Characterization of dipeptide isomers by tandem mass spectrometry of their mono- versus dilithiated complexes

The Li+ complexes of the isomeric dipeptide pairs PheGly/GlyPhe, PheAla/AlaPhe, and TrpAla/AlaTrp, namely, [Pep + Li]+, and of the corresponding lithium carboxylates, namely, [Pep - H + 2Li]+, are produced in the gas phase by desorption ionization, and their unimolecular chemistry is probed by tandem mass spectrometry experiments at various activation conditions. At low internal energies, monolithiated isomers dissociate to the same products, formed through a mixed anhydride intermediate in which the sequence information is lost. Isomerization to the mixed anhydride is less competitive at higher internal energies, which start promoting sequence-specific fragmentations. On the other hand, dilithiated isomers (they contain a permanent COO-Li+ salt bridge) do not rearrange to an anhydride and give rise to substantially different fragmentation patterns; structurally diagnostic c1- and y1-type fragments are observed at all internal energies, allowing for unequivocal sequence assignment. The mono- and dilithiated peptides undergo loss of their aromatic side chain to form distonic radical ions carrying Li+ charge(s) and one unpaired electron at an alpha-C atom of the peptide backbone. The yield of such metal-bound peptide radicals is particularly high from the dilithiated complexes, [Pep - H + 2Li]+. Upon activation, the Li+ ions become mobile and can be shuttled to the various basic sites of the dipeptides, where they may initiate backbone fragmentation or the elimination of small neutral molecules.     

Carbohydrate structural isomers analyzed by sequential mass spectrometry

Consistent with the goals of a comprehensive carbohydrate sequencing strategy, we extend earlier reports to include the characterization of structural (constitutional) isomers. Protocols were developed around ion trap instrumentation providing sequential mass spectrometry (MSn) and supported with automation and related computational tools. These strategies have been built on the principle that for a single structure all product spectra upon sequential fragmentation are reproducible and each stage represents a rational spectrum of its precursor; i.e., all major fragments should be accounted for. Anomalous ions at any stage are clues indicating the presence of structural isomers. Gas-phase isolation and subsequent fragmentation of such ions provide an opportunity to specifically resolve selected structures for their detailed characterization. Importantly, some isomers were not detected following MS2 and required multiple (MSn>2) stages for their characterization. Derivatization remains critical to position substructures in a glycan array since product ions carry fragmentation "scars" throughout the MSn tree. Equally as important are the pathway relationships between each stage and the greater yield of fragments with the smaller number of oscillators. Applications were directed to the structural isomers in ovalbumin and IgG, where, in the latter case, several previously unreported glycans were detected. Procedures were supported with bioinformatics tools for assimilating structure from the MSn data sets.     

Characterization of cellobiohydrolase I N-glycans and differentiation of their phosphorylated isomers by capillary electrophoresis-Q-Trap mass spectrometry

A capillary electrophoresis-mass spectrometric (CE-MS) method is described for the simultaneous analysis of uncharged and charged glycans. The glycans were labeled with the negatively charged tag 8-aminopyrene-1,3,6-trisulfonate by reductive amination and separated in an ammonium acetate buffer. A Q-Trap instrument was used for mass spectrometric detection. The CE-MS method was first optimized using maltooligosaccharides and ribonuclease B N-glycans and then applied to the characterization of enzymatically released N-glycans from the glycoprotein cellobiohydrolase I. The method, as developed, allowed differentiation of phosphorylated isomers and MS/MS provided useful structural information. Further structural evidence was obtained by studying the methylated glycans in off-line ESI-MS/MS experiments and by using a combination of chemical and enzymatic sequencing.     

Differentiation of complex lipid isomers by radical-directed dissociation mass spectrometry

Contemporary lipidomics protocols are dependent on conventional tandem mass spectrometry for lipid identification. This approach is extremely powerful for determining lipid class and identifying the number of carbons and the degree of unsaturation of any acyl-chain substituents. Such analyses are however, blind to isomeric variants arising from different carbon-carbon bonding motifs within these chains including double bond position, chain branching, and cyclic structures. This limitation arises from the fact that conventional, low energy collision-induced dissociation of even-electron lipid ions does not give rise to product ions from intrachain fragmentation of the fatty acyl moieties. To overcome this limitation, we have applied radical-directed dissociation (RDD) to the study of lipids for the first time. In this approach, bifunctional molecules that contain a photocaged radical initiator and a lipid-adducting group, such as 4-iodoaniline and 4-iodobenzoic acid, are used to form noncovalent complexes (i.e., adduct ions) with a lipid during electrospray ionization. Laser irradiation of these complexes at UV wavelengths (266 nm) cleaves the carbon-iodine bond to liberate a highly reactive phenyl radical. Subsequent activation of the nascent radical ions results in RDD with significant intrachain fragmentation of acyl moieties. This approach provides diagnostic fragments that are associated with the double bond position and the positions of chain-branching in glycerophospholipids, sphingomyelins and triacylglycerols and thus can be used to differentiate isomeric lipids differing only in such motifs. RDD is demonstrated for well-defined lipid standards and also reveals lipid structural diversity in olive oil and human very-low density lipoprotein.     

Characterization of polyphosphates by electrospray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was applied for the characterization of inorganic polyphosphates [orthophosphate, pyrophosphate, tripolyphosphate, trimetaphosphate, and tetrapolyphosphatel. The high selectivity of ESI-MS allows the detection of different polyphosphate species without preseparation by ion chromatography or capillary electrophoresis. Furthermore, ESI-MS does not require the incorporation of UV-absorbing chromophores into the analytical method for the detection of phosphates, unlike conventional UV-chromatographic methods. Limits of detection by ESI-MS were estimated to range from approximately 1 to 10 ng/mL. The quantification of polyphosphate samples as single-component and multicomponent mixtures was investigated. Linear signal response for single-component samples ranged from the limit of detection to approximately 10 microg/mL Quantification of polyphosphate in streamwater is demonstrated using the standard addition method. The effect of multi-polyphosphate components and salts on signal response was also studied. For concentrations less than 2.0 microg/mL, signal response from a tetrapolyphosphate sample was comparable to those obtained from tetrapolyphosphate-tripolyphosphate mixtures. Signal response obtained from tetrapolyphosphate in the presence of tripolyphosphate or NH4NO3 at higher concentrations (approximately 50 microg/mL and 35 microg/mL, respectively) was significantly lower relative to single-component standards (approximately 40%-70%).     

Differentiation of hydroxyproline isomers and isobars in peptides by tandem mass spectrometry

The isomeric 3- and 4-hydroxyprolines are isobaric with the isomers leucine and isoleucine, and all four have, therefore, the same "residue mass" of 113. Secondary fragmentation processes were found that differentiate the hydroxyproline isomers from each other and from the leucines. Variants of synthetic bradykinin containing one or two hydroxyproline moieties were prepared by using manual Edman degradation and/or enzymatic methods. The tandem mass spectra of these peptides were recorded. The C-terminal wn fragment ions allow the differentiation of 4-hydroxyproline from the 3-isomer and isoleucine, while the N-terminal an ions containing 4-hydroxyproline undergo H2O elimination to differentiate this amino acid from the 3-isomer and leucine. Lys-C digestion of a mussel adhesive protein produced a set of decapeptides varying in the degree of hydroxylation of proline and tyrosine. Heterogeneity with respect to 3-hydroxyproline and 4-hydroxyproline at a certain position in these peptides was assessed by tandem mass spectrometry based on the wn ion series in the CID spectra of these Lys-C peptides. Some N-terminal ions further allow for the differentiation of these two isomeric species.     

Cobalt nanoparticles prepared by three different methods

This work aimed to study cobalt nanoparticles (Co-NPs) preparation using three different methods in order to evaluate the effect of synthesis variables that can influence the nanoparticle size distribution and particle shape. The synthesised nanoparticles were characterised by Transmission Electron Microscopy. The first synthesis employed decomposition of Co₂(CO)₈, at high temperatures. This procedure resulted in spherical nanoparticles with low size distribution. The size of Co-NPs could be tuned by modification of precursor/surfactant, nevertheless the stirring and injection time influenced the size distribution. Using polyol process, at high temperatures, it was produced undefined-shape nanoparticles. This result suggests that the solution composition, i.e. the amount of trioctylphosphine and oleic acid was not suitable to control both size and shape of nanoparticles. Finally, the method based on reduction with NaBH₄ resulted spherical nanoparticles with tiny sizes, indicating that in this case a variation on amount of reductant would be more efficient on the particle size control than a variation in concentration of oleic acid. These results indicated that, for each method, a different variable exists for the control of the distribution size and the shape of the formed particles.     

Identification and quantitation of unsaturated fatty acid isomers by electrospray ionization tandem mass spectrometry: a shotgun lipidomics approach

Identification and quantification of unsaturated fatty acid (FA) isomers in a biological system are significant in the study of lipid metabolism and catabolism, membrane biophysics, and pathogenesis of diseases but are challenging in lipidomics. We developed a novel approach for identification and quantitation of unsaturated FA isomers by exploiting two facts: (1) unsaturated FA anions yield fragment ion(s) from loss of CO(2) or H(2)O from the anions upon collision-induced dissociation; and (2) the fragment ions yielded from discrete FA isomers have distinct profiles of the fragment ion intensity vs. collision conditions. These distinct profiles likely result from the differential interactions of the negative charge of the fragment ion with the electron clouds of the double bonds due to their different distances in discrete FA isomers. The novel approach was also extended to analyze the double bond isomers of FA chains present in phospholipids by multistage tandem mass spectrometry. Collectively, we developed a new approach for identification and quantification of the double bond isomers of endogenous FA species or FA chains present in intact phospholipid species. We believe that this approach should further advance the lipidomic power for identification of the biochemical mechanisms underlying metabolic diseases.     

Characterization of hydrophobic peptides by atmospheric pressure photoionization-mass spectrometry and tandem mass spectrometry

The use of photoionization at atmospheric pressure shows great potential for the mass analysis of large apolar or hydrophobic peptides. Mass spectra that were obtained using this technique showed mainly singly charged ions. While polar peptides spectra do not produce fragment ions, others lead to B-type or C-type in-source fragmentation. These dissociation reactions, which could involve electron capture dissociation processes in the case of the C-type ions, are observed for hydrophobic peptides. Both the compatibility of this ionization mode with reversed- or normal-phase liquid chromatographic separation and its sensitivity allow liquid chromatography coupling to both mass spectrometry and tandem mass spectrometry for the analyses of hydrophobic peptide mixtures. Atmospheric pressure photoionization seems to be an interesting alternative method to study hydrophobic peptides that are not easily ionizable by more classical ionization techniques such as electrospray ionization and matrix-assisted laser desorption/ionization.     

Characterization of polyubiquitin chain structure by middle-down mass spectrometry

Ubiquitin (Ub) is a 76 amino acid polypeptide that modifies a wide range of proteins in the types of monomer or polymers, and functional consequence of ubiquitination is modulated by the length and topologies of polyUb chains. Whereas polyUb chains are usually analyzed by fully trypsin digestion and mass spectrometry (MS), we present here a middle-down strategy to characterize the structure of polyUb chains by high-resolution mass spectrometry (MS). Under optimized condition, native folded polyUb is partially trypsinized exclusively at the R74 residue, generating a large Ub fragment (1-74 residues termed UbR74) and its ubiquitinated form with a diglycine tag (UbR74-GG). The molar ratio between UbR74 and UbR74-GG reflects the length of homogeneous polyUb chains (i.e., 1:1 for the dimer, 1:2 for the trimer, 1:3 for the tetramer, and so on). Moreover, lysine residues in ubiquitin used for chain linkages are detectable by MS/MS and MS/MS/MS of large GG-tagged Ub fragments. The strategy was validated using a number of ubiquitin polymers, including K48-linked human di-Ub, K63-linked human tetra-Ub, as well as His-tagged polyUb chains purified from yeast under native condition. The potential of this strategy to analyze polyUb chains with mixed linkages (e.g., forked chains) is also discussed. Together, this middle-down MS strategy provides a novel complementary method for studying the length and linkages of complex polyUb chain structures.     

Characterization of alkali-treated collagen gels prepared by different crosslinkers

We have developed a naturally-derived crosslinker named malic acid derivative (MAD). In the present study, we prepared alkali-treated collagen (AlCol) gels with different crosslinkers including MAD and commercially available crosslinkers such as 1-ethyl-3-(3(′-dimethylaminopropyl) carbodiimide (EDC) and glutaraldehyde (GA). There are named as AlCol-MAD, AlCol-EDC, and AlCol-GA. We then compared their physicochemical properties. The residual amino groups in AlCol-MAD were not detected at MAD concentrations higher than 30 mM. On the other hand, the residual amino groups in AlCol-EDC and AlCol-GA were detected at crosslinker concentrations of 30 mM. The swelling ratios of AlCol-MAD, AlCol-EDC, and AlCol-GA decreased with increasing crosslinker concentration. Enzymatic degradation rate of AlCol-GA was slower than that of AlCol-MAD and AlCol-EDC. The cytotoxicity of MAD was clearly lower than that of EDC and GA. The number of adhered L929 on AlCol-MAD was higher than on AlCol-EDC and AlCol-GA after incubation for 1 day. After the culture for 3 and 7 days, excellent growth of L929 was observed on AlCol-MAD. These results suggested that MAD was excellent crosslinker for the reactivity with amino groups and cytocompatibility. Therefore, the resulting AlCol-MAD has potential for various biomedical applications like tissue engineering scaffolds and carrier for drug delivery systems.     

Characterization of N-linked oligosaccharides by electrospray and tandem mass spectrometry.

Electrospray and tandem mass spectrometry are used to characterize underivatized oligosaccharides that have been digested from asparagine side chains of glycoproteins. Oligosaccharides that contain sialic acids were detected with the best sensitivity in the negative-ion detection mode whereas those that do not contain sialic acid were detected with the best sensitivity in the positive-ion detection mode. The positive-ion abundances of oligosaccharides were greatly enhanced in electrospray mass spectra by adding 10 mM sodium acetate or ammonium acetate to the sample solvent. Tandem mass spectrometry was used to determine primary structural features of the oligosaccharides. Methodology that has been developed on branched high-mannose, hybrid, and complex carbohydrate standards was applied to a mixture of oligosaccharides that were digested with N-glycanase from the glycoprotein, ovalbumin. The composition and relative abundances of individual oligosaccharides obtained from the electrospray mass spectrum compare favorably to those obtained by anion-exchange chromatography/pulsed amperometric detection and by gel permeation chromatography of the oligosaccharides after radiolabelling the reducing end of the carbohydrates. The oligosaccharide content of ovalbumin was independently determined from the heterogeneity observed in the electrospray mass spectrum of the intact 44-kDa glycoprotein. Comparison of the oligosaccharide compositions determined before and after enzymatic digestion shows a selective digestion of high-mannose and low molecular weight oligosaccharides by N-glycanase.     

Characterization of phosphoantigens by high-performance anion-exchange chromatography-electrospray ionization ion trap mass spectrometry and nanoelectrospray ionization ion trap mass spectrometry

New phosphorylated microbial metabolites referred to as phosphoantigens activate immune responses in humans. Although these molecules have leading applications in medical research, no direct method allows their rapid and unambiguous structural identification. Here, we interfaced online HPAEC (high performance anion-exchange chromatography) with ESI-ITMS (electrospray ionization ion trap mass spectrometry) to identify such pyrophosphorylated molecules. A self-regenerating anion suppressor located upstream of electrospray ionization enabled the simultaneous detection of pyrophosphoester by conductimetry, UV and MS. By HPAEC-ITMS and HPAEC-ITMS2, a single run permitted characterization of reference phosphoantigens and of related structures. Although all compounds were resolved by HPAEC, MS enabled their detection and identification by [M-H]- and fragment ions. Isobaric phosphoantigen analogues were also separated by HPAEC and distinguished by MS2. The relevance of this device was demonstrated for phosphoantigens analysis in human urine and plasma. Furthermore, identification of natural phosphoantigens by automatically generated 2D mass spectra from nano-ESI-ITMS is presented. This last technique permits the simultaneous performance of molecular screening of natural phosphoantigen extracts and their identification.     

Simultaneous characterization of perfluoroalkyl carboxylate, sulfonate, and sulfonamide isomers by liquid chromatography-tandem mass spectrometry

A comprehensive method was developed to simultaneously separate and detect perfluorinated acid (PFA) and PFA-precursor isomers using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A linear perfluorooctyl stationary phase and acidified mobile phase increased separation efficiency, relative to alkyl stationary phases, for the many perfluoroalkyl carboxylate (PFCA), perfluoroalkyl sulfonate (PFSA), and perfluorooctyl sulfonamide (PFOSA) isomers and in combination with their distinct MS/MS transitions allowed full resolution of most isomers in standards. Utilizing the absence of the "9-series" and "0-series" product ions, several perfluorooctane sulfonate (C8F17SO3-, PFOS) isomers were structurally elucidated. In human serum, only perfluorooctane sulfonamide (C8F17SO2NH2, FOSA) and PFOS consisted of significant quantities of branched isomers, whereas PFCAs were predominantly linear. Interferences that coelute with the m/z 499 --> 80 transition of PFOS on alkyl stationary phases were simultaneously separated and identified as taurodeoxycholate isomers, removal of which permitted the use of the more sensitive m/z 80 product ion and a resulting 20-fold decrease in PFOS detection limits compared to the m/z 499 --> 99 transition (0.8 pg versus 20 pg using m/z 80 and 99, respectively). Interferences in human serum which caused a 10-20-fold over-reporting of perfluorohexane sulfonate (C6F13SO3-, PFHxS) concentrations on alkyl stationary phases were also simultaneously separated from linear PFHxS and identified as endogenous steroid sulfates. PFOSA isomers, generated with human microsomes, had different rates of metabolism, suggesting that the perfluoroalkyl branching pattern may affect the biological properties of individual isomers. This fact, and for reasons of improved accuracy and sensitivity, investigators are urged to utilize more efficient separation methods capable of isomer characterization in perfluoroalkyl research.     

Characterization of Enterobacteria using MALDI-TOF mass spectrometry

A method is proposed for the rapid classification of Gram-negative Enterobacteria using on-slide solubilization and trypsin digestion of proteins, followed by MALDI-TOF MS analysis. Peptides were identified from tryptic digests using microsequencing by tandem mass spectrometry and database searches. Proteins from the outer membrane family (OMP) were consistently identified in the Enterobacteria Escherichia coli, Enterobacter cloacae, Erwinia herbicola, and Salmonella typhimurium. Database searches indicate that these OMP peptides observed are unique to the Enterobacteria order.     

Negative ion mass spectrometry of sialylated carbohydrates: discrimination of N-acetylneuraminic acid linkages by MALDI-TOF and ESI-TOF mass spectrometry

Negative ion MALDI and electrospray fragmentation spectra were recorded from 12 sialylated carbohydrates ranging from trisaccharides to biantennary N-linked glycans. D-Arabinosazone was found to be the most satisfactory MALDI matrix for these compounds. Fragmentation mechanisms were investigated with the aid of several synthesized analogues of the sugars labeled with 13C and 2H. The substitution position of the sialic acid (alpha2-->3 or alpha2-->6) was found to have a dramatic effect on the overall fragmentation pattern of these compounds, and several features of the spectra were identified that allowed the substitution pattern to be determined. In particular, the appearance of an ion at m/z 306 appeared to be diagnostic of the presence of an alpha2-->6-linked sialic acid. Selection and further fragmentation of the in-source (conevoltage) fragment ion corresponding to the trisaccharide Neu5Acalpha2-->3(or 6)Galbeta1-->4GlcNAc from larger, N-linked glycans, ionized by electrospray, gave fragmentation patterns identical to those of the reference trisaccharides, thus providing a method for confirming the sialic acid linkage.     

Characterization of archaeological beeswax by electron ionization and electrospray ionization mass spectrometry

To better detect and identify beeswax in ancient organic residues from archaeological remains, we developed a new analytical methodology consisting of the analysis of (i) the trimethylsilylated organic extract by GC/MS and (ii) the crude extract by ESI-MS. Selective scanning modes, such as SIM or MRM, permit separate quantification of each chemical family (fatty acids, monoesters, monohydroxyesters, and diesters) and allow an improvement in sensitivity and selectivity, allowing the crude extract to be treated without further purification. GC/MS (SIM) was revealed to be a powerful method for the detection of components, with a detection limit down to a total lipid extract in the range of approximately 50 ng in a complex matix, such as archaeological degraded material, whereas ESI-MS/MS is instead used for the detection of nonvolatile biomarkers. Identification by GC/MS (SIM) and ESI-MS/ MS (MRM) of more than 50 biomarkers of beeswax in an Etruscan cup at the parts-per-million level provides the first evidence for the use of this material by the Etruscans as fuel or as a waterproof coating for ceramics.     

Characterization of photooxidized self-assembled monolayers and bilayers by spontaneous desorption mass spectrometry

We show that selected self-assembled monolayers (SAMs) and bilayers are readily characterized by the application of controlled photooxidation and spontaneous desorption mass spectrometry (SDMS) in the negative ion mode. Additionally, SDMS is used to characterize organic and inorganic anionic species adsorbed to the surface of a positively charged SAM surface, 2-aminoethanethiol (AET). Prominent peaks are observed that correspond both to the sulfonate form of each SAM and bilayer and to the anion form of each molecule adsorbed to AET. In addition, fragments of the oxidized thin films were also observed at m/z 80 (SO3-) and 97 (HSO4-). Other prominent fragment peaks more characteristic of the molecule are also seen in the mass spectra.     

Characterization of variable regions of monoclonal antibodies by top-down mass spectrometry

A technique for rapid characterization of variable regions of monoclonal antibodies (mAb) is described. Several intact mAbs were analyzed on a Thermo-Fisher LTQ-Orbitrap high-resolution mass spectrometer (MS) by in-source fragmentation. In-source fragmentation has the unique advantage of fragmenting all charge states of a protein at the same time and, thus, greatly improves the sensitivity of the fragment ions over a true MS/MS experiment, where a single charge state is isolated and fragmented. In addition, immediate fragmentation of the protein before tertiary structure formation may also facilitate protein fragmentation. This technique has been proved very useful for top-down analysis of large proteins. In-source fragmentation of mAbs generated a series of fragment ions. In addition to some small b and y ions from the light chain and heavy chain in the low m/z region, a series of b ions corresponding to N-terminal 106-120 residues of both heavy chain and light chain were observed. The cleavage sites for these b ions happen to be near the linker regions between the variable domains and the constant domains of these antibodies. These b ions, therefore, correspond to the entire variable region of each chain. Similar results were obtained for all mAbs analyzed, including both immunoglobulin G1 and G2 molecules. To further characterize the variable regions, these b ions were isolated and fragmented by collision-induced dissociation in the linear trap, followed by mass analysis in the orbitrap. Large number of product ions was observed from these b ions. Many of these product ions are internal fragments between the two disulfide-linked cysteine residues. To demonstrate the capability of the technique, several mAbs were force-oxidized by treating with tert-butyl hydroperoxide, followed by mass spectrometric analysis. In-source fragmentation and MS/MS of the variable region b ions clearly identified the locations of the oxidized methionine.