more affordable external calibrations. Finally, simultaneous quantification of three different samples labeled with different lanthanides was successfully performed demonstrating the potential of MeCAT combined with ICP‐MS for multiplexing. Electrospray ionization mass spectrometry techniques provided the structural information needed for the identification of the labeled species. Copyright © 2012 John Wiley & Sons, Ltd.     

IRMPD and ECD fragmentation of intermolecular cross-linked peptides

Despite the increasing number of studies using mass spectrometry for three dimensional analyses of proteins (MS3D), the identification of cross-linked peptides remains a bottleneck of the method. One of the main reasons for this is the lack of knowledge about the fragmentation of these species. Intermolecular cross-linked peptides are considered the most informative species present in MS3D experiment, since different peptides are connected by a cross-linker, the peptides chain can be either from a single protein, providing information about protein folding, or from two different proteins in a complex, providing information about binding partners, complex topology and interaction sites. These species tend to be large and highly charged in ESI, making comprehensive fragmentation by CID MS/MS problematic. On the other hand, these highly charged peptides are very suitable for dissociation using both infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD). Herein, we report the fragmentation study of intermolecular cross-linked peptides using IRMPD and ECD. Using synthetic peptides and different commercial cross-linkers, a series of intermolecular cross-linked peptides were generate, and subsequently fragmented by IRMPD and ECD in a FT-ICR-MS instrument. Due to the high mass accuracy and resolution of the FT-ICR, the fragment ions could be attributed with high confidence. The peptides sequence coverage and fragmentation features obtained from IRMPD and ECD were compared for all charge states.     

Tandem mass spectrometry investigation of ADP-ribosylated kemptide

Bacterial adenosine diphosphate-ribosyltransferases (ADPRTs) are toxins that play a significant role in pathogenicity by inactivating host proteins through covalent addition of ADP-ribose. In this study we used ADP-ribosylated Kemptide (LRRASLG) as a standard to examine the effectiveness of three common tandem mass spectrometry fragmentation methods for assignment of amino acid sequence and site of modification. Fragmentation mechanisms investigated include low-energy collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron-capture dissociation (ECD); all were performed on a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer. We show that ECD, but neither CID nor IRMPD, of ADP-ribosylated Kemptide produces tandem mass spectra that are interpretable with regard to amino acid sequence assignment and site of modification. Examination of CID and IRMPD tandem mass spectra of ADP-ribosylated Kemptide revealed that fragmentation was primarily focused to the ADP-ribose region, generating several potential diagnostic ions for use in discovery of ADP-ribosylated proteins. Because of the lower relative sensitivity of ECD during data-dependent acquisition to CID, we suggest a 2-fold strategy where CID and IRMPD are first used to detect ADP-ribosylated peptides, followed by sequence assignment and location of modification by ECD analysis.     

MeCAT—new iodoacetamide reagents for metal labeling of proteins and peptides

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.     

Sequencing of peptide‐derived Amadori products by the electron capture dissociation method

The electron capture dissociation (ECD) of peptide‐derived Amadori products has been successfully applied for their sequencing. In contrast to the collision induced dissociation (CID), based on the vibrational excitation of peptides, the ECD method does not produce ions formed by fragmentation of the hexose moiety, that facilitates interpretation of the obtained spectra. The fragmentation spectrum is dominated by c_n and z·_n ions, providing the sufficient information for sequencing of peptides and establishing the location of glycated Lys residues in the peptide chain. The ECD experiments were conducted on a series of synthetic peptides and unseparated digests of glycated ubiquitin. Copyright © 2009 John Wiley & Sons, Ltd.     

Increased sequence coverage of thymine‐rich oligodeoxynucleotides by infrared multiphoton dissociation compared to collision‐induced dissociation

Infrared multiphoton dissociation (IRMPD) of thymine‐rich oligodeoxynucleotides in a linear ion‐trap mass spectrometer affords far more extensive fragmentation than conventional collision‐induced dissociation (CID). For oligodeoxynucleotides containing one non‐thymine base, CID results primarily in cleavage on the 3′ side of the non‐thymine nucleobase, whereas IRMPD results in cleavages between all the nucleobases and thus provides complete sequence coverage. Furthermore, for oligodeoxynucleotides containing a single non‐thymine base, it is shown that the full series of diagnostic sequence ions observed in the IRMPD mass spectra arise from secondary dissociation of the two primary products formed from the initial cleavage site located next to the non‐thymine base. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of carbonylation sites in apomyoglobin after exposure to 4‐hydroxy‐2‐nonenal by solid‐phase enrichment and liquid chromatography–electrospray ionization tandem mass spectrometry

Identification of protein carbonylation because of covalent attachment of a lipid peroxidation end‐product was performed by combining proteolytic digestion followed by solid‐phase hydrazide enrichment and liquid chromatography (LC)–electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using both collision‐induced dissociation (CID) and electron capture dissociation (ECD). To evaluate this approach, we selected apomyoglobin and 4‐hydroxy‐2‐nonenal (4‐HNE) as a model protein and a representative end‐product of lipid peroxidation, respectively. Although the characteristic elimination of 4‐HNE (156 Da) in CID was found to serve as a signature tag for the modified peptides, generation of nearly complete fragment ion series because of efficient peptide backbone cleavage (in most cases over 75%) and the capability to retain the labile 4‐HNE moiety of the tryptic peptides significantly aided the elucidation of primary structural information and assignment of exact carbonylation sites in the protein, when ECD was employed. We have concluded that solid‐phase enrichment with both CID‐ and ECD‐MS/MS are advantageous during an in‐depth interrogation and unequivocal localization of 4‐HNE‐induced carbonylation of apomyoglobin that occurs via Michael addition to its histidine residues. Copyright © 2010 John Wiley & Sons, Ltd.     

Conversion of 3‐nitrotyrosine to 3‐aminotyrosine residues facilitates mapping of tyrosine nitration in proteins by electrospray ionization–tandem mass spectrometry using electron capture dissociation

Protein tyrosine nitration is associated with oxidative stress and various human diseases. Tandem mass spectrometry has been the method of choice for the identification and localization of this posttranslational modification to understand the underlying mechanisms and functional consequences. Due to the electron predator effect of the nitro group limiting fragmentation of the peptide backbone, electron‐based dissociation has not been applicable, however, to nitrotyrosine‐containing peptides. A straightforward conversion of the nitrotyrosine to the aminotyrosine residues is introduced to address this limitation. When tested with nitrated ubiquitin and human serum albumin as model proteins in top‐down and bottom‐up approaches, respectively, this chemical derivatization enhanced backbone fragmentation of the corresponding nitroproteins and nitropeptides by electron capture dissociation (ECD). Increased sequence coverage has been obtained by combining in the bottom‐up strategy the conversion of nitrotyrosine to aminotyrosine and introducing, in addition to trypsin, a further digesting enzyme of complementary specificity, when protein nitration was mapped by liquid chromatography–electrospray ionization tandem mass spectrometry using both collision‐induced dissociation (CID) and ECD. Copyright © 2012 John Wiley & Sons, Ltd.     

Electron capture dissociation mass spectrometry of metallo-supramolecular complexes

The electron capture dissociation (ECD) of metallo-supramolecular dinuclear triple-stranded helicate Fe₂L₃⁴⁺ ions was determined by Fourier transform ion cyclotron resonance mass spectrometry. Initial electron capture by the di-iron(II) triple helicate ions produces dinuclear double-stranded complexes analogous to those seen in solution with the monocationic metal centers Cu^I or Ag^I. The gas-phase fragmentation behavior [ECD, collision-induced dissociation (CID), and infrared multiphoton dissociation (IRMPD)] of the di-iron double-stranded complexes, (i.e., MS³ of the ECD product) was compared with the ECD, CID, and IRMPD of the Cu^I and Ag^I complexes generated from solution. The results suggest that iron-bound dimers may be of the form Fe₂^IL₂²⁺ and that ECD by metallo-complexes allows access, in the gas phase, to oxidation states and coordination chemistry that cannot be accessed in solution.     

The Radical Ion Chemistry of S-Nitrosylated Peptides

The radical ion chemistry of a suite of S-nitrosopeptides has been investigated. Doubly and triply-protonated ions of peptides NYCGLPGEYWLGNDK, NYCGLPGEYWLGNDR, NYCGLPGERWLGNDR, NACGAPGEKWAGNDK, NYCGLPGEKYLGNDK, NYGLPGCEKWYGNDK and NYGLPGEKWYGCNDK were subjected to electron capture dissociation (ECD), and collision-induced dissociation (CID). The peptide sequences were selected such that the effect of the site of S-nitrosylation, the nature and position of the basic amino acid residues, and the nature of the other amino acid side chains, could be interrogated. The ECD mass spectra were dominated by a peak corresponding to loss of ^?NO from the charge-reduced precursor, which can be explained by a modified Utah-Washington mechanism. Some backbone fragmentation in which the nitrosyl modification was preserved was also observed in the ECD of some peptides. Molecular dynamics simulations of peptide ion structure suggest that the ECD behavior was dependent on the surface accessibility of the protonated residue. CID of the S-nitrosylated peptides resulted in homolysis of the S?CN bond to form a long-lived radical with loss of ^?NO. The radical peptide ions were isolated and subjected to ECD and CID. ECD of the radical peptide ions provided an interesting comparison to ECD of the unmodified peptides. The dominant process was electron capture without further dissociation (ECnoD). CID of the radical peptide ions resulted in cysteine, leucine, and asparagine side chain losses, and radical-induced backbone fragmentation at tryptophan, tyrosine, and asparagine residues, in addition to charge-directed backbone fragmentation.     

Comparison of infrared multiphoton dissociation and collision-induced dissociation of supercharged peptides in ion traps

The number and types of diagnostic ions obtained by infrared multiphoton dissociation (IRMPD) and collision-induced dissociation (CID) were evaluated for supercharged peptide ions created by electrospray ionization of solutions spiked with m-nitrobenzyl alcohol. IRMPD of supercharged peptide ions increased the sequence coverage compared with that obtained by CID for all charge states investigated. The number of diagnostic ions increased with the charge state for IRMPD; however, this trend was not consistent for CID because the supercharged ions did not always yield the greatest number of diagnostic ions. Significantly different fragmentation pathways were observed for the different charge states upon CID or IRMPD with the latter yielding far more immonium ions and often fewer uninformative ammonia, water, and phosphoric acid neutral losses. Pulsed-Q dissociation resulted in an increase in the number of internal product ions, a decrease in sequence-informative ions, and reduced overall ion abundances. The enhanced sequence coverage afforded by IRMPD of supercharged ions was demonstrated for a variety of model peptides, as well as for a tryptic digest of cytochrome c.     

Characterization of Tyrosine Nitration and Cysteine Nitrosylation Modifications by Metastable Atom-Activation Dissociation Mass Spectrometry

The fragmentation behavior of nitrated and S-nitrosylated peptides were studied using collision induced dissociation (CID) and metastable atom-activated dissociation mass spectrometry (MAD-MS). Various charge states, such as 1+, 2+, 3+, 2–, of modified and unmodified peptides were exposed to a beam of high kinetic energy helium (He) metastable atoms resulting in extensive backbone fragmentation with significant retention of the post-translation modifications (PTMs). Whereas the high electron affinity of the nitrotyrosine moiety quenches radical chemistry and fragmentation in electron capture dissociation (ECD) and electron transfer dissociation (ETD), MAD does produce numerous backbone cleavages in the vicinity of the modification. Fragment ions of nitrosylated cysteine modifications typically exhibit more abundant neutral losses than nitrated tyrosine modifications because of the extremely labile nature of the nitrosylated cysteine residues. However, compared with CID, MAD produced between 66% and 86% more fragment ions, which preserved the labile –NO modification. MAD was also able to differentiate I/L residues in the modified peptides. MAD is able to induce radical ion chemistry even in the presence of strong radical traps and therefore offers unique advantages to ECD, ETD, and CID for determination of PTMs such as nitrated and S-nitrosylated peptides.     

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides

In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification. Here, we have investigated the electron capture dissociation (ECD) and collision-induced dissociation (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains.     

Quantification of intact covalently metal labeled proteins using ESI‐MS/MS

Mass spectrometric methods matured from the successful qualitative characterization of proteins in complex mixtures into methods for quantitative proteomics often based on chemical tags with stable isotope labeling. In the study presented here, we extended the application of lanthanide‐ion‐based tags from the quantification using inductively coupled plasma‐MS into the quantification of labeled intact proteins using electrospray ionization (ESI)‐MS and ESI‐MS/MS. We applied the metal chelate tag MeCAT‐iodoacetamide (IA) (1,4,7,10‐tetraazacyclododecane N,N′,N″,N″ ′‐tetra acetic acid with a IA reactive site). Labeled proteins were separated using C3‐reversed phase‐high‐performance liquid chromatography interfaced to ESI‐MS. We could prove that even large proteins were completely labeled at all available cysteine residues using MeCAT‐IA with only a small excess of reagent. Fragmentation of labeled proteins either using infrared multiphoton dissociation in Fourier transform ion cyclotron resonance‐MS or higher‐energy collision dissociation with an Orbitrap gave characteristic fragments. We used these fragments to quantify several intact proteins avoiding digestion. To demonstrate the applicability, human serum albumin was quantified in blood serum. The high‐performance liquid chromatography/ESI‐MS/MS quantification data were validated using inductively coupled plasma‐MS. Because the metal within the tag may be any of the lanthanides, multiplexing capabilities are inherent. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of protostane triterpenoids in Alisma orientalis by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

A reliable and sensitive ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) method has been optimized and established for analysis of protostane triterpenoids in a commonly used traditional Chinese herbal medicine Alisma orientalis (Sam.) Juzep. The separation of crude extract of A. orientalis was achieved on a Waters ACQUITY HSS T3 column (100 mm × 2.1 mm, 1.8 µm) eluting with 0.1% (v/v) formic acid/acetonitrile. A total of 20 protostane triterpenoids including 19 known compounds and a new one were well separated within 7 min. The collision‐induced dissociation (CID) tandem mass spectrometric (MS/MS) fragmentation patterns of protostane triterpenoids was firstly reported in this study. The hydrogen rearrangement at the C‐23‐OH leads to dissociation of the bond between C‐23 and C‐24 in the protostane triterpenoid skeleton during the CID process. This dissociation was the characteristic CID fragmentation pathway of this class of triterpenoids, and was useful for further differentiation of some positional isomers which contain an acetyl unit on the C‐23 or C‐24 position. The identities of isolated compounds were identified by comparing their retention times and CID fragmentation behaviors with those of reference standards or tentatively assigned by matching the empirical molecular formulae with those reported in the literature. It is concluded that this newly established UPLC/Q‐TOF‐MS method is a powerful approach for structural elucidation of protostane triterpenoids isolated from A. orientalis. Copyright © 2010 John Wiley & Sons, Ltd.     

Free radical-induced site-specific peptide cleavage in the gas phase: Low-energy collision-induced dissociation in ESI- and MALDI mass spectrometry

Protein identification is routinely accomplished by peptide sequencing using mass spectrometry (MS) after enzymatic digestion. Site-specific chemical modification may improve peptide ionization efficiency or sequence coverage in mass spectrometry. We report herein that amino group of lysine residue in peptides can be selectively modified by reaction with a peroxycarbonate and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID) in electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) MS. Selective modification of lysine residue in peptides by our strategy can induce specific peptide cleavage at or near the lysine site. Studies using deuterated analogues of modified lysine indicate that fragmentation of the modified peptides involves apparent free-radical processes that lead to peptide chain fragmentation and side-chain loss. The formation of a-, c-, or z-types of ions in MS is reminiscent of the proposed free-radical mechanisms in low-energy electron capture dissociation (ECD) processes that may have better sequence coverage than that of the conventional CID method. This site-specific cleavage of peptides by free radical- promoted processes is feasible and such strategies may aid the protein sequencing analysis and have potential applications in top-down proteomics.     

Combined infrared multiphoton dissociation and electron capture dissociation with a hollow electron beam in Fourier transform ion cyclotron resonance mass spectrometry

An electron injection system based on an indirectly heated ring-shaped dispenser cathode has been developed and installed in a 7 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This new hardware design allows high-rate electron capture dissociation (ECD) to be carried out by a hollow electron beam coaxial with the ion cyclotron resonance (ICR) trap. Infrared multiphoton dissociation (IRMPD) can also be performed with an on-axis IR-laser beam passing through a hole at the centre of the dispenser cathode. Electron and photon irradiation times of the order of 100 ms are required for efficient ECD and IRMPD, respectively. As ECD and IRMPD generate fragments of different types (mostly c, z and b, y, respectively), complementary structural information that improves the characterization of peptides and proteins by FTICR mass spectrometry can be obtained. The developed technique enables the consecutive or simultaneous use of the ECD and IRMPD methods within a single FTICR experimental sequence and on the same ensemble of trapped ions in multistage tandem (MS/MS/MS or MS(n)) mass spectrometry. Flexible changing between ECD and IRMPD should present advantages for the analysis of protein digests separated by liquid chromatography prior to FTICRMS. Furthermore, ion activation by either electron or laser irradiation prior to, as well as after, dissociation by IRMPD or ECD increases the efficiency of ion fragmentation, including the w-type fragment ion formation, and improves sequencing of peptides with multiple disulfide bridges. The developed instrumental configuration is essential for combined ECD and IRMPD on FTICR mass spectrometers with limited access into the ICR trap.     

Organization of nucleobase‐functionalized β‐peptides investigated by soft electrospray ionization mass spectrometry

The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI‐MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR‐MS) as well as a quadrupole/time‐of‐flight tandem mass spectrometer (QqToF‐MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical β‐peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of β‐peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR‐MS and QqToF‐MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)‐MS/MS and infrared multiphoton dissociation (IRMPD)‐MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd.