Ion-Neutral Collision Cross Section as a Function of the Static Dipole Polarizability and the Ionization Energy of the Ion.

Ion mobility spectrometry is becoming more and more popular as a fast, efficient, and sensitive tool for the separation and identification of ionized molecules in the gas phase. An ion traveling through a drift tube at atmospheric pressure under the influence of an electric field collides with the buffer gas molecules. The mobility of the ion depends inversely on the ion-neutral collision cross section. In the simplest hard-sphere approximation, the collision cross section is the area of the conventional geometric cross section. However, deviations are expected because of the physical interactions between the colliding species. More than a century ago, Langevin described a model for the interaction between a point-charge ion and a polarizable atom (molecule). Since then, the model has been modified many times to include better approximations of the interaction potential, usually preserving the point-charge nature of the ion. Although more advanced approaches allow for considering polarizable ions with dissimilar sizes and shapes, still explicit analytical dependencies on the properties of the ion remain elusive. In this work, an extended version of the Langevin model is proposed and solved using algebraic perturbation theory. A simple analytical expression of the collision cross section depending explicitly on both the static dipole polarizability and the ionization energy of the ion is found. The equation is validated using ion mobility data. Surprisingly, even low-level calculations of the polarizability tensors produce results that are consistent with the experimental observations. This fact makes the equation very attractive for helping applications in different areas, such as the deconvolution of mobilograms of protomers, ion-molecule chemical kinetics, and others.

Unexpected Gas-Phase Nitrogen-Oxygen Smiles Rearrangement: Collision-Induced Dissociation of Deprotonated 2-(N-Methylanilino)ethanol and Morpholinylbenzoic Acid Derivatives.

A nitrogen-oxygen Smiles rearrangement was reported to occur after collisional activation of the PhN(R)CH2CH2O- (R = alkyl) anion, which undergoes a five-membered ring rearrangement to form a phenoxide ion C6H5O-. When R = H, such a Smiles rearrangement is unlikely since the negative charge is more favorably located on the nitrogen atom than the oxygen atom; hence, alternative neutral losses dominate the fragmentation. For example, collisional activation of deprotonated 2-anilinoethanol (PhN-CH2CH2OH) leads to the formation of an anilide anion (C6H5NH-, m/z 92) rather than a phenoxide ion (C6H5O-, m/z 93.0343). However, when the amino hydrogen of 2-anilinoethanol is substituted by a methyl group, i.e., 2-(N-methylanilino)ethanol, a Smiles rearrangement does occur, leading to the phenoxide ion, as the negative charge can only reside on the oxygen atom. To confirm the Smiles rearrangement mechanism, 2-(N-methylanilino)ethanol-18O was synthesized and subjected to collisional activation, leading to an intense peak at m/z 95.0385, which corresponds to the 18O phenoxide ion ([C6H518O]-). The abundance of the phenoxide ion is sensitive to substituents on the N atom, as demonstrated by the observation that an ethyl substituent results in the rearrangement ion with a much lower abundance. The nitrogen-oxygen Smiles rearrangement also occurs for various morpholinylbenzoic acid derivatives with a multistep mechanism, where the phenoxide ion is found to be predominantly formed after loss of CO2, proton transfers, breaking of the morpholine ring, and Smiles rearrangement. The Smiles mechanism is also supported by density functional theory calculations and other observations.

CID Fragmentation of Deprotonated N-Acyl Aromatic Sulfonamides. Smiles-Type and Nitrogen-Oxygen Rearrangements.

The NIST tandem mass spectral library (2020 version) includes over 800 aromatic sulfonamides. In negative mode, upon collisional activation most benzenesulfonamides lose a neutral SO2 molecule leading to an anilide anion (C6H5NH-, m/z 92). However, for deprotonated N-benzoyl aromatic sulfonamides, the phenoxide ion (C6H5O-, m/z 93.0343) is the principal product ion. A variety of N-acylbenzenesulfonamide derivatives were also found to overwhelmingly produce the phenoxide ion as the most intense product ion. A mechanism is proposed in which, at low energy, a carbonyl oxygen atom (C═O) is transferred to a benzene ring, known as a Smiles-type rearrangement (the amide oxygen atom attacks the arylsulfonyl group at the ipso position), in parallel and determining the reaction at high energy a nitrogen-oxygen rearrangement mechanism leads to the formation of the phenoxide ion. Tandem mass spectra of deprotonated N-benzoyl-18O-benzenesulfonamide and N-thiobenzoyl-p-toluenesulfonamide confirmed the rearrangement since base peaks at m/z 95.0384 and 123.0270 which correspond to an 18O phenoxide ion ([C6H518O]-) and a 4-methylbenzenethiolate anion ([CH3C6H4S]-) were observed, respectively. The parallel mechanism is supported by the strong correlation between the observed product ion intensities and the corresponding activation energies obtained by Density Functional Theory calculations. This is an example of a relatively simple ion with a complex path to fragmentation, being a cautionary tale for indiscriminate use of in silico spectra in place of actual measurement.

Creation and filtering of a recurrent spectral library of CHO cell metabolites and media components.

This paper reports the first implementation of a new type of mass spectral library for the analysis of Chinese hamster ovary (CHO) cell metabolites that allows users to quickly identify most compounds in any complex metabolite sample. We also describe an annotation methodology developed to filter out artifacts and low-quality spectra from recurrent unidentified spectra of metabolites. CHO cells are commonly used to produce biological therapeutics. Metabolic profiles of CHO cells and media can be used to monitor process variability and look for markers that discriminate between batches of product. We have created a comprehensive library of both identified and unidentified metabolites derived from CHO cells that can be used in conjunction with tandem mass spectrometry to identify metabolites. In addition, we present a workflow that can be used for assigning confidence to a NIST MS/MS Library search match based on prior probability of general utility. The goal of our work is to annotate and identify (when possible), all liquid chromatography-mass spectrometry generated metabolite ions as well as create automatable library building and identification pipelines for use by others in the field.

Metabolomics Test Materials for Quality Control: A Study of a Urine Materials Suite.

There is a lack of experimental reference materials and standards for metabolomics measurements, such as urine, plasma, and other human fluid samples. Reasons include difficulties with supply, distribution, and dissemination of information about the materials. Additionally, there is a long lead time because reference materials need their compositions to be fully characterized with uncertainty, a labor-intensive process for material containing thousands of relevant compounds. Furthermore, data analysis can be hampered by different methods using different software by different vendors. In this work, we propose an alternative implementation of reference materials. Instead of characterizing biological materials based on their composition, we propose using untargeted metabolomic data such as nuclear magnetic resonance (NMR) or gas and liquid chromatography-mass spectrometry (GC-MS and LC-MS) profiles. The profiles are then distributed with the material accompanying the certificate, so that researchers can compare their own metabolomic measurements with the reference profiles. To demonstrate this approach, we conducted an interlaboratory study (ILS) in which seven National Institute of Standards and Technology (NIST) urine Standard Reference Material®s (SRM®s) were distributed to participants, who then returned the metabolomic data to us. We then implemented chemometric methods to analyze the data together to estimate the uncertainties in the current measurement techniques. The participants identified similar patterns in the profiles that distinguished the seven samples. Even when the number of spectral features is substantially different between platforms, a collective analysis still shows significant overlap that allows reliable comparison between participants. Our results show that a urine suite such as that used in this ILS could be employed for testing and harmonization among different platforms. A limited quantity of test materials will be made available for researchers who are willing to repeat the protocols presented here and contribute their data.

Hybrid Search: A Method for Identifying Metabolites Absent from Tandem Mass Spectrometry Libraries.

Metabolomics has a critical need for better tools for mass spectral identification. Common metabolites may be identified by searching libraries of tandem mass spectra, which offers important advantages over other approaches to identification. But tandem libraries are not nearly complete enough to represent the full molecular diversity present in complex biological samples. We present a novel hybrid search method that can help identify metabolites not in the library by similarity to compounds that are. We call it "hybrid" searching because it combines conventional, direct peak matching with the logical equivalent of neutral-loss matching. A successful hybrid search requires the library to contain "cognates" of the unknown: similar compounds with a structural difference confined to a single region of the molecule, that does not substantially alter its fragmentation behavior. We demonstrate that the hybrid search is highly likely to find similar compounds under such circumstances.

Mass Spectrometry Fingerprints of Small-Molecule Metabolites in Biofluids: Building a Spectral Library of Recurrent Spectra for Urine Analysis.

A large fraction of ions observed in electrospray liquid chromatography-mass spectrometry (LC-ESI-MS) experiments of biological samples remain unidentified. One of the main reasons for this is that spectral libraries of pure compounds fail to account for the complexity of the metabolite profiling of complex materials. Recently, the NIST Mass Spectrometry Data Center has been developing a novel type of searchable mass spectral library that includes all recurrent unidentified spectra found in the sample profile. These libraries, in conjunction with the NIST tandem mass spectral library, allow analysts to explore most of the chemical space accessible to LC-MS analysis. In this work, we demonstrate how these libraries can provide a reliable fingerprint of the material by applying them to a variety of urine samples, including an extremely altered urine from cancer patients undergoing total body irradiation. The same workflow is applicable to any other biological fluid. The selected class of acylcarnitines is examined in detail, and derived libraries and related software are freely available. They are intended to serve as online resources for continuing community review and improvement.

Cross-Ring Fragmentation Patterns in the Tandem Mass Spectra of Underivatized Sialylated Oligosaccharides and Their Special Suitability for Spectrum Library Searching.

Reference spectral library searching, while widely used to identify compounds in other areas of mass spectrometry, is not commonly used in glycomics. Building on a study by Cotter and coworkers on analysis of sialylated oligosaccharides using atmospheric pressure-matrix-assisted laser-induced tandem mass spectrometry (MS/MS), we show that library search methods enable the automated differentiation of such sialylated oligosaccharide isomers using MS/MS derived from electrospray collision-induced dissociation in ion trap and beam-type fragmentation mass spectrometers. We compare MS/MS spectra of five sets of native sialylated oligosaccharide isomers and show a spectral library search method that can distinguish between these isomers using the precursor ion [M+2X-H]+, where X=Li, Na, or K. Sialic acid linkage (α2,3 vs. α2,6) is known to have a dramatic effect on the fragmentation of the sialylated compounds. We found that 2,4A3 cross-ring fragment at the terminal monosaccharide in sialyllactoses, sialyllactosamines, and sialyl pentasaccharides is highly abundant in the MS/MS spectra of [M+2X-H]+ species of α2,6-NeuAc glycans, while (2,4A3-H2O) fragment is highly abundant in α2,3-NeuAc moiety. The 2,4A3-H2O peak is specific to NeuAc-α2,3-Gal-ß1,4-Y (Y=GlcNAc or Glc). To our knowledge, this observation was not reported previously. Theoretical calculations reveal major conformational differences between α2,6-NeuAc and α2,3-NeuAc structures that provide reasonable explanations for the observed fragmentation patterns. Other singly-charged ions ([M+X]+) do not show similar cross-ring cleavages. Implemented in a searchable library, these spectral differences provide a facile method to distinguish sialyl isomers without derivatization. We also found good spectral matching across instruments. MS/MS spectra and tools are available at http://chemdata.nist.gov/glycan/spectra . Graphical Abstract.

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.

Retention projection enables accurate calculation of liquid chromatographic retention times across labs and methods.

Identification of small molecules by liquid chromatography-mass spectrometry (LC-MS) can be greatly improved if the chromatographic retention information is used along with mass spectral information to narrow down the lists of candidates. Linear retention indexing remains the standard for sharing retention data across labs, but it is unreliable because it cannot properly account for differences in the experimental conditions used by various labs, even when the differences are relatively small and unintentional. On the other hand, an approach called "retention projection" properly accounts for many intentional differences in experimental conditions, and when combined with a "back-calculation" methodology described recently, it also accounts for unintentional differences. In this study, the accuracy of this methodology is compared with linear retention indexing across eight different labs. When each lab ran a test mixture under a range of multi-segment gradients and flow rates they selected independently, retention projections averaged 22-fold more accurate for uncharged compounds because they properly accounted for these intentional differences, which were more pronounced in steep gradients. When each lab ran the test mixture under nominally the same conditions, which is the ideal situation to reproduce linear retention indices, retention projections still averaged 2-fold more accurate because they properly accounted for many unintentional differences between the LC systems. To the best of our knowledge, this is the most successful study to date aiming to calculate (or even just to reproduce) LC gradient retention across labs, and it is the only study in which retention was reliably calculated under various multi-segment gradients and flow rates chosen independently by labs.

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.

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.

Metabolite profiling of a NIST Standard Reference Material for human plasma (SRM 1950): GC-MS, LC-MS, NMR, and clinical laboratory analyses, libraries, and web-based resources.

Recent progress in metabolomics and the development of increasingly sensitive analytical techniques have renewed interest in global profiling, i.e., semiquantitative monitoring of all chemical constituents of biological fluids. In this work, we have performed global profiling of NIST SRM 1950, "Metabolites in Human Plasma", using GC-MS, LC-MS, and NMR. Metabolome coverage, difficulties, and reproducibility of the experiments on each platform are discussed. A total of 353 metabolites have been identified in this material. GC-MS provides 65 unique identifications, and most of the identifications from NMR overlap with the LC-MS identifications, except for some small sugars that are not directly found by LC-MS. Also, repeatability and intermediate precision analyses show that the SRM 1950 profiling is reproducible enough to consider this material as a good choice to distinguish between analytical and biological variability. Clinical laboratory data shows that most results are within the reference ranges for each assay. In-house computational tools have been developed or modified for MS data processing and interactive web display. All data and programs are freely available online at http://peptide.nist.gov/ and http://srmd.nist.gov/ .

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.

Formation of y + 10 and y + 11 ions in the collision-induced dissociation of peptide ions.

Tandem mass spectra of peptide ions, acquired in shotgun proteomic studies of selected proteins, tissues, and organisms, commonly include prominent peaks that cannot be assigned to the known fragmentation product ions (y, b, a, neutral losses). In many cases these persist even when creating consensus spectra for inclusion in spectral libraries, where it is important to determine whether these peaks represent new fragmentation paths or arise from impurities. Using spectra from libraries and synthesized peptides, we investigate a class of fragment ions corresponding to y(n-1) + 10 and y(n-1) + 11, where n is the number of amino acid residues in the peptide. These 10 and 11 Da differences in mass of the y ion were ascribed before to the masses of [+ CO - H(2)O] and [+ CO - NH(3)], respectively. The mechanism is suggested to involve dissociation of the N-terminal residue at the CH-CO bond following loss of H(2)O or NH(3). MS(3) spectra of these ions show that the location of the additional 10 or 11 Da is at the N-terminal residue. The y(n-1) + 10 ion is most often found in peptides with N-terminal proline, asparagine, and histidine, and also with serine and threonine in the adjacent position. The y(n-1) + 11 ion is observed predominantly with histidine and asparagine at the N-terminus, but also occurs with asparagine in positions two through four. The intensities of the y(n-1) + 10 ions decrease with increasing peptide length. These data for y(n-1) + 10 and y(n-1) + 11 ion formation may be used to improve peptide identification from tandem mass spectra.

Tryptic y(++) fragment ion distributions are guided by Coulombic repulsion.

Ideal tryptic peptides contain only a single basic residue, located at the C-terminus. Collisional fragmentation of their doubly- or triply-protonated ions generates doubly-charged y(++) fragment ions with modest intensities. The size distribution of the y(++) fragments, when averaged over many spectra, corresponds closely to the expectations from charge-directed backbone cleavage and a Coulomb-Boltzmann distribution of mobile protons. This observation should be helpful in developing mechanistic models for y(++) formation.