Identification of unknowns in atmospheric pressure ionization mass spectrometry using a mass to structure search engine

This study evaluates a new model for identifying unknown compounds in atmospheric pressure ionization mass spectrometry based on a mass-to-structure (MTS) paradigm. In this method, rudimentary ESI spectrum interpretation is required to recognize key spectral features such as MH (+), MNa (+), and MNH 4 (+), which lead to the unknown's monoisotopic mass. The unknown's mass is associated directly with known organic compounds using an Access 2003 database containing records of 19,438 substances assembled from common sources such as the Merck Index, pesticide and pharmaceutical compilations, and chemical catalogues. A user-defined mass tolerance (+/-0.001-0.5 Da) is set according to the instrument mass accuracyunit mass resolution data require a wide mass tolerance ( approximately 0.5 Da) while tolerances for accurate mass data can be as narrow as +/-0.001 Da. Candidate structures retrieved with the MTS Search Engine appear in a report window providing formulas, mass error, and Internet links. This paper provides examples of structure elucidation with 15 organic compounds based on ESI mass spectra from both unit mass resolution (e.g., quadrupole ion trap and triple-stage quadrupole) and accurate mass instruments (e.g., TOF and Q-TOF). Orthogonal information (e.g., isotope ratios and fragmentation data) is complementary and useful for ranking candidates and confirming assignments. The MTS Search Engine identifies unknowns quickly and efficiently, and supplements existing interpretation schemes for unknown identification.     

Baseline mass resolution of peptide isobars: a record for molecular mass resolution

Baseline resolution of two peptides, RVMRGMR and RSHRGHR, of neutral monoisotopic mass, approximately 904 Da, has been achieved by microelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry at a mass resolving power of approximately 3 300 000. The elemental compositions of these molecules differ by N40 vs. S2H8 (0.000 45 Da), which is less than one electron's mass (0.000 55 Da)! This result establishes a new record for the smallest resolved mass difference between any two molecules. This achievement is made possible by a combination of high magnetic field (9.4 T), large-diameter (4-in.) Penning trap, and low ion density. The implications for proteomics based on accurate mass measurements are discussed briefly.     

Two- and three-dimensional van krevelen diagrams: a graphical analysis complementary to the kendrick mass plot for sorting elemental compositions of complex organic mixtures based on ultrahigh-resolution broadband fourier transform ion cyclotron resonance mass measurements

Ultrahigh-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry has resolved and identified the elemental compositions of over 10000 organic constituents of coal and petroleum crude oil. A plot of Kendrick mass defect versus Kendrick nominal mass sorts compounds into homologous series according to compound class (i.e., numbers of N, O, and S heteroatoms), type (number of rings plus double bonds), and degree of alkylation (number of CH(2) groups), to yield unique elemental assignments from ultrahigh-resolution mass measurements in the 200-900 Da range. Interpretation of such a vast compilation requires a simple (preferably graphical) means to differentiate between complex organic mixtures of different origin or processing. In an extension of the recently revived van Krevelen plot, each elemental composition is projected onto two or three axes according to its H/C, O/C, and/or N/C atomic ratios. The H/C ratio separates compounds according to degree of saturation, whereas O/C or N/C ratios separate according to O and N classes. We show that the three-dimensional van Krevelen diagram can completely separate different classes in pyridine-extracted coal or petroleum samples and can also graphically distinguish fossil fuels according to their nature (coal vs petroleum), maturation (coals of different rank), and processing (the same coal at two stages of liquefaction). The van Krevelen diagram thus appears well suited to amplifying and exposing compositional differences within and between complex organic mixtures.     

Resolution of 11,000 compositionally distinct components in a single electrospray ionization Fourier transform ion cyclotron resonance mass spectrum of crude oil

Mass spectrometry is well-suited for complex mixture analysis, because unlike other types of spectroscopy, the number of mass spectral peaks per analyte is of order one. Here, we extend significantly the upper limit for the number of chemically distinct components resolved and identified in a single step. Specifically, electrospray selectively ionizes only the basic compounds (i.e., a small fraction of the entire chemical composition) in a sample of South American crude oil. Nevertheless, their positive ion Fourier transform ion cyclotron resonance mass spectrum (average mass resolving power of approximately 350,000 from 225 to 1000 Da) contains more than 11,100 resolved peaks, of which >75% may be assigned to a unique elemental composition (CcHhOoNnS8). Mass scale expansion and graphical representations reveal increased heteroatom diversity, aromatic rings, and alkyl substitution with increased mass. These results set a new standard and illustrate the potential of high-resolution mass spectrometry for analysis of compositionally complex chemical mixtures.     

Determination of elemental compositions from mass peak profiles of the molecular ion (m) and the m + 1 and m + 2 ions

The relative abundances of M + 1 and M + 2 ions help to identify the elemental composition of the molecular ion (M). But scan speed, sensitivity, and resolution limitations of mass spectrometers have impeded determination of these abundances. Mass peak profiling from selected ion recording data (MPPSIRD) provided faster sampling and enhanced sensitivity, which permitted use of higher resolution. M + 2 profiles having only a few percent of the ion abundance of M were monitored at 20?000 resolution. The relative abundances, exact masses, and shapes of M, M + 1, and M + 2 mass peak profiles were determined. By applying five criteria based on these quantities, elemental compositions were determined even for ions too large (up to 766 Da) to be uniquely assigned from their exact mass and accuracy limits alone. A profile generation model (PGM) was written to predict these resolution-dependent quantities by considering all M + 1 and M + 2 ions for each candidate composition. The model also provided assurance that no other compositions were possible. Characterization of the M + 1 and M + 2 profiles by MPPSIRD and the PGM greatly expanded the practical ability of high-resolution mass spectrometry to determine elemental compositions.     

Application of mass spectrometry to the characterization of polymers

Recent advances in soft ionization techniques for mass spectrometry of polymeric materials make it possible to determine the mass of intact molecular ions exceeding 1 × 10⁶ Da. Developments in high resolution mass spectrometers have additionally led to impressive advances in our ability to characterize polymers. The entire molecular mass distribution of a polymer sample can be accurately measured. From the molecular mass, the molecular formulae and information regarding polymer composition and end-groups can be deduced. The two techniques which have received the most attention are matrix-assisted laser desorption/ionization and electrospray ionization. In recent work, these techniques have been combined with chromatographic separations, and a series of mass spectra are acquired for each fraction of the distribution. This simplifies the analysis by reducing the number of components present in each mass spectrum, and additionally improves quantitation.     

De novo peptide sequencing by two-dimensional fragment correlation mass spectrometry

A novel concept of two-dimensional fragment correlation mass spectrometry and its application to peptide sequencing is described. The daughter ion (MS2) spectrum of a peptide contains the sequence information of the peptide. However, deciphering the MS2 spectrum, and thus deriving the peptide sequence is complex because of the difficulty in distinguishing the N-terminal fragments (e.g., b series) from the C-terminal fragments (e.g., y series). By taking a granddaughter ion (MS3) spectrum of a particular daughter ion, all fragment ions of the opposite terminus are eliminated in the MS3 spectrum. However, some internal fragments of the peptide will appear in the MS3 spectrum. Because internal fragments are rarely present in the MS2 spectrum, the intersection (a spectrum containing peaks that are present in both spectra) of the MS2 and MS3 spectra should contain only fragments of the same terminal type. A two-dimensional plot of the MS2 spectrum versus the intersection spectra (2-D fragment correlation mass spectrum) often gives enough information to derive the complete sequence of a peptide. This paper describes this novel technique and its application in sequencing cytochrome c and apomyoglobin. For a tryptic digest of cytochrome c, approximately 78% of the protein sequence was determined. For the Glu-C/tryptic digest of apomyoglobin, approximately 66% of the protein sequence was determined.     

Identification of intact proteins in mixtures by alternated capillary liquid chromatography electrospray ionization and LC ESI infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.

Here we propose a novel method for rapidly identifying proteins in complex mixtures. A list of candidate proteins (including provision for posttranslational modifications) is obtained by database searching, within a specified mass range about the accurately measured mass (e.g., +/- 0.1 Da at 10 kDa) of the intact protein, by capillary liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS). On alternate scans, LC ESI infrared multiphoton dissociation (IRMPD) FT-ICR MS yields mostly b and y fragment ions for each protein, from which the correct candidate is identified as the one with the highest "hit" score (i.e., most b and y fragments matching the candidate database protein amino acid sequence masses) and sequence "tag" score (based on a series of fragment sequences differing in mass by 1 or 2 amino acids). The method succeeds in uniquely identifying each of a mixture of five proteins treated as unknowns (melittin, ubiquitin, GroES, myoglobin, carbonic anhydrase II), from more than 1000 possible database candidates within a +/- 500 Da mass window. We are also able to identify posttranslational modifications of two of the proteins (mellitin and GroES). The method is simple, rapid, and definitive and is extendable to a mixture of affinity-selected proteins, to identify proteins with a common biological function.     

Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurements

The throughput of proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations as well as the subsequent mass spectrometric analysis; at present, analysis times can range anywhere from hours (high throughput) to days or longer (low throughput). We have explored the basis for proteomics analyses conducted on the order of minutes using high-speed capillary RPLC combined through on-line electrospray ionization interface with high-accuracy mass spectrometry (MS) measurements. Short 0.8-microm porous C18 particle-packed 50-microm-i.d. capillaries were used to speed the RPLC separations while still providing high-quality separations. Both time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) MS were applied for identifying peptides using the accurate mass and time (AMT) tag approach. Peptide RPLC relative retention (elution) times that were generated by solvent gradients that differed by at least 25-fold were found to provide relative elution times that agreed to within 5%, which provides the basis for using peptide AMT tags for higher throughput proteomics measurements. For fast MS acquisition speeds (e.g., 0.2 s for TOF and either approximately 0.3 or approximately 0.6 s for FTICR), peptide mass measurement accuracies of better than +/-15 ppm were obtained with the high-speed RPLC separations. The ability to identify peptides and the overall proteome coverage was determined by factors that include the separation peak capacity, the sensitivity of the MS (with fast scanning), and the accuracy of both the mass measurements and the relative RPLC peptide elution times. The experimental RPLC relative elution time accuracies of 5% (using high-speed capillary RPLC) and mass measurement accuracies of better than +/-15 ppm allowed for the confident identification of >2800 peptides and >760 proteins from >13,000 different putative peptides detected from a Shewanellaoneidensis tryptic digest. Initial results for both RPLC-ESI-TOF and RPLC-ESI-FTICR MS were similar, with approximately 2000 different peptides from approximately 600 different proteins identified within 2-3 min. For <120-s proteomic analysis, TOF MS analyses were more effective, while FTICR MS was more effective for the >150-s analysis due to the improved mass accuracies attained using longer spectrum acquisition times.     

Identification, Composition, and Asymmetric Formation Mechanism of Glycidyl Methacrylate/Butyl Methacrylate Copolymers up to 7000 Da from Electrospray Ionization Ultrahigh-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Glycidyl methacrylate (GMA) and butyl methacrylate (BMA) have the same nominal mass (142 Da) but differ in exact mass by 0.036 Da (CH(4) vs O). Therefore, copolymers formed from the two isobaric monomers exhibit a characteristic isobaric distribution due to different monomer compositions. Here, we show that electrospray ionization FT-ICR mass spectrometry at 9.4 T resolves the isobaric components of copolymers as large as 7000 Da with a resolving power (m/Δm(50%)) of ～500?000 in a gel permeation chromatography fractionated polymer sample. That resolution provides for complete and unequivocal component analysis of such copolymers of the size used for high solid content automobile coatings. All five possible copolymer products predicted by the polymerization mechanism are resolved and identified in the mass spectrum. Two of those polymer series (each with saturated end group) were previously unresolved by mass spectrometry because they differ in mass from the two other unsaturated products by only 0.0089 Da. Finally, analysis of the asymmetrical isobaric distribution for the copolymer n-mers, (GMA)(m)(BMA)(n)(-)(m), 0≤ m ≤ n, in which species with adjacent values of m differ from each other in mass by 36 mDa (i.e., the mass difference, CH(4) vs O, between GMA and BMA) proves that GMA is less reactive than BMA in the polymerization process.     

Ultrahigh mass resolution and accurate mass measurements as a tool to characterize oligomers in secondary organic aerosols

Organic aerosols are a major fraction, often more than 50%, of the total atmospheric aerosol mass. The chemical composition of the total organic aerosol mass is poorly understood, although hundreds of compounds have been identified in the literature. High molecular weight compounds have recently gained much attention because this class of compounds potentially represents a major fraction of the unexplained organic aerosol mass. Here we analyze secondary organic aerosols, generated in a smog chamber from alpha-pinene ozonolysis with ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). About 450 compounds are detected in the mass range of m/z 200-700. The mass spectrum is clearly divided into a low molecular weight range (monomer) and a high molecular weight range, where dimers and trimers are distinguishable. Using the Kendrick mass analysis, the elemental composition of about 60% of all peaks could be determined throughout the whole mass range. Most compounds have high O:C ratios between 0.4 and 0.6. Small compounds (i.e., monomers) have a higher maximum O:C ratio than dimers and trimers, suggesting that condensation reactions with, for example, the loss of water are important in the oligomer formation process. A program developed in-house was used to determine exact mass differences between peaks in the monomer, dimer, and trimer mass range to identify potential monomer building blocks, which form the co-oligomers observed in the mass spectrum. A majority of the peaks measured in the low mass region of the spectrum (m/z < 300) is also found in the calculated results. For the first time the elemental composition of the majority of peaks over a wide mass range was determined using advanced data analysis methods for the analysis of ultra-high-resolution MS data. Possible oligomer formation mechanisms in secondary organic aerosols were investigated.     

Periodicity of monoisotopic mass isomers and isobars in proteomics

We report trends in the theoretically derived number of compositionally distinct peptides (i.e., peptides made up of different amino acid residues) up to a nominal mass of 1000. A total of 21 amino acid residues commonly found in proteomics studies are included in this study, 19 natural, nonisomeric amino acid residues as well as oxidated methione and acetamidated cysteine. The number of possibilities is found to increase in an exponential fashion with increasing nominal mass, and the data show a periodic oscillation that starts at mass ~200 and continues throughout to 1000. Note that similar effects are reported in the companion article on fragment ions from electron capture/transfer dissociation (ECD/ETD) (Mao et al. Anal. Chem.2011, DOI: 10.1021/ac201619t). The spacing of this oscillation is ~15 mass units at lower masses and ~14 mass units at higher nominal masses. This correlates with the most common mass differences between the amino acid building blocks. In other words, some mass differences are more common than others, thus determining the periodicity in this data. From an analytical point of view, nominal masses with a larger number of compositionally distinct peptides include a substantial number of isomers, which cannot be separated based on mass. Consequently, even ultrahigh mass accuracy (i.e., 0.5 ppm) does not lead to a substantially enhanced rate of identification. Conversely, for adjacent nominal masses with a lower number of isomers, moderately accurate mass (i.e., 10 ppm) gives a higher degree of certainty in identification. These effects are limited to the mass range between 200 and 500 Da. At higher masses, the percentage of uniquely identified peptides drops off to close to zero, independent of nominal mass, due the inherently high number of isomers. While the exact number of isobars/isomers at each nominal mass depends on the amino acid building blocks that are considered, the periodicity in the data is found to be remarkably robust; for instance, inclusion of phosphorylated residues barely affects the pattern at lower masses (i.e., <500 Da).     

Baseline resolution of isobaric phosphorylated and sulfated peptides and nucleotides by electrospray ionization FTICR ms: another step toward mass spectrometry-based proteomics

Electrospray ionization broadband FTICR mass spectrometry at a mass resolving power, m/delta m50% > or = 400,000 has achieved the first direct mass spectral resolution of phosphorylated and sulfated peptides (or nucleotides) of the same nominal mass. The elemental composition difference in each case is PH versus S (9.5 mDa), requiring a minimum mass resolving power ((m2 - m1)/ml) of 118,000 (C terminal amidated cholecystekinin fragment 26-33 (CCK-8), DY(PO3H2)MGWMDF-NH2 versus DY(SO3H)MGWMDF-NH2) or 65,400 (adenosine triphosphate vs 3-phosphoadenosine 5'-phosphosulfate). The isobaric mass doublets were detected in broadband mode (400 < m/z <1400) in the presence of dozens of other species. It is therefore now possible to distinguish phosphorylated from sulfated peptides, even when both species are present at the same time in a protein digest.     

High-accuracy molecular mass determination of proteins using matrix-assisted laser desorption mass spectrometry

A method for obtaining protein molecular masses with an accuracy of approximately +/- 0.01% by matrix-assisted laser desorption using an internal calibrant is described. The technique allows accurate mass determinations of protein sample sizes as small as 1 pmol. High concentrations of organic and inorganic contaminants (e.g. 1 M urea) do not strongly affect either the signal intensity or the mass assignment. The ability to assign an accurate molecular mass to a protein is contingent on the observation of clearly resolved protonated molecule ions in the mass spectrum.     

Multistage accurate mass spectrometry: a "basket in a basket" approach for structure elucidation and its application to a compound from combinatorial synthesis

A "basket in a basket" method based on a multistage accurate mass spectrometric (MAMS) technique was developed and demonstrated by obtaining a unique elemental composition of a compound (with a molecular weight of 517) from combinatorial synthesis. The accurate masses for the parent and the fragment ions were obtained with up to five stages of MAMS using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). This approach requires only input of elements used in the synthetic processes and some constraints about unusual light elements, such as fluorine, while the compositions of the parent ions and their fragments are obtained for structure elucidation. Conversely, accuracy of better than 0.02 ppm (assuming elements C, H, N, O, S, and F are involved) would be required in order to define a unique composition for the same mass using a direct accurate mass measurement because the number of possible elemental compositions increases sharply as the mass increases. Similarly, due to the uncertainty in determining elemental compositions of fragments and complexity of possible internal fragmentation, tandem mass spectrometry may not provide enough information for structure elucidation of unknown compounds, especially of the organic molecules in the mass range of 300-1000 Da, typically encountered in combinatorial lead generation. The application of MAMS to combinatorial drug discovery is particularly advantageous since the built-in chemical information from the synthesis can be used as constraints. The implementation of a nanoelectrospray ionization technique makes this approach practical for characterization of small quantities of compounds typically available from lead generation processes.     

Multiplexed ion mobility spectrometry-orthogonal time-of-flight mass spectrometry

Ion mobility spectrometry (IMS) coupled to orthogonal time-of-flight mass spectrometry (TOF) has shown significant promise for the characterization of complex biological mixtures. The enormous complexity of biological samples (e.g., from proteomics) and the need for both biological and technical analysis replicates imposes major challenges for multidimensional separation platforms with regard to both sensitivity and sample throughput. A major potential attraction of the IMS-TOF MS platform is separation speeds exceeding that of conventional condensed-phase separations by orders of magnitude. Known limitations of the IMS-TOF MS platforms that presently mitigate this attraction include the need for extensive signal averaging due to factors that include significant ion losses in the IMS-TOF interface and an ion utilization efficiency of less than approximately 1% with continuous ion sources (e.g., ESI). We have developed a new multiplexed ESI-IMS-TOF mass spectrometer that enables lossless ion transmission through the IMS-TOF as well as a utilization efficiency of >50% for ions from the ESI source. Initial results with a mixture of peptides show a approximately 10-fold increase in signal-to-noise ratio with the multiplexed approach compared to a signal averaging approach, with no reduction in either IMS or TOF MS resolution.     

FTMS structure elucidation of natural products: application to muraymycin antibiotics using ESI multi-CHEF SORI-CID FTMS(n), the top-down/bottom-up approach,and HPLC ESI capillary-skimmer CID FTMS

The molecular formulas for the structures and substructures of muraymycin antibiotics A1 (C52H90N14O19, MW 1214) and B1 (C49H83N11O18, MW 1113) were determined using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The muraymycin A1 and B1 structures were elucidated by utilizing capillary-skimmer fragmentation with up to five stages of mass spectrometry (MS5). Multi-CHEF, a multiple ion isolation method, was used at each stage of MS(n) to isolate a parent ion and up to four reference ions, for exact-mass calibration. The parent ions were fragmented by SORI-CID and the product ions internally calibrated with average absolute mass errors less than 1 ppm at each stage in the fragmentation processes. Using the top-down/bottom-up approach, the molecular formulas for the antibiotics were determined by summing the elemental formulas of the neutral losses, obtained by measuring the mass differences (<500 Da) between the genetically related sequential parent ion masses in the MS(n) spectra, with the unique elemental formula of the lowest parent ion mass (<500 Da). The structures of 12 additional compounds in the muraymycin complex were elucidated using HPLC ESI capillary-skimmer CID FTMS by correlating their fragmentation patterns with those of muraymycins A1 and B1. Sequential neutral losses of an aminosugar, a valine, a uridine, and an ester fatty acid from the muraymycin parent ions provided diagnostic fragments for characterization.     

Web and database software for identification of intact proteins using "top down" mass spectrometry

For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/).     

Compositional space boundaries for organic compounds

An upper elemental compositional boundary for fossil hydrocarbons has previously been established as double-bond equivalents (i.e., DBE = rings plus double bonds) not exceeding 90% of the number of carbons. For heteroatom-containing fossil compounds, the 90% rule still applies if each N atom is counted as a C atom. The 90% rule eliminates more than 10% of the possible elemental compositions at a given mass for fossil database molecules. However, some synthetic compounds can fall outside the upper boundary defined for naturally occurring compounds. Their inclusion defines an "absolute" upper boundary as DBE (rings plus double bonds to carbon) equal to carbon number plus one, and applies to all organic compounds including fullerenes and other molecules containing no hydrogen. Finally, the DBE definition can fail for molecules with particular atomic valences. Therefore, we also present a generalized DBE definition that includes atomic valence to enable calculation of the correct total number of rings, double bonds, and triple bonds for heteroatom-containing compounds.