High-resolution Fourier-transform Ion Cyclotron Resonance Research Articles

Combination of long-term 13CO2 labeling and isotopolog profiling allows turnover analysis of photosynthetic pigments in Arabidopsis leaves

BackgroundLiving cells maintain and adjust structural and functional integrity by continual synthesis and degradation of metabolites and macromolecules. The maintenance and adjustment of thylakoid membrane involve turnover of photosynthetic pigments along with subunits of protein complexes. Quantifying their turnover is essential to understand the mechanisms of homeostasis and long-term acclimation of photosynthetic apparatus. Here we report methods combining whole-plant long-term 13CO2 labeling and liquid chromatography - mass spectrometry (LC–MS) analysis to determine the size of non-labeled population (NLP) of carotenoids and chlorophylls (Chl) in leaf pigment extracts of partially 13C-labeled plants.ResultsThe labeling chamber enabled parallel 13CO2 labeling of up to 15 plants of Arabidopsis thaliana with real-time environmental monitoring ([CO2], light intensity, temperature, relative air humidity and pressure) and recording. No significant difference in growth or photosynthetic pigment composition was found in leaves after 7-d exposure to normal CO2 (~ 400 ppm) or 13CO2 in the labeling chamber, or in ambient air outside the labeling chamber (control). Following chromatographic separation of the pigments and mass peak assignment by high-resolution Fourier-transform ion cyclotron resonance MS, mass spectra of photosynthetic pigments were analyzed by triple quadrupole MS to calculate NLP. The size of NLP remaining after the 7-d 13CO2 labeling was ~ 10.3% and ~ 11.5% for all-trans- and 9-cis-β-carotene, ~ 21.9% for lutein, ~ 18.8% for Chl a and 33.6% for Chl b, highlighting non-uniform turnover of these pigments in thylakoids. Comparable results were obtained in all replicate plants of the 13CO2 labeling experiment except for three that were showing anthocyanin accumulation and growth impairment due to insufficient water supply (leading to stomatal closure and less 13C incorporation).ConclusionsOur methods allow 13CO2 labeling and estimation of NLP for photosynthetic pigments with high reproducibility despite potential variations in [13CO2] between the experiments. The results indicate distinct turnover rates of carotenoids and Chls in thylakoid membrane, which can be investigated in the future by time course experiments. Since 13C enrichment can be measured in a range of compounds, long-term 13CO2 labeling chamber, in combination with appropriate MS methods, facilitates turnover analysis of various metabolites and macromolecules in plants on a time scale of hours to days.

Temperature and moisture alter organic matter composition across soil fractions

Understanding the complex interplay of biotic and abiotic controls on soil organic carbon (SOC) stabilization and aggregate formation is a vital and evolving research field, with implications for C and climate change modeling. Here, we delve into the effects of temperature and moisture treatments on aggregate SOC composition. Aggregate fractions representing different levels of physical protection for SOC were isolated three times during a 6-month incubation with 2x2 factorial temperature and moisture treatments (22 or 30 °C, 45% or 65% water-filled pore space). The chemical composition within each fraction was analyzed using high-resolution Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to evaluate the prevalence of different classes of C compounds by fraction and temperature and moisture treatments. In addition, a partial least square regression (PLSR) was used to explore potential correlations between relative abundance of C compound classes and C content within aggregate fractions. We found that organic matter in the macro- (>250 µm) and micro-aggregates (53 to 250 µm) was relatively enriched in lipid-, carbohydrates-, and protein--like compounds compared to silt and clay fractions (<53 µm). Organic matter in silt and clay was, on the other hand, relatively enriched in compounds with no specific classification, with overall high aromaticity. Broadly, simpler, low-molecular-weight C storage was altered by both temperature and moisture, while complex C storage was especially altered by moisture, within aggregates. Univariate effects of temperature and moisture on specific compound classes varied by soil fraction, but across fractions temperature increased the relative abundance of condensed- and unsaturated hydrocarbon-, tannin-, and amino sugar-like compounds and decreased the relative abundance of protein-like compounds. Moisture increased tannin-, condensed hydrocarbon-like compounds, and the overall aromaticity, and had the most pronounced effect in fractions occluded within macroaggregates, suggesting that substrate diffusion and pore connectivity within the aggregate environment drive the composition of C protected within aggregates. The PLSR indicated that treatments promoted different compounds to contribute to C accrual. Under dry conditions, condensed hydrocarbon-like compounds were associated with microaggregates, while amino sugar-like compounds were associated with macroaggregates and coarse particulate organic matter (POM), and lipid-like and aliphatic compounds were associated with silt and clay. Temperature effects on PLSR results were most visible in silt and clay fractions, where carbohydrate- and tannin-like compounds were associated with C content. This suggests that warmer conditions under climate change may more substantially alter mineral-associated C content, while changing water regimes will alter C content in physically protected environments, with the most significant changes under cool and moist conditions. Overall, our data reveal distinct resource pools in different climate and aggregate environments, a baseline for our understanding of SOC accrual and soil structure under different conditions. More research into how microbes process physically protected SOC under altered environments may fine-tune our ability to predict soil functions including water behavior and nutrient release.

A quantitative light-isotope measurement system for climate and energy applications

We describe the design, operation, and performance of a new instrumental configuration capable of quantitative determinations with sub-picomole accuracy of dilute concentrations of low mass species, such as He4, He3, Ne20, and Ar40, in a balance of stable hydrogen (H2, DH, and D2) gas. This inexpensive system may realize important applications in fields ranging from climate studies to hydrogen fusion energy research, thereby providing an expanded availability of this diagnostic within emerging energy systems research and development. These spectra, calibration curves, and determinations were obtained by using a novel method for the purification and subsequent removal of the hydrogen matrix gas, and an extensively modified commercial Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometer with an electron impact (EI) ionizer. These high-resolution FT-ICR mass spectrometers have routinely achieved a resolution, R = m/Δm better than 10,000 Da at mass-3, with a mass resolution that scales as 1/m. These devices have easily resolved D2 from He4, and DH from He3. The performance of this upgraded instrument has demonstrated the ability to detect impurities from tiny air leaks, such as Ar40 and Ne20, in the presence of the hydrogen matrix gas. While no concentration measurements of radioactive species have been attempted to date with this system, it is expected to easily resolve DT from D2H (a 0.0059 Da mass difference) and HT from all other mass-4 species.

First screening of biocides, persistent organic pollutants, pharmaceutical and personal care products in Antarctic phytoplankton from Deception Island by FT-ICR-MS

In recent years, the Antarctic territory has seen a rise in the number of tourists and scientists. This has led to an increase in the anthropogenic footprint in Antarctic ecosystems, namely in terms of emerging contaminants, such as Biocides, Persistent Organic Pollutants (POPs) as well as Pharmaceutical and Personal Care Products (PPCPs). Yet scarce information on the presence of these emerging contaminants is available for trophic compartments, especially the phytoplankton community. Using high resolution Fourier-transform ion cyclotron-resonance mass spectrometry (FT-ICR-MS), an untargeted screening of the metabolome of the phytoplankton community was performed. Seventy different contaminant compounds were found to be present in phytoplankton collected at two sites in Port Foster Bay at Deception Island. These emerging contaminants included 1 polycyclic aromatic hydrocarbon (PAH), 10 biocides (acaricides, fungicides, herbicides, insecticides and nematicides), 11 POPs (flame retardants, paints and dyes, polychlorinated biphenyl (PCB), phthalates and plastic components), 5 PCPs (cosmetic, detergents and dietary compounds), 40 pharmaceutical compounds and 3 illicit drugs. Pharmaceutical compounds were, by far, the largest group of emerging contaminants found in phytoplankton cells (anticonvulsants, antihypertensives and beta-blockers, antibiotics, analgesic and anti-inflammatory drugs). The detection of several of these potentially toxic compounds at the basis of the marine food web has potentially severe impacts for the whole ecosystem trophic structure. Additionally, the present findings also point out that the guidelines proposed by the Antarctic Treaty and Protocol on Environmental Protection to the Antarctic Treaty should be revisited to avoid the proliferation of these and other PPCPs in such sensitive environments.

Coupled Biotic-Abiotic Processes Control Biogeochemical Cycling of Dissolved Organic Matter in the Columbia River Hyporheic Zone

A critical component of assessing the impacts of climate change on watershed ecosystems involves understanding the role that dissolved organic matter (DOM) plays in driving whole ecosystem metabolism. The hyporheic zone—a biogeochemical control point where ground water and river water mix—is characterized by high DOM turnover and microbial activity and is responsible for a large fraction of lotic respiration. Yet, the dynamic nature of this ecotone provides a challenging but important environment to parse out different DOM influences on watershed function and net carbon and nutrient fluxes. We used high-resolution Fourier-transform ion cyclotron resonance mass spectrometry to provide a detailed molecular characterization of DOM and its transformation pathways in the Columbia river watershed. Samples were collected from ground water (adjacent unconfined aquifer underlying the Hanford 300 Area), Columbia river water, and its hyporheic zone. The hyporheic zone was sampled at five locations to capture spatial heterogeneity within the hyporheic zone. Our results revealed that abiotic transformation pathways (e.g., carboxylation), potentially driven by abiotic factors such as sunlight, in both the ground water and river water are likely influencing DOM availability to the hyporheic zone, which could then be coupled with biotic processes for enhanced microbial activity. The ground water profile revealed high rates of N and S transformations via abiotic reactions. The river profile showed enhanced abiotic photodegradation of lignin-like molecules that subsequently entered the hyporheic zone as low molecular weight, more degraded compounds. While the compounds in river water were in part bio-unavailable, some were further shown to increase rates of microbial respiration. Together, river water and ground water enhance microbial activity within the hyporheic zone, regardless of river stage, as shown by elevated putative amino-acid transformations and the abundance of amino-sugar and protein-like compounds. This enhanced microbial activity is further dependent on the composition of ground water and river water inputs. Our results further suggest that abiotic controls on DOM should be incorporated into predictive modeling for understanding watershed dynamics, especially as climate variability and land use could affect light exposure and changes to ground water essential elements, both shown to impact the Columbia river hyporheic zone.

Challenges and opportunities for on-line monitoring of chlorine-produced oxidants in seawater using portable membrane-introduction Fourier transform-ion cyclotron resonance mass spectrometry.

The present study reports the first evaluation of a MIMS device equipped with a high-resolution Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS) for comprehensive speciation of chlorine-produced oxidants (CPO) in seawater. A total of 40 model compounds were studied: 4 inorganic haloamines (mono-, di-, and trichloramine and monobromamine), 22 organic N-haloamines, 12 N-haloamino acids, and 2 free oxidants (HOCl/ClO- and HOBr/BrO-). The main key factors influencing the analytes' introduction and their detection were optimized. Under optimized conditions, the rise and fall times of the MIMS signal ranged from 8 to 79min and from 7 to 73min, respectively, depending on the compound. Free oxidants and N-haloamino acids, which are ionic or too polar at seawater pH, hardly crossed the membrane, and MIMS analysis was thus unsuitable. Nevertheless, better enrichment and therefore better sensitivity were achieved with organic N-haloamines than with inorganic haloamines. The observed detection limits ranged from tens of μM to sub-μM levels. Oxidant decomposition occurred inside the MIMS device, at a higher rate for N-bromamines than for chlorinated analogues.Graphical abstract.

Untargeted Metabolite Mapping in 3D Cell Culture Models Using High Spectral Resolution FT-ICR Mass Spectrometry Imaging

Multicellular tumor spheroids (MTS) are a well-established model system for drug development and are a valuable in vitro research tool for use prior to employing animal models. These 3D-cell cultures are thought to display chemical gradients of oxygen and nutrients throughout their structure, giving rise to distinct microenvironments in radial layers, thus, mimicking the pathophysiological environment of a tumor. Little is known about the localized distributions of metabolites within these microenvironments. To address this, here we utilize high spectral resolution Fourier-transform ion cyclotron resonance (FT-ICR), MALDI mass spectrometry imaging (MSI) to image the distribution of endogenous metabolites in breast cancer MCF-7 spheroids. We show that known specific metabolite markers (adenosine phosphates and glutathione) indicate that the central region of these cell culture models experiences increased hypoxic and oxidative stress. By using discriminatory analysis, we have identified which m/z values localize toward the outer proliferative or central hypoxic regions of an MTS. Elemental formulae were assigned with sub-ppm mass accuracy, allowing metabolite assignment. Using this information, we have mapped these metabolites back to distinct pathways to improve our understanding of the molecular environment and biochemistry of these tumor models.

Identifying precursors and aqueous organic aerosol formation pathways during the SOAS campaign

Abstract. Aqueous multiphase chemistry in the atmosphere can lead to rapid transformation of organic compounds, forming highly oxidized, low-volatility organic aerosol and, in some cases, light-absorbing (brown) carbon. Because liquid water is globally abundant, this chemistry could substantially impact climate, air quality, and health. Gas-phase precursors released from biogenic and anthropogenic sources are oxidized and fragmented, forming water-soluble gases that can undergo reactions in the aqueous phase (in clouds, fogs, and wet aerosols), leading to the formation of secondary organic aerosol (SOAAQ). Recent studies have highlighted the role of certain precursors like glyoxal, methylglyoxal, glycolaldehyde, acetic acid, acetone, and epoxides in the formation of SOAAQ. The goal of this work is to identify additional precursors and products that may be atmospherically important. In this study, ambient mixtures of water-soluble gases were scrubbed from the atmosphere into water at Brent, Alabama, during the 2013 Southern Oxidant and Aerosol Study (SOAS). Hydroxyl (OH⚫) radical oxidation experiments were conducted with the aqueous mixtures collected from SOAS to better understand the formation of SOA through gas-phase followed by aqueous-phase chemistry. Total aqueous-phase organic carbon concentrations for these mixtures ranged from 92 to 179 µM-C, relevant for cloud and fog waters. Aqueous OH-reactive compounds were primarily observed as odd ions in the positive ion mode by electrospray ionization mass spectrometry (ESI-MS). Ultra high-resolution Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) spectra and tandem MS (MS–MS) fragmentation of these ions were consistent with the presence of carbonyls and tetrols. Products were observed in the negative ion mode and included pyruvate and oxalate, which were confirmed by ion chromatography. Pyruvate and oxalate have been found in the particle phase in many locations (as salts and complexes). Thus, formation of pyruvate/oxalate suggests the potential for aqueous processing of these ambient mixtures to form SOAAQ.

Biodegradation of carbamazepine and clarithromycin by Trichoderma harzianum and Pleurotus ostreatus investigated by liquid chromatography – high-resolution tandem mass spectrometry (FTICR MS-IRMPD)

In this study, the capability of pharmaceutical biodegradation of fungus Trichoderma harzianum was evaluated through the comparison with the well-known biodegradation capability of white-rot fungus Pleurotus ostreatus. The study was performed in aqueous phase under aerobic conditions, using two of the most frequently detected drugs in water bodies: carbamazepine and clarithromycin, with concentrations commonly found in treated wastewater (4μg/l and 0.03μg/l respectively). For the first time, we demonstrated that T. harzianum is able to remove carbamazepine and clarithromycin. The analyses were performed by reversed-phase liquid chromatography/mass spectrometry, using high-resolution Fourier-transform ion cyclotron resonance mass spectrometry upon electrospray ionization in positive ion mode. The high selectivity and mass accuracy provided by high-resolution mass spectrometry, allowed us to identify some unknown metabolites. On the basis of our study, the major metabolites detected in liquid culture treated by T. harzianum were: 14-hydroxy-descladinosyl- and descladinosyl-clarithromycin, which are pharmacologically inactive products not dangerous for the environment.

Collision cross section measurements for biomolecules within a high-resolution FT-ICR cell: theory.

In this study, an energetic hard-sphere ion-neutral collision model was proposed to bridge-link ion collision cross section (CCS) with the image current collected from a high-resolution Fourier transform ion cyclotron resonance (FT-ICR) cell. By investigating the nonlinear effects induced by high-order electric fields and image charge forces, the energetic hard-sphere collision model was validated through experiments. Suitable application regions for the energetic hard-sphere collision model, as well as for the conventional Langevin and hard-sphere collision models, were also discussed. The energetic hard-sphere collision model was applied in the extraction of ion CCSs from high-resolution FT-ICR mass spectra. Discussions in the present study also apply to FT-Orbitraps and FT-quadrupole ion traps.

Spatial segmentation of MALDI FT-ICR MSI data: a powerful tool to explore the head and neck tumor in situ lipidome.

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) is a well-established analytical technique for determining spatial localization of lipids in biological samples. The use of Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometers for the molecular imaging of endogenous compounds is gaining popularity, since the high mass accuracy and high mass resolving power enables accurate determination of exact masses and, consequently, a more confident identification of these molecules. The high mass resolution FT-ICR imaging datasets are typically large in size. In order to analyze them in an appropriate timeframe, the following approach has been employed: the FT-ICR imaging datasets were spatially segmented by clustering all spectra by their similarity. The resulted spatial segmentation maps were compared with the histologic annotation. This approach facilitates interpretation of the full datasets by providing spatial regions of interest. The application of this approach, which has originally been developed for MALDI-TOF MSI datasets, to the lipidomic analysis of head and neck tumor tissue revealed new insights into the metabolic organization of the carcinoma tissue.

Advanced analytical mass spectrometric techniques and bioassays to characterize untreated and ozonated oil sands process-affected water.

Oil sands process-affected water (OSPW) is a toxic and poorly biodegradable mixture of sand, silt, heavy metals, and organics. In this study, qualitative and quantitative comparisons of naphthenic acids (NAs) were done using ultraperformance liquid chromatography time-of-flight mass spectrometry (UPLC TOF-MS), Fourier transform ion cyclotron resonance (FT-ICR) MS, and ion mobility spectrometry (IMS). The unique combination of these analyses allowed for the determination and correlation of NAs, oxidized NAs, and heteroatom (sulfur or nitrogen) NAs. Despite its lower resolution, UPLC-TOF MS was shown to offer a comparable level of reliability and precision as the high resolution FT-ICR MS. Additionally, the impacts of ozonation (35 mg/L utilized ozone dose) and subsequent NAs degradation on OSPW toxicity were assessed via a collection of organisms and toxicity end points using Vibrio fischeri (nonspecific), specific fish macrophage antimicrobial responses, and fish olfactory responses. Fish macrophages exposed to ozonated OSPW for 1 week showed higher production of reactive oxygen and nitrogen intermediates; however, after 12 weeks the responses were reduced significantly. Fish olfactory tests suggested that OSPW interfered with their perception of odorants. Current results indicate that the quantification of NAs species, using novel analytical methods, can be combined with various toxicity methods to assess the efficiency of OSPW treatment processes.

Intact protein mass spectrometry and top-down proteomics

American Society for Mass Spectrometry Sanibel meeting on top-down mass spectrometrySt Pete Beach, FL, USA, 24–27 January 2013Top-down mass spectrometry involves analysis of intact proteins, typically using electrospray ionization, as multiple charging enhances dissociation and thus identification by comparison of precursor and product ion masses with protein sequence databases. Traditionally a low-throughput, precision technology performed on high-resolution Fourier-transform ion cyclotron resonance mass analyzers, top-down proteomics aims to increase throughput for whole proteome analysis while preserving the inherent value of an intact protein mass measurement. This years’ American Society for Mass Spectrometry Sanibel meeting brought together established scientists who have demonstrated the viability of the top-down approach and its applicability to virtually all segments of the proteome, mixing them with researchers from diverse areas and with the common interest of advancing top-down into the high-throughput proteomics mainstream. Advances in instrumentation including the orbitrap analyzer, ionization mechanisms, dissociation strategies and informatics, as well as a wide variety of applications, were discussed in depth, leading to the inescapable conclusion that the future for top-down is bright.

Characterization of Bio‐oil from Hydrothermal Liquefaction of Organic Waste by NMR Spectroscopy and FTICR Mass Spectrometry

Solid wastes of organic origins are potential feedstocks for the production of liquid biofuels, which could be suitable alternatives to fossil fuels for the transport and heating sectors, as well as for industrial use. By hydrothermal liquefaction, the wet biomass is partially transformed into a water-immiscible, oil-like organic matter called bio-oil. In this study, an integrated NMR spectroscopy/mass spectrometry approach has been developed for the characterization of the hydrothermal liquefaction of bio-oil at the molecular level. (1)H and (13)C NMR spectroscopy were used for the identification of functional groups and gauging the aromatic carbon content in the mixture. GC-MS analysis revealed that the volatile fraction was rich in fatty acids, as well as in amides and esters. High-resolution Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) has been applied in a systematic way to fully categorize the bio-oil in terms of different classes of components, according to their molecular formulas. Most importantly, for the first time, by using this technique, and for the liquefaction bio-oil characterization in particular, FT-MS data have been used to develop a methodology for the determination of the aromatic versus aliphatic carbon and nitrogen content. It is well known that, because they resist hydrogenation and represent sources of polluting species, both aromatic molecules and nitrogen-containing species raise concerns for subsequent upgrading of bio-oil into a diesel-like fuel.

Identification of unsaturated N‐acylhomoserine lactones in bacterial isolates of Rhodobacter sphaeroides by liquid chromatography coupled to electrospray ionization‐hybrid linear ion trap‐Fourier transform ion cyclotron resonance mass spectrometry

The identification of two unsaturated N-acylhomoserine lactones (AHLs) produced by Rhodobacter sphaeroides bacteria, based on liquid chromatography (LC) coupled to a hybrid quadrupole linear ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer upon electrospray ionization (ESI), is presented. Besides the confirmation of the signaling molecule already described in the literature, i.e. (Z)-N-tetradec-7-enoyl-homoserine lactone (C(14:1)-HSL), we have discovered the occurrence, at low, yet significant levels, of another monounsaturated compound, C(12:1) -HSL, which may extend the number of small diffusible chemical signals known for R. sphaeroides. Both unsaturated AHLs were identified by high-resolution FTICR mass spectrometry in extracts of bacterial culture media and the occurrence of a C=C bond was assessed upon their conversion into bromohydrins. Collision-induced dissociation (CID) spectra were then collected on the LTQ mass analyzer. A careful comparison of tandem MS spectra of monounsaturated (i.e., C(12:1)-HSL and C(14:1)-HSL) and saturated AHLs (i.e. C(12)-HSL and C(14)-HSL) led to the emphasis of two series of product ions, exhibiting 14 Da spaced m/z ratios. Both series were referred to progressive fragmentations at the aliphatic end of the AHL acyl chains, followed by neutral losses of terminal alkenes (i.e. CH(2)=CH(CH(2))(n)H). In particular, the series located at the higher end of the explored m/z range (>200 Da), observed only for monounsaturated species, enabled the location of the C=C bond between carbons 7 and 8 of the acyl chain.

Deciphering sulfoglycolipids of Mycobacterium tuberculosis

For 4 decades, in vivo and in vitro studies have suggested that sulfoglycolipids (SGLs) play a role in the virulence or pathogenesis of the tubercle bacilli. However, the SGL structure and biosynthesis pathway remain only partially elucidated. Using the modern tools of structural analysis, including MALDI-time-of-flight MS, MS/MS, and two-dimensional NMR, we reevaluated the structure of the different SGL acyl (di-, tri-, and tetra-acylated) forms of the reference strain Mycobacterium tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains previously described to intracellularly accumulate di-acylated SGL. We report here the identification of new acyl forms: di-acylated SGL esterified by simple fatty acids only, as well as mono-acylated SGL bearing a hydroxyphthioceranoic acid, which were characterized in the wild-type strain. In a clinical strain, a complete family of mono-acylated SGLs was characterized in high abundance for the first time. For the mmpL8 mutant, SGLs were found to be esterified i) by an oxophthioceranoic acid, never observed so far, and ii) at nonconventional positions in the case of the unexpected tri-acylated forms. Our results further confirm the requirement of MmpL8 for the complete assembly of the tetra-acylated forms of SGL and also provide, by the discovery of new intermediates, insights in terms of the possible SGL biosynthetic pathways.

Enhanced digestion efficiency, peptide ionization efficiency, and sequence resolution for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.

Solution-phase hydrogen/deuterium exchange (HDX) monitored by high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry offers a rapid method to study protein conformations and protein-protein interactions. Pepsin is usually used to digest proteins in HDX and is known for lack of cleavage specificity. To improve digestion efficiency and specificity, we have optimized digestion conditions and cleavage preferences for pepsin and protease type XIII from Aspergillus saitoi. A dilution series of the proteases was used to determine the digestion efficiency for several test proteins. Protease type XIII prefers to cleave on the C-terminal end of basic amino acids and produced the highest number of fragments and the best sequence coverage compared to pepsin or protease type XVIII from Rhizhopus. Furthermore, protease type XIII exhibited much less self-digestion than pepsin and thus is superior for HDX experiments. Many highly overlapped segments from protease type XIII and pepsin digestion, combined with high-resolution FTICR mass spectrometry, provide high sequence resolution (to as few as one or two amino acids) for the assignment of amide hydrogen exchange rate. Our H/D exchange results correlate well with the secondary and tertiary structure of myoglobin. Such assignments of highly overlapped fragments promise to greatly enhance the accuracy and sequence resolution for determining conformational differences resulting from ligand binding or protein-protein interactions.

Self-Association of Organic Acids in Petroleum and Canadian Bitumen Characterized by Low- and High-Resolution Mass Spectrometry

We examine solution-phase aggregation for a whole crude oil, whole bitumen, and bitumen distillate fractions by negative-ion electrospray ionization [(−) ESI] detected by both high-resolution [Fourier transform ion cyclotron resonance (FT-ICR)] and low-resolution [linear quadrupole ion trap (LTQ)] mass spectrometry (MS). Aggregate formation for both crude oil and bitumens is concentration-dependent. At high concentrations (i.e., >1 mg/mL), the disruption of noncovalent interactions between heteromultimers by low-energy collision-activated dissociation (CAD) yields LTQ dissociation mass spectra with molecular-weight distributions identical to those observed by FT-ICR MS analysis at lower concentrations for purely monomeric species. These materials can exist as aggregates in solution even at high dilution (less than 0.1 mg/mL). We demonstrate the concentration and boiling point dependence for multimerization of polar acidic species in the Athabasca bitumen and bitumen distillates. Interestingly, the lowest boiling distillation cut (375−400 °C) displays the highest aggregation tendency, with dimers at concentrations as low as 0.05 mg/mL. Higher boiling point distillation cuts display a decreased aggregation tendency with an increasing cut point. High-resolution negative-ESI FT-ICR MS of the bitumen distillation fractions reveals the elemental composition, and thus the class, type, and carbon number of the multimeric species. Acidic heteroatomic classes for the distillation cut multimers include O4, S1O4, O3, S1O3, N1O2, and N1S1O2. The most abundant multimers for the 375−400 °C distillation cut are O4 species, whereas the 450−475 °C cut contains N1O2 multimers in the highest relative abundance. Changes in multimer heteroatom content as a function of the monomer composition and distillation cut suggest that aggregation depends upon the chemical functionalities of the monomer species.

Charge ratio analysis method to interpret high resolution electrospray Fourier transform—ion cyclotron resonance mass spectra

The charge ratio analysis method (CRAM) is a new approach for the interpretation of high resolution Fourier transform ion cyclotron resonance (FT-ICR) electrospray mass spectral data. The high resolution capability of FT-MS provides resolved isotopic peaks of multiply charged ions of biopolymers enabling their accurate and monoisotopic molecular mass determination. It does, however, require that the correct charge and isotope composition of these ions be assigned in order for this accuracy to be realized. The unique feature of the CRAM in processing the FT-ICR data is that the charge states of ions are identified from analysis of the ratios of m/ z values of isotopic peaks of different multiply charged ions. In addition, the CRAM process correlates the isotopic peaks of different multiply charged ions that share the same isotopic composition. As the size of biopolymers increases, their isotope patterns become more uniform and more difficult to discern from one another. This impacts on the correct matching of a theoretical isotope distribution to experimental data particularly in the case of biopolymers of unknown elemental compositions. The significance of the CRAM is demonstrated in terms of correlating theoretical isotopic distributions to experimental data where this correlation could not always be achieved based on the relative intensities of isotopic peaks alone. While for high resolution FT-ICR mass spectral data, the ion charge can be otherwise determined from the reciprocal of the m/ z difference between adjacent isotopic peaks, the CRAM approach is superior and determines ion charge with several orders of magnitude higher accuracy. The CRAM has been applied to high resolution FT-ICR mass spectral for several proteins (ubiquitin, cytochrome c, transthyretin, lysozyme and calmodulin) to demonstrate the general utility of this approach and its application to proteomics. The results have been discussed in terms of internally calibrated ions versus external calibration where the CRAM approach was superior in both cases.

Assigning product ions from complex MS/MS spectra: The importance of mass uncertainty and resolving power

This study offers a unique insight into the mass accuracy and resolving power requirements in MS/MS analyses of complex product ion spectra. In the examples presented here, accurate mass assignments were often difficult because of multiple isobaric interferences and centroid mass shifts. The question then arose whether the resolving power of a medium-resolution quadrupole time-of flight (QqTOF) is sufficient or high-resolution Fourier-transform ion cyclotron resonance (FT-ICR) is required for unambiguous assignments of elemental compositions. For the comparison, two paralytic shellfish poisons (PSP), saxitoxin (STX) and neosaxitoxin (NEO), with molecular weights of 299 and 315 g x mol(-1), respectively, were chosen because of the high peak density in their MS/MS spectra. The assessment of QqTOF collision-induced dissociation spectra and FT-ICR infrared multiphoton dissociation spectra revealed that several intrinsic dissociation pathways leading to isobaric fragment ions could not be resolved with the QqTOF instrument and required FT-ICR to distinguish very close mass differences. The second major source of interferences was M + 1 species originating from coactivated 13C12Cc-1 ion contributions of the protonated molecules of the PSPs. The problem in QqTOF MS results from internal mass calibration when the MH+ ions of analyte and mass calibrant are activated at the same time in the collision or trapping cell. Although FT-ICR MS readily resolved these interfering species, the QqTOF did not provide resolving power >20,000 (full width at half maximum) required to separate most isobaric species. We were able to develop a semi-internal QqTOF calibration technique that activated only the isolated 12C isotope species of the protonated molecules, thus reducing the M + 1 interferences significantly. In terms of overall automated elemental formulas assignment, FT-ICR MS achieved the first formula hit for 100% of the product ions, whereas the QqTOF MS hit rate was only 56 and 65% for STX and NEO product ions, respectively. External mass calibration from commercial FT-ICR and QqTOF instruments gave similar results.