On the benefits of acquiring peptide fragment ions at high measured mass accuracy

The advantages and disadvantages of acquiring tandem mass spectra by collision-induced dissociation (CID) of peptides in linear ion trap Fourier-transform hybrid instruments are described. These instruments offer the possibility to transfer fragment ions from the linear ion trap to the FT-based analyzer for analysis with both high resolution and high mass accuracy. In addition, performing CID during the transfer of ions from the linear ion trap (LTQ) to the FT analyzer is also possible in instruments containing an additional collision cell (i.e., the "C-trap" in the LTQ-Orbitrap), resulting in tandem mass spectra over the full m/z range and not limited by the ejection q value of the LTQ. Our results show that these scan modes have lower duty cycles than tandem mass spectra acquired in the LTQ with nominal mass resolution, and typically result in fewer peptide identifications during data-dependent analysis of complex samples. However, the higher measured mass accuracy and resolution provides more specificity and hence provides a lower false positive ratio for the same number of true positives during database search of peptide tandem mass spectra. In addition, the search for modified and unexpected peptides is greatly facilitated with this data acquisition mode. It is therefore concluded that acquisition of tandem mass spectral data with high measured mass accuracy and resolution is a competitive alternative to "classical" data acquisition strategies, especially in situations of complex searches from large databases, searches for modified peptides, or for peptides resulting from unspecific cleavages.     

Neutral fragment mass spectra via ambient thermal dissociation of peptide and protein ions

A novel method for the fragmentation of peptide and protein ions at atmospheric pressure outside the mass spectrometer is described. Peptide/protein ions generated by electrosonic spray ionization (ESSI) are carried through a heated coiled metal tube where they fragment. Fragment ions of types a, b, and y are observed for peptides such as angiotensin II and bradykinin. In the case of phosphopeptides, informative b and y ions which preserve the labile phosphate groups are observed in the negative ion mode, which is potentially useful in the location of phosphorylation sites in proteins through chemical analysis of phosphopeptides. The thermal dissociation method extends to proteins such as ubiquitin and myoglobin, giving rise to y-type and other fragment ions. The most important feature of this method is that it also allows characterization of the neutral fragments arising from thermal dissociation by use of on-line corona discharge ionization. This neutral re-ionization experiment is much easier to perform outside the mass spectrometer than as conventionally done, in vacuum. It yields increased structural information from the resulting mass spectra in both the positive and the negative ion modes.     

Surface-induced dissociation of peptide ions in Fourier-transform mass spectrometry

Peptide molecular ion species up to m/z 3055 introduced into a Fourier-transform mass spectrometer can be made to undergo extensive fragmentation by electrically floating the ion cell. The proportion of ions dissociated increases with increasing voltage, with 48 eV producing the highest absolute abundance of fragment ions above m/z 200. At this energy, spectra closely resemble those from photodissociation at 193 nm, indicating an internal energy deposition of 6–7 eV; change of product abundances with kinetic energy resembles a conventional breakdown curve. The precursor ions apparently are electrostatically attracted to strike screen wires across the ion cell entrance, producing daughter ions of low kinetic energy.

     

Evidence for structural variants of a- and b-type peptide fragment ions using combined ion mobility/mass spectrometry

Tandem mass spectrometry (MS/MS) of peptides plays a key role in the field of proteomics, and an understanding of the fragmentation mechanisms involved is vital for data interpretation. Not all the fragment ions observed by low-energy collision-induced dissociation of protonated peptides are readily explained by the generally accepted structures for a- and b-ions. The possibility of a macrocyclic structure for b-type ions has been recently proposed. In this study, we have undertaken investigations of linear protonated YAGFL-NH(2), N-acetylated-YAGFL-NH(2), and cyclo-(YAGFL) peptides and their fragments using a combination of ion mobility (IM) separation and mass spectrometry. The use of IM in this work both gives insight into relative structural forms of the ion species and crucial separation of isobaric species. Our study provides compelling evidence for the formation of a stable macrocyclic structure for the b(5) ion generated by fragmentation of protonated linear YAGFL-NH(2). Additionally we demonstrate that the a(4) ion fragment of protonated YAGFL-NH(2) has at least two structures; one of which is attributable to a macrocyclic structure on the basis of its subsequent fragmentation. More generally, this work emphasizes the value of combined IM-MS/MS in probing the detailed fragmentation mechanisms of peptide ions, and illustrates the use of combined ion mobility/collisional activation/mass spectrometry analysis in achieving an effective enhancement of the resolution of the mobility separator.     

Hybrid tandem mass spectrometry of electrospray-produced peptide ions

The collision-induced decomposition spectra of electrospray-produced molecule ions were investigated on a hybrid tandem mass spectrometer. The molecular mass of the peptides investigated ranged from m/z 409 (as the singly protonated molcule ion) to 1758 (as the singly, doubly and triply protonated molecules). The effect of changing the center-of-mass collision energy by changing either the laboratory collision energy or the mass of the target gas was investigated. The spectra of all ions investigated showed a large number of sequence ions representative of the peptides' structures.     

The internal energy of product ions formed in mass spectral reactions

Major factors which determine the distribution of internal energy of a primary product ion A⁺, P(E)_A+, at formation are delineated in terms of the quasi-equilibrium theory and a variety of experimental evidence is offered to support these conclusions. These major factors include: the P(E) of the molecular ion, the rate constants as a function of energy, κ(E), for M⁺·→A⁺ + N⁰ and for competing reactions of M⁺·, and the partitioning of excess internal energy between A⁺ and N⁰. The ‘fluctuation effect’ on this partitioning makes P(E)_A+ relatively insensitive to many structural and energy changes.     

Doubly charged fragment ions in the field ionization mass spectra of alkylbenzenes

It is shown that the radical [C₆H₅C_mH_2m]²⁺ fragment ions found in the field ionization mass spectra of alkylbenzenes are formed via a different adsorption state of the singly charged species than in the case of the formation of [M]²⁺ molecular ions. It is further demonstrated that the primary fragmentation of molecules by the cleavage of C? C bonds results not only from decompositions of molecular ions in the gas phase but also from surface reactions.     

Origin of adduct ions in the electron-capture mass spectrum of tetracyanoethylene

The high-pressure electron-capture (HPEC) mass spectrum of tetracyanoethylene (TCNE) is dominated by unexpected hydrogen atom and hydrocarbon radical adduct ions when methane is used as the buffer gas. The origin of these unexpected ions was investigated by three separate mass spectrometric experiments: the electron-capture (EC) rate constant of TCNE was determined and integrated into a previously developed kinetic model of HPEC ion source events; electron impact mass spectra of TCNE were obtained following exposure of the ion source surfaces to irradiated methane and irradiated carbon dioxide; and TCNE was determined by gas chromatographic introduction into the HPEC ion source with multiple ion monitoring. All of these experiments suggest that the reactions leading to the major adduct ions observed in the HPEC mass spectrum of TCNE are initiated by alteration of TCNE on the walls of the ion source rather than in the gas phase.     

Rapid mass spectral identification of contryphans. Detection of characteristic peptide ions by fragmentation of intact disulfide-bonded peptides in crude venom

The mass spectrometric cleavage of intact disulfide-bonded peptides in conus venom has been investigated. Contryphans containing a single disulfide bond are shown to fragment preferentially at X-Pro bonds, giving rise to linearized, unsymmetrical cystine peptides, which subsequently fragment by multiple pathways at the disulfide bridge. Cleavage at the disulfide bond can be initiated by initial loss of the CalphaH or CbetaH proton, resulting in distinct product ions, with the subsequent loss of elemental sulfur, H2S or H2S2. Contryphans from Conus amadis, Conus loroisii, and Conus striatus are presented as examples, in which detailed assignment of the product ions resulting from tandem mass spectrometric analysis of the intact disulfide is also accomplished. Characteristic fragments arising from conserved contryphan sequences can be used as diagnostic, permitting rapid identification of this class of peptides in crude venom. The observed fragment ions obtained for contryphans in diverse cone snail species are also compared.     

Studies of peptide a- and b-type fragment ions using stable isotope labeling and integrated ion mobility/tandem mass spectrometry

The structures of peptide a- and b-type fragment ions were studied using synthetic peptides including a set of isomeric peptides, differing in the sequence location of an alanine residue labeled with ¹⁵N and uniformly with ¹³C. The pattern of isotope labeling of second-generation fragment ions derived via a _n and b _n ions (where n=4 or 5) suggested that these intermediates existed in part as macrocyclic structures, where alternative sites of ring opening gave rise to different linear forms whose simple cleavage might give rise to the observed final products. Similar conclusions were derived from combined ion mobility/tandem MS analyses where different fragmentation patterns were observed for isomeric a- or b-type ions that display different ion mobilities. These analyses were facilitated by a new approach to the processing of ion mobility/tandem MS data, from which distinct and separate product ion spectra are derived from ions that are incompletely separated by ion mobility. Finally, an example is provided of evidence for a macrocyclic structure for b _n ions where n=8 or 9.     

Structure and fragmentation of b2 ions in peptide mass spectra

In a number of cases the b2 ion observed in peptide mass spectra fragments directly to the a1 ion. The present study examines the scope of this reaction and provides evidence as to the structure(s) of the b2 ions undergoing fragmentation to the a1 ion. The b2 ion H-Ala-Gly+ fragments, in part, to the a1 ion, whereas the isomeric b2 ion H-Gly-Ala+ does not fragment to the a1 ion. Ab initio calculations of ion energies show that this different behavior can be rationalized in terms of protonated oxazolone structures for the b2 ions provided one assumes a reverse activation energy of approximately 1 eV for the reaction b2-->a2; such a reverse activation energy is consistent with experimental kinetic energy release measurements. Experimentally, the H-Aib-Ala+ b2 ion, which must have a protonated oxazolone structure, fragments extensively to the a1 ion. We conclude that the proposal by Eckart et al. (J. Am. Soc. Mass Spectrom. 1998, 9, 1002) that the b2 ions which undergo fragmentation to a1 ions have an immonium ion structure is not necessary to rationalize the results, but that the fragmentation does occur from a protonated oxazolone structure. It is shown that the b2-->a1 reaction occurs extensively when the C-terminus residue in the b2 ion is Gly and with less facility when the C-terminus residue is Ala. When the C-terminus residue is Val or larger, the b2-->a1 reaction cannot compete with the b2-->a2 fragmentation reaction. Some preliminary results on the fragmentation of a2 ions are reported.     

Formation of c1 fragment ions in collision-induced dissociation of glutamine-containing peptide ions: a tip for de novo sequencing

A c1 ion was observed with significant yield in the tandem mass (MS/MS) spectra of peptide ions containing glutamine as the second amino acid residue from the N-terminus. The c1 fragment was generated independently of the N-terminal residue of the peptide, but its abundance was strongly dependent on the side-chain identity. This ion is not a common fragmentation product in low-energy collision-induced dissociation of peptide ions, but it assists in identification of the first two amino acid residues, often difficult due to a low or absent signal from the heaviest y ion. A consecutive fragmentation mechanism is proposed, involving a b2 ion with a six-membered ring as an intermediate, to explain the exceptional stability of the c1 fragment ion. The utility of this information is discussed, especially in de novo sequencing of peptide ions.     

Development of a dedicated peptide tandem mass spectral library for conservation science

In recent years, the use of liquid chromatography tandem mass spectrometry (LC–MS/MS) on tryptic digests of cultural heritage objects has attracted much attention. It allows for unambiguous identification of peptides and proteins, and even in complex mixtures species-specific identification becomes feasible with minimal sample consumption. Determination of the peptides is commonly based on theoretical cleavage of known protein sequences and on comparison of the expected peptide fragments with those found in the MS/MS spectra. In this approach, complex computer programs, such as Mascot, perform well identifying known proteins, but fail when protein sequences are unknown or incomplete. Often, when trying to distinguish evolutionarily well preserved collagens of different species, Mascot lacks the required specificity. Complementary and often more accurate information on the proteins can be obtained using a reference library of MS/MS spectra of species-specific peptides. Therefore, a library dedicated to various sources of proteins in works of art was set up, with an initial focus on collagen rich materials. This paper discusses the construction and the advantages of this spectral library for conservation science, and its application on a number of samples from historical works of art.     

Negative ion electrospray ionization mass spectral study of dicarboxylic acids in the presence of halide ions

The negative ion electrospray ionization (ESI) mass spectra of a series of dicarboxylic acids, a pair of isomeric (cis/trans) dicarboxylic acids and two pairs of isomeric (positional) substituted benzoic acids, including a pair of hydroxybenzoic acids, were recorded in the presence of halide ions (F(-), Cl(-), Br(-) and I(-)). The ESI mass spectra contained [M--H](-) and [M+X](-) ions, and formation of these ions is found to be characteristic of both the analyte and the halide ion used. The analytes showed a greater tendency to form adduct ions with Cl(-) under ESI conditions compared with the other halide ions used. The isomeric compounds yielded distinct spectra by which the isomers could be easily distinguished. The collision-induced dissociation mass spectra of [M+X](-) ions reflected the gas-phase basicities of both the halide ion and [M--H](-) ion of the analyte. However, the relative ordering of gas-phase basicities of all analyte [M--H](-) and halide ions could not account for the dominance of chloride ion adducts in ESI mass spectra of the analytes mixed with equimolar quantities of the four halides.     

Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic fragment ions for protein ubiquitination

In this work, synthetic peptides were used to determine the fragmentation behavior of ubiquitinated peptides and to find ions diagnostic for peptide ubiquitination. The ubiquitin-calmodulin peptide1 was chosen as the model peptide for naturally occurring ubiquitinated proteins cleaved with endoproteinase gluC. In addition, the fragmentation behavior of model ubiquitinated peptides produced by tryptic digestion was also of great interest since the standard protocols for proteomics-based protein identification use trypsin as the protease. Attachment of ubiquitin to a target protein results in a branched structure, but only ions from the ubiquitin side chain (and the lysine to which it is attached) can be used as diagnostic ions, since fragment ions that contain other amino acids from the parent protein will vary in mass. Characteristic b-type fragment ions from the gluC cleavage of the ubiquitin side chain (designated as b ions) were found which involve only the ubiquitin tail (b2, b3, b4, b5 and b6 ions at m/z 189.06, 302.12, 439.18, 552.30 and 651.30, respectively). Maximum production of these ions occurred at a collision energy of 45 eV in a Q-TOF instrument. Although a non-ubiquitinated peptide may produce isobaric fragment ions, it is unlikely that it can produce these ions in combination. With liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments, ubiquitinated peptides can readily be determined by surveying the reconstructed or extracted ion chromatograms of the diagnostic fragment ions for common peaks. Characteristic ions resulting from tryptic cleavage of the side chain were found in cleavage products with a missed cleavage, resulting in a LRGG- tag instead of a GG- tag. For the LRGG-tagged peptide, diagnostic MS/MS fragment ions (at m/z 270.17 and 384.21) from the ubiquitin tail (b2 and b4, respectively) were found, along with an internal fragment ion (LRGGK-28) at m/z 484.30. These ions should prove useful in precursor-ion scanning experiments for identifying peptides modified by attachment of ubiquitin, and for locating the site of ubiquitin attachment.     

Factors influencing the formation of some characteristic fragment ions in the mass spectra of 16-trimethylsilyloxy androstanes

The mass spectra of the trimethylsilyl derivatives of several steroids containing a 16-hydroxy function are presented. The mechanisms giving rise to various characteristic fragment ions were investigated with the aid of deuterium and ¹⁸O-labeling.     

Measurement of initial translational energies of peptide ions in laser desorption/ionization mass spectrometry

The initial kinetic energy distribution of [Arg]-vasopressin molecular ions generated by matrix-assisted UV laser desorption/ionization was measured using a delayed ion extraction, linear time-of-flight mass spectrometer. Energy distributions of the nicotinic acid matrix ions, with or without the presence of peptide, were also measured. These were compared with the kinetic energy distribution of gramicidin-S ions using IR laser desorption. The measured molecular ion kinetic energy distribution from vasopressin is much broader than that from gramicidin-S, and is characterized by a high-energy tail that most likely results from entrainment of anlayte ions in the higher velocity matrix ions and fragments as they leave the surface.