Yamil Simón-Manso

Electrospray tandem quadrupole fragmentation of quinolone drugs and related ions. On the reversibility of water loss from protonated molecules.

Selected reaction monitoring (SRM) of quinolone drugs showed different sensitivities in aqueous solution vs. biological extract. The authors suggested formation of two singly protonated molecules with different behavior, one undergoing loss of H(2)O and the other loss of CO(2), so that SRM transitions might depend on the ratios of these forms generated by the electrospray. These surprising results prompted us to re-examine several quinolone drugs and some simpler compounds to further elucidate the mechanisms. We find that the relative contributions of loss of H(2)O vs. loss of CO(2) in tandem mass spectrometric (MS/MS) experiments depend not only on molecular structure and collision energy, but also, in certain cases, on the cone voltage. We further find that many product ions formed by loss of H(2)O can reattach a water molecule in the collision cell, whereas ions formed by loss of CO(2) do not. Since reattachment of H(2)O can occur after water loss in the cone region and prior to selection of the precursor ion, this effect leads to the dependence of MS/MS spectra on the cone voltage used in creating the precursor ion, which explains the formerly observed effect on SRM ratios. Our results support the earlier conclusion that varying amounts of two ions of the same m/z value are responsible for problems in the analysis of these drugs, but the origin is in dehydration/rehydration reactions. Thus, SRM transitions for certain complex compounds may be comparable only when monitored under equivalent ion-forming conditions, including the voltage used in the production of the protonated molecules in the electrospray ionization (ESI) source.

Effect of N-Terminal Glutamic Acid and Glutamine on Fragmentation of Peptide Ions

A prominent dissociation path for electrospray generated tryptic peptide ions is the dissociation of the peptide bond linking the second and third residues from the ammo-terminus. The formation of the resulting b2 and yn−2 fragments has been rationalized by specific facile mechanisms. An examination of spectral libraries shows that this path predominates in diprotonated peptides composed of 12 or fewer residues, with the notable exception of peptides containing glutamine or glutamic acid at the N-terminus. To elucidate the mechanism by which these amino acids affect peptide fragmentation, we synthesized peptides of varying size and composition and examined their MS/MS spectra as a function of collision voltage in a triple quadrupole mass spectrometer. Loss of water from N-terminal glutamic acid and glutamine is observed at a lower voltage than any other fragmentation, leading to cyclization of the terminal residue. This cyclization results in the conversion of the terminal amine group to an imide, which has a lower proton affinity. As a result, the second proton is not localized at the N-terminus but is readily transferred to other sites, leading to fragmentation near the center of the peptide. Further confirmation was obtained by examining peptides with N-terminal pyroglutamic acid and N-acetyl peptides. Peptides with N-terminal proline maintain the trend of forming b2 and yn−2 because their ring contains an imine rather than imide and has sufficient proton affinity to retain the proton at the N-terminus.

Collisional Energy Dependence of Peptide Ion Fragmentation

The energy dependence of fragmentation in a collision cell was measured for 2400 protonated peptide ions derived from the digestion of 24 proteins. The collision voltage at which the sum of the fragment ion abundances was equal to the remaining parent ion (V1/2) was the principal measure of fragmentation effectiveness. Each class of peptides was characterized by a linear relation between V1/2 and m/z whose slope depended on the peptide class and, with little adjustment, intersected the origin. Peptide ions where the number of protons is no greater than the number of arginine residues show the greatest slope, V1/2/(m/z)=0.0472 (all slopes in units of V Da−1 e). For peptides where the number of protons is greater than the number of arginines, but not greater than the total number of basic residues, the slope decreases to 0.0414 for singly charged ions, 0.0382 for doubly charged, 0.0346 for triply charged, and 0.0308 for more highly charged ions. With one mobile proton, the slope is about 0.029 for singly and doubly charged ions and slightly lower for more highly charged ions. With two or more mobile protons the slope is 0.0207. By removing m/z dependence, the deviation of V1/2 from a line provides a relative measure of the ease of fragmentation of an ion in each class. This information can guide the selection of optimal conditions for tandem mass spectrometry studies in collision cells for selected peptide ions as well as aid in comparing the reactivity of ions differing in m/z and charge state.

Analysis of human plasma metabolites across different liquid chromatography/mass spectrometry platforms: Cross-platform transferable chemical signatures

RATIONALE The metabolite profiling of a NIST plasma Standard Reference Material (SRM 1950) on different liquid chromatography/mass spectrometry (LC/MS) platforms showed significant differences. Although these findings suggest caution when interpreting metabolomics results, the degree of overlap of both profiles allowed us to use tandem mass spectral libraries of recurrent spectra to evaluate to what extent these results are transferable across platforms and to develop cross-platform chemical signatures. METHODS Non-targeted global metabolite profiles of SRM 1950 were obtained on different LC/MS platforms using reversed-phase chromatography and different chromatographic scales (conventional HPLC, UHPLC and nanoLC). The data processing and the metabolite differential analysis were carried out using publically available (XCMS), proprietary (Mass Profiler Professional) and in-house software (NIST pipeline). RESULTS Repeatability and intermediate precision showed that the non-targeted SRM 1950 profiling was highly reproducible when working on the same platform (relative standard deviation (RSD) <2%); however, substantial differences were found in the LC/MS patterns originating on different platforms or even using different chromatographic scales (conventional HPLC, UHPLC and nanoLC) on the same platform. A substantial degree of overlap (common molecular features) was also found. A procedure to generate consistent chemical signatures using tandem mass spectral libraries of recurrent spectra is proposed. CONLUSIONS Different platforms rendered significantly different metabolite profiles, but the results were highly reproducible when working within one platform. Tandem mass spectral libraries of recurrent spectra are proposed to evaluate the degree of transferability of chemical signatures generated on different platforms. Chemical signatures based on our procedure are most likely cross-platform transferable.

Unexpected peaks in tandem mass spectra due to reaction of product ions with residual water in mass spectrometer collision cells

RATIONALE Certain product ions in electrospray ionization tandem mass spectrometry are found to react with residual water in the collision cell. This reaction often leads to the formation of ions that cannot be formed directly from the precursor ions, and this complicates the mass spectra and may distort MRM (multiple reaction monitoring) results. METHODS Various drugs, pesticides, metabolites, and other compounds were dissolved in acetonitrile/water/formic acid and studied by electrospray ionization mass spectrometry to record their MS(2) and MS(n) spectra in several mass spectrometers (QqQ, QTOF, IT, and Orbitrap HCD). Certain product ions were found to react with residual water in collision cells. The reaction was confirmed by MS(n) studies and the rate of reaction was determined in the IT instrument using zero collision energy and variable activation times. RESULTS Examples of product ions reacting with water include phenyl and certain substituted phenyl cations, benzoyl-type cations formed from protonated folic acid and similar compounds by loss of the glutamate moiety, product ions formed from protonated cyclic siloxanes by loss of methane, product ions formed from organic phosphates, and certain negative ions. The reactions of product ions with residual water varied greatly in their rate constant and in the extent of reaction (due to isomerization). CONCLUSIONS Various types of product ions react with residual water in mass spectrometer collision cells. As a result, tandem mass spectra may contain unexplained peaks and MRM results may be distorted by the occurrence of such reactions. These often unavoidable reactions must be taken into account when annotating peaks in tandem mass spectra and when interpreting MRM results. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

Loss of H2 and CO from protonated aldehydes in electrospray ionization mass spectrometry

RATIONALE Electrospray ionization mass spectrometry (ESI-MS) of many protonated aldehydes shows loss of CO as a major fragmentation pathway. However, we find that certain aldehydes undergo loss of H2 followed by reaction with water in the collision cell. This complicates interpretation of tandem mass (MS/MS) spectra and affects multiple reaction monitoring (MRM) results. METHODS 3-Formylchromone and other aldehydes were dissolved in acetonitrile/water/formic acid and studied by ESI-MS to record their MS(2) and MS(n) spectra in several mass spectrometers (QqQ, QTOF, ion trap (IT), and Orbitrap HCD). Certain product ions were found to react with water and the rate of reaction was determined in the IT instrument using zero collision energy and variable activation times. Theoretical calculations were performed to help with the interpretation of the fragmentation mechanism. RESULTS Protonated 3-formylchromones and 3-formylcoumarins undergo loss of H2 as a major fragmentation route to yield a ketene cation, which reacts with water to form a protonated carboxylic acid. In general, protonated aldehydes which contain a vicinal group that forms a hydrogen bridge with the formyl group undergo significant loss of H2. Subsequent losses of CO and C3O are also observed. Theoretical calculations suggest mechanistic details for these losses. CONCLUSIONS Loss of H2 is a major fragmentation channel for protonated 3-formychromones and certain other aldehydes and it is followed by reaction with water to produce a protonated carboxylic acid, which undergoes subsequent fragmentation. This presents a problem for reference libraries and raises concerns about MRM results.

Reaction of arylium ions with the collision gas N2 in electrospray ionization mass spectrometry

RATIONALE The tandem mass spectra of many compounds contained peaks which could not have arisen from the precursor ion. Such peaks were found to be due to reaction of arylium ions with N2 in the collision cell. Therefore, this reaction was studied in detail with representative compounds. METHODS Various classes of compounds were dissolved in acetonitrile/water/formic acid and studied by electrospray ionization mass spectrometry to record their MS(2) and pseudo-MS(3) spectra in a QqQ mass spectrometer and their accurate m/z values in an Orbitrap Elite instrument. Arylium ions were found to react with N2 in the collision cell. The reaction was confirmed by pseudo-MS(3) studies, by comparison with authentic diazonium ions, and by the pressure dependence of the product ion survival yield. RESULTS Reactions of arylium ions with N2 were observed with p-toluenesulfonic acid, o-toluenesulfonamide, phenylphosphonic acid, phenol, aniline, aminonaphthalenes, benzoic acid, benzophenone, and other compounds. By using a QqQ mass spectrometer, we observed that the protonated compounds produce arylium ions, which then react with N2 to form diazonium ions. The diazonium ion was produced with N2 but not with Ar in the collision cell, and its abundance increased with increasing N2 pressure. CONCLUSIONS Arylium ions generated from a wide variety of compounds in electrospray ionization tandem mass spectrometry may react with N2 to form diazonium ions. The abundance of the diazonium ions is affected by collision energy and N2 pressure. This reaction should be considered when annotating peaks in MS/MS libraries. Published in 2015. This article is a U.S. Government work and is in the public domain in the USA.

Dipole Orientation and Surface Cluster Size Effects on Chemisorption-Induced Magnetism : A DFT Study of the Interaction of Gold-Thiopolypeptide

A nanosystem formed by a high electric dipole moment thiopolypeptide alpha-helix, consisting of eight l-glycine units, chemisorbed on the (111) surface of Au23 and Au55 clusters, with the S as the linking atom, was studied using the wave function broken symmetry UDFT method. We have found a strong correlation between the orientation of the electric dipole of the alpha-helix and charge transfer and the magnetic behavior of the adsorbate-cluster system. Upon chemisorption, dipole moments may be quenched or enhanced, with respect to the gas phase value, with the strongest reduction corresponding to the magnetic state. A reduction of the alpha-helixs electric dipole with the net charge transfer from the Au surface was obtained for the more stable state. In this state description, it may happen that the calculated spin densities of the chemisorbed alpha-helix and its free radical form are similar. The magnetic properties are strongly dependent on the size of the Au cluster and on its electronic structure with respect to nuclei positions. In general, the localized spin density per atom increases and the magnetization of the extended system decreases with cluster size, a trend found experimentally for organic monolayers with a similar type of adsorbate we consider here.

Pairwise alignment of chromatograms using an extended Fisher–Rao metric

A conceptually new approach for aligning chromatograms is introduced and applied to examples of metabolite identification in human blood plasma by liquid chromatography-mass spectrometry (LC-MS). A square-root representation of the chromatograms derivative coupled with an extended Fisher-Rao metric enables the computation of relative differences between chromatograms. Minimization of these differences using a common dynamic programming algorithm brings the chromatograms into alignment. Application to a complex sample, National Institute of Standards and Technology (NIST) Standard Reference Material 1950, Metabolites in Human Plasma, analyzed by two different LC-MS methods having significantly different ranges of elution time is described.

Developing qualitative LC-MS methods for characterization of Vaccinium berry Standard Reference Materials

Standard Reference Materials (SRMs) offer the scientific community a stable and homogenous source of material that holds countless application possibilities. Traditionally, the National Institute of Standards and Technology (NIST) has provided SRMs with associated quantitative information (certified values) for a select group of targeted analytes as measured in a solution or complex matrix. While the current needs of the SRM community are expanding to include non-quantitative data, NIST is attempting to broaden the scope of how and what information is offered to the SRM community by providing qualitative information about biomaterials, such as chromatographic fingerprints and profiles of untargeted identifications. In this work, metabolomic and proteomic profiling efforts were employed to characterize a suite of six Vaccinium berry SRMs. In the discovery phase, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data was matched to mass spectral libraries; a subsequent validation phase based on multiple-reaction monitoring LC-MS/MS relied on both retention time matching of authentic standards along with fragmentation data for a qualitative overview of the most prominent organic compounds present. Definitive and putative identifications were determined for over 70 metabolites based on reporting guidelines set forth by the Metabolomics Standards Initiative (Metabolomics 3(3):211–221, 2007), and the capability of electrospray ionization mass spectrometry (ESI-MS) to profile untargeted metabolites within a complex matrix using mass spectral matching is demonstrated. Bottom-up proteomic analyses were possible using peptide databases translated from expressed sequence tags (ESTs). Homology searches provided identification of novel Vaccinium proteins based on homology to related genera. Chromatographic fingerprints of these berry materials were acquired for supplemental qualitative information to be provided to users of these SRMs. An unbounded set of qualitative data about a biomaterial is a valuable complement to quantitative information traditionally provided in NIST Certificates of Analysis.