Fourier Transform Ion Cyclotron Resonance MS Research Articles

Native Top-Down Mass Spectrometry for Characterizing Sarcomeric Proteins Directly from Cardiac Tissue Lysate.

Native top-down mass spectrometry (nTDMS) has emerged as a powerful structural biology tool that can localize post-translational modifications (PTMs), explore ligand-binding interactions, and elucidate the three-dimensional structure of proteins and protein complexes in the gas-phase. Fourier-transform ion cyclotron resonance (FTICR) MS offers distinct capabilities for nTDMS, owing to its ultrahigh resolving power, mass accuracy, and robust fragmentation techniques. Previous nTDMS studies using FTICR have mainly been applied to overexpressed recombinant proteins and protein complexes. Here, we report the first nTDMS study that directly analyzes human heart tissue lysate by direct infusion FTICR MS without prior chromatographic separation strategies. We have achieved comprehensive nTDMS characterization of cardiac contractile proteins that play critical roles in heart contraction and relaxation. Specifically, our results reveal structural insights into ventricular myosin light chain 2 (MLC-2v), ventricular myosin light chain 1 (MLC-1v), and alpha-tropomyosin (α-Tpm) in the sarcomere, the basic contractile unit of cardiac muscle. Furthermore, we verified the calcium (Ca2+) binding domain in MLC-2v. In summary, our nTDMS platform extends the application of FTICR MS to directly characterize the structure, PTMs, and metal-binding of endogenous proteins from heart tissue lysate without prior separation methods.

High-Resolution Mass Spectrometry-Based Chemical Fingerprinting of Baijiu, a Traditional Chinese Liquor.

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, coupled with electrospray ionization (ESI) or atmospheric-pressure photoionization (APPI), was employed for chemical fingerprinting of baijiu, a traditional Chinese liquor. Baijiu is the most consumed distilled alcoholic beverage globally, with over 10 billion liters sold annually. It is a white (transparent) spirit that exhibits similarities to dark spirits such as whisky or rum in terms of aroma and mouthfeel. In this study, direct-infusion FT-ICR mass spectrometry was used to analyze 10 commercially available baijiu liquors, enabling the examination of both volatile and nonvolatile constituents without the need for tedious sample extractions or compound derivatizations. The chemical fingerprints obtained by FT-ICR MS revealed substantial compositional diversity among different baijiu liquors, reflecting variations in the raw materials and production methods. The main compounds identified included a variety of acids, esters, aldehydes, lactones, terpenes, and phenolic compounds. The use of ESI and APPI provided complementary compositional information; while ESI demonstrated greater selectivity toward polar, aliphatic sample constituents, APPI also ionized semipolar and nonpolar (aromatic) ones.

Analysis of Volatile and Nonvolatile Constituents in Gin by Direct-Infusion Ultrahigh-Resolution ESI/APPI FT-ICR Mass Spectrometry

Gin is one of the most consumed distilled alcoholic spirits worldwide, with more than 400 million liters sold every year. It is most often produced through redistillation of agricultural ethanol in the presence of botanicals, most notably juniper berries, which give gin its characteristic flavor. Due to its natural ingredients, gin is a complex mixture of hundreds of volatile and nonvolatile chemical constituents. In this work, ultrahigh-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry was used for the compositional analysis of 16 commercially produced gins. Two complementary ionization methods, namely, electrospray ionization (ESI) and atmospheric-pressure photoionization (APPI), were employed to cover a wider compositional space. Each gin provided unique chemical fingerprints by ESI and APPI, which allowed semiquantitative analysis of 135 tentatively identified compounds, including terpene hydrocarbons, terpenoids, phenolics, fatty acids, aldehydes, and esters. Most of these compounds have not been previously reported in gins. While chemical fingerprints were rather similar between most products, some products contained unique compounds due to their special natural ingredients or the production methods applied. For instance, a barrel-matured gin contained a high content of syringaldehyde and sinapaldehyde, which are typical phenolic aldehydes originated from oak wood. In addition, the relative abundance of vanillin, vanillic acid, gallic acid, coniferyl aldehyde, and syringaldehyde was clearly higher than in the other gin samples. Ultrahigh-resolution FT-ICR MS serves as a powerful tool for direct chemical fingerprinting of gin or any other distilled spirit, which can be used for rapid product quality screening, product optimization, or possible counterfeit product discovery.

Large-Scale Interlaboratory DI-FT-ICR MS Comparability Study Employing Various Systems.

Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics. Extended comparisons employing different FT-ICR MS instruments with qualitative direct infusion analysis are scarce since the majority of detected compounds cannot be quantified. The extent to which observations can be reproduced by different laboratories remains unknown. We set up a preliminary study which encompassed a set of 17 laboratories around the globe, diverse in instrumental characteristics and applications, to analyze the same sets of extracts from commercially available standard human blood plasma and Standard Reference Material (SRM) for blood plasma (SRM1950), which were delivered at different dilutions or spiked with different concentrations of pesticides. The aim of this study was to assess the extent to which the outputs of differently tuned FT-ICR mass spectrometers, with different technical specifications, are comparable for setting the frames of a future DI-FT-ICR MS ring trial. We concluded that a cluster of five laboratories, with diverse instrumental characteristics, showed comparable and representative performance across all experiments, setting a reference to be used in a future ring trial on blood plasma.

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.

Imaging Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of oxaliplatin derivatives in human tissue sections

Mass Spectrometry Imaging is an effective technology that allows to determine the in-situ distribution of endogen and/or exogen small molecules. It is a rapidly emerging approach for visualizing drugs and their metabolites within biological tissues. Matrix-Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry Imaging (MSI) coupled to high resolving power analyzer (e.g. TOF) was already investigated for metallodrug localization and metabolization studies, but was proved to suffer from a lack of sensitivity and resolution, leading to poor coverage and assignment. To counter these technological limitations, the use of ultra-high resolving power analyzer such as Fourier Transform Ion Cyclotron Resonance (FTICR) could be revealed as a technique of choice. The high field FTICR MS provides ultra-high resolving power and mass accuracy that allows exhaustive molecule coverage and non-ambiguous molecular formula assignments. Platinum derivatives, such as oxaliplatin, are widely used as therapeutic agents for cancer treatment. The assessment of their intake, distribution and metabolism within the organs is important to know the risks associated with their use. In this study, MALDI FTICR MSI analyses were performed to better understand the penetration and metabolization of platinum derivatives in ovaries of women treated by Hyperthermic Intraperitoneal Chemotherapy (HIPEC) for peritoneal metastasis of colorectal or appendicular origin. Twelve ovary sections, from six ovary samples in six women donors, before and after treatment, were analyzed with 120 μm spatial resolution. For the first time, the high resolving power (220,000 at m/z 457) and sub-ppm accuracy (<1 ppm) of the FTICR combined with an Isotopic Fine Structure study enabled to distinguish two Pt-isobaric species derived from oxaliplatin in biological tissues. One of these, which is unknown, was specifically localized at the contour of the ovary.

Molecular Characterization of Aged Bitumen with Selective and Nonselective Ionization Methods by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. 2. Statistical Approach on Multiple-Origin Samples

Bitumen is a very viscous and complex mixture primarily used as a binder for asphalt construction. During its service life, this binder is subjected to oxidative aging, which leads to asphalt stiffness and thermal cracking of the pavement structure. The behavior of bitumen toward oxidative aging is greatly dependent on its intrinsic properties and particularly on its content of nitrogen, oxygen, and sulfur functional groups. This is of fundamental importance as the origin of the crude will influence its resistance to oxidation after several years of use in a road. It is then essential to correlate the native composition of bitumen and their corresponding oxidized products to explain changes in the physicochemical behavior of asphalt binders. Pressure aging vessel is probably the most widely used technique to simulate long-time aging of bitumen. This artificial aging technique leads to accelerated aging under high-pressure and -temperature conditions. To understand such mechanisms, characterization at the molecular level is required. Nowadays, Fourier transform ion cyclotron resonance (FTICR) is the most-used technique for the analysis of petroleum products. Bitumen samples from three different origins were subjected to PAV aging and analyzed by FTICR-MS. Coupling of electrospray ionization (ESI) and atmospheric pressure photo ionization (APPI) sources were used to provide an exhaustive characterization of the samples. Principal component analysis (PCA), which is a multivariate statistical approach, was used to separate samples according to aging and origin. Indeed, ESI coupled to PCA allowed a significant separation of samples according to their PAV-aging on the PC1 axis. PC1 scores were then used as an intensity scale to carry out a modified double bond equivalent versus carbon number (DBE vs C#) mapping. This technique allowed the characterization of specific aging markers in Oz and OzSy families for each bitumen sample. In addition, PC1 coordinates were used to develop a predictive model by principal component regression (PCR). Significant differences in sensitivity to oxidation were obtained for the three bitumen samples. Concerning APPI results, less significant differences were obtained for aging compared to those obtained by ESI. PCA coupled to APPI was also used for modified DBE vs C# maps, separating samples according to their origin. Significant differences were obtained in each bitumen sample in the different compound classes (HC, S1, N1, O1, etc.). Overall, this study demonstrates the great interest of coupling FTICR MS data to a PCA statistical approach to characterize oxidative aging of bitumen samples according to their origin.

Molecular Characterization of NSO Compounds and Paleoenvironment Implication for Saline Lacustrine Oil Sands by Positive-Ion Mass Spectrometry Coupled with Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry.

NSO compounds mainly exist in geological bodies in the form of nonhydrocarbons and asphaltenes with abundant geological and geochemical information. Combined with the gas chromatography mass spectrometry (GC–MS) technology, positive-ion electrospray ionization Fourier-transform ion cyclotron resonance MS (FT-ICR MS) was used to understand the composition and distribution characteristics of NSO compounds in the oil sands of the Dongpu Depression and to explore their paleoenvironmental significance. The results show that n-alkanes are characterized by an even carbon number and phytane dominance, suggesting a saline lacustrine environment. Certain abundance of nC37 and β-carotane, high gammacerane content, and low diasterane content are detected in the analyzed samples, reflecting the saline-reducing paleoenvironment for the organic matter. Nine types of heteroatom compounds are detected: N1, N1O1, N1S1, O1, O1S1, O2, O2S1, S1, and S2. The main compounds are S1 and N1 compounds, followed by O1S1 compounds. The double-bond equivalent (DBE) value of S1 compounds is mainly distributed between 3 and 12, and the carbon number is mainly distributed between 18 and 35. The DBE value of N1 compounds is mainly distributed between 4 and 14, and the carbon number is mainly distributed in the range 15–35. Among the S1 compounds, DBE3 compounds (thiophenes) have relatively more sulfur-containing carotenoids (C40). The abundance of C40 S1 and the ratio of pyridine and its homologue DBE4–8/DBE9–12 N1 compounds show a good contrast with the paleoenvironment indicators of gammacerane/C30 hopane and diasterane/regular sterane. They can be used as a reference for the paleoenvironment index. Maturity is another factor affecting the distribution of NSO heteroatoms in the oil sands. NSO compounds are enriched in the DBE area with higher condensation, and the main peak carbon shifts forward. As the maturity increases, the relative abundance of N1 compounds increases, the aromatization intensifies, and carbon is broken into short chains. The research results shed light on the potential application of NSO compounds in petroleum exploration based on FT-ICR MS.

Negative-mode mass spectrometry in the analysis of invertebrate, fungal, and protist N-glycans.

The approaches for analysis of N-glycans have radically altered in the last 20 years or so. Due to increased sensitivity, mass spectrometry has become the predominant method in modern glycomics. Here, we summarize recent studies showing that the improved resolution and detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has contributed greatly to the discovery of a large range of anionic and zwitterionic N-glycan structures across the different kingdoms of life, whereby MALDI-TOF MS in negative mode is less widely performed than in positive mode. However, its use enables the detection of key fragments indicative of certain sugar modifications such as sulfate, (methyl) phosphate, phosphoethanolamine, (methyl)aminoethylphosphonate, glucuronic, and sialic acid, thereby enabling certain isobaric glycan variations to be distinguished. As we also discuss in this review, complementary approaches such as negative-mode electrospray ionization-MS/MS, Fourier-transform ion cyclotron resonance MS, and ion mobility MS yield, respectively, cross-linkage fragments, high accuracy masses, and isomeric information, thus adding other components to complete the jigsaw puzzle when defining unusual glycan modifications from lower organisms.

Study on the Pretreatment Process and Removal Rules of Sulfur-Containing Compounds for Medium- and Low-Temperature Coal Tar.

The heteroatoms (sulfur and nitrogen) and metals (ferrum and calcium) in coal tar can easily cause the corrosion of hydrogenation equipment, catalyst poisoning, and environmental pollution. These should be removed before coal tar is hydrogenated. In this study, with the acid refining method, the effects of three polyether demulsifiers (i.e., PD1, PD2, and PD3), polyamine carboxylate demetallizers (i.e., PCD1, PCD2, and PCD3), and separation temperature on the removal of ferrum, calcium, sulfur, and nitrogen in medium- and low-temperature coal tar were determined. PD2 was selected, and the added amount was 200 μg·g–1. When the PD2 demulsifier was added alone or PD2 demulsifier with various demetallization agents was added, heteroatoms in coal tar could be effectively removed. For the experiments and analysis, the pretreatment conditions of coal tar were as follows: the addition amount of the PD2 demulsifier was 200 μg·g–1, the addition amount of the PCD3-type demetallization agent was 400 μg·g–1, and the stirring temperature was 80 °C. Before and after pretreatment, the methods of inductively coupled plasma–atomic emission spectrometer, gas chromatography–mass spectrometry (MS), and Fourier transform-ion cyclotron resonance MS were used in the present study to explore and analyze the distribution, occurrence form, and removal law of sulfur in coal tar. As revealed from the results, sulfur compounds in coal tar <360 °C fraction (light coal tar fraction, LF) before being pretreated had a lower content, which existed as benzothiophene and dibenzothiophene largely. Sulfur compounds S1 and S2 achieved the maximum relative abundance in >360 °C fraction (heavy coal tar fraction, HF). After the compounds were pretreated, the sulfur removal rate reached 40.0% in LF, and the sulfur compounds were primarily removed. For HF, the sulfur removal rate reached 20.1%. In addition, S1 compounds within the dibenzothiophene derivatives exhibiting more side chains and a larger condensation degree were basically removed. S2 compounds, mainly linked to several quinolines or more aromatic rings and thioether-aliphatic amine sulfur compounds exhibiting small molecular weight and simple structures, were relatively easy to remove. The SO class (e.g., the sulfones and thiophene-ketone group) was more difficult to remove.

Cross-validated Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Quantitation Protocol for a Pharmaceutical Drug and Its Drug-Target Effects in the Brain Using Time-of-Flight and Fourier Transform Ion Cyclotron Resonance Analyzers.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an established tool in drug development, which enables visualization of drugs and drug metabolites at spatial localizations in tissue sections from different organs. However, robust and accurate quantitation by MALDI-MSI still remains a challenge. We present a quantitative MALDI-MSI method using two instruments with different types of mass analyzers, i.e., time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) MS, for mapping levels of the in vivo-administered drug citalopram, a selective serotonin reuptake inhibitor, in mouse brain tissue sections. Six different methods for applying calibration standards and an internal standard were evaluated. The optimized method was validated according to authorities’ guidelines and requirements, including selectivity, accuracy, precision, recovery, calibration curve, sensitivity, reproducibility, and stability parameters. We showed that applying a dilution series of calibration standards followed by a homogeneously applied, stable, isotopically labeled standard for normalization and a matrix on top of the tissue section yielded similar results to those from the reference method using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The validation results were within specified limits and the brain concentrations for TOF MS (51.1 ± 4.4 pmol/mg) and FTICR MS (56.9 ± 6.0 pmol/mg) did not significantly differ from those of the cross-validated LC–MS/MS method (55.0 ± 4.9 pmol/mg). The effect of in vivo citalopram administration on the serotonin neurotransmitter system was studied in the hippocampus, a brain region that is the principal target of the serotonergic afferents along with the limbic system, and it was shown that serotonin was significantly increased (2-fold), but its metabolite 5-hydroxyindoleacetic acid was not. This study makes a substantial step toward establishing MALDI-MSI as a fully quantitative validated method.

Base-catalyzed oxidative depolymerization of softwood kraft lignin

Lignin is an important aromatic biopolymer with the structure based on phenolic units. Depolymerized lignin materials can be useful substitutes for phenol in phenol-formaldehyde resins. Our aim was to examine the potential of the base-catalyzed oxidation method to provide reactive oligomeric lignin fragments from softwood kraft lignin through the cleavage of the ether bonds. Three different bases, namely, NaOH, KOH, and NH4OH, were used as the catalyst in an aerobic oxidation, in which the influence of the reaction time was examined for 2 h and 6 h. Oxidized lignin materials were characterized with high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry and nuclear magnetic resonance spectroscopy (13C, 1H-13C HSQC, and 31P NMR). The presence of methanol in the oxidation gas phase and formation of carboxylic acids indicate that some of the lignin structures were degraded in the oxidation. Guaiacyl units, the major phenolic structure of softwood kraft lignin, survived the oxidation. The oxidatively depolymerized lignin materials showed a reactivity close to that of pristine lignin in a reaction with formaldehyde, as determined by differential scanning calorimetry (DSC). Overall, the FT-ICR MS, NMR, and DSC results proved that the oxidized lignin materials maintained their oligomeric nature and possess the reactivity with formaldehyde.

A new optimization strategy for MALDI FTICR MS tissue analysis for untargeted metabolomics using experimental design and data modeling.

Ultra-high-resolution imaging mass spectrometry using matrix-assisted laser desorption ionization (MALDI) MS coupled to a Fourier transform ion cyclotron resonance (FTICR) mass analyzer is a powerful technique for the visualization of small molecule distribution within biological tissues. The FTICR MS provides ultra-high resolving power and mass accuracy that allows large molecular coverage and molecular formula assignments, both essential for untargeted metabolomics analysis. These performances require fine optimizations of the MALDI FTICR parameters. In this context, this study proposes a new strategy, using experimental design, for the optimization of ion transmission voltages and MALDI parameters, for tissue untargeted metabolomics analysis, in both positive and negative ionization modes. These experiments were conducted by assessing the effects of nine factors for ion transmission voltages and four factors for MALDI on the number of peaks, the weighted resolution, and the mean error within m/z 150-1000 mass range. For this purpose, fractional factorial designs were used with multiple linear regression (MLR) to evaluate factor effects and to optimize parameter values. The optimized values of ion transmission voltages (RF amplitude TOF, RF amplitude octopole, frequency transfer optic, RF frequency octopole, deflector plate, funnel 1, skimmer, funnel RF amplitude, time-of-flight, capillary exit), MALDI parameters (laser fluence, number of laser shots), and detection parameters (data size, number of scans) led to an increase of 32% and 18% of the number of peaks, an increase of 8% and 39% of the resolution, and a decrease of 56% and 34% of the mean error in positive and negative ionization modes, respectively. Graphical abstract.

Structural Analysis of Monoclonal Antibodies by Ultrahigh Resolution MALDI In-Source Decay FT-ICR Mass Spectrometry.

The emergence of complex protein therapeutics in general and monoclonal antibodies (mAbs) in particular have stimulated analytical chemists to develop new methods and strategies for their structural characterization. Mass spectrometry plays a key role in providing information on the primary amino acid sequence, post-translational modifications, and other structure characteristics that must be monitored during the manufacturing process and subsequent quality control assessment. In this study, we present a novel method that allows structural characterization of mAbs based on MALDI in-source decay (ISD) fragmentation, coupled with Fourier transform ion cyclotron resonance (FT-ICR) MS. The method benefits from higher resolution of absorption mode FT mass spectra, compared to magnitude mode, which enables simultaneous identification of ISD fragments from both the heavy and light chains with a higher confidence in a wide mass range up to m/z 13 500. This method was applied to two standard mAbs, namely NIST mAb and trastuzumab, in preparation for method application in an interlaboratory study on mAbs structural analysis coordinated by the Consortium for Top-Down Proteomics. Extensive sequence coverage was obtained from the middle-down analysis (IdeS- and GingisKHAN-digested mAbs) that complemented the top-down analysis of intact mAbs. In addition, MALDI FT-ICR MS of IdeS-digested mAbs allowed isotopic-level profiling of proteoforms with regard to heavy chain N-glycosylation.

Top-Down Deep Sequencing of Ubiquitin Using Two-Dimensional Mass Spectrometry.

Two-dimensional mass spectrometry (2DMS) allows data independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors without isolation prior to fragmentation. Developments in computer capabilities and implementations in Fourier transform ion cyclotron resonance (FTICR) MS over the past decade have allowed the technique to become a useful analytical tool for bottom-up proteomics (BUP) and, more recently, in top-down protein analysis (TDP). In this work, a new method of TDP is developed using 2D FTICR MS, called MS/2D FTICR MS or MS/2DMS. In MS/2DMS, an entire protein is initially fragmented in a hexapole collision cell, e.g., with collisionally activated dissociation (CAD). The primary fragments are then sent to the ICR cell, where 2DMS is performed with infrared multiphoton dissociation (IRMPD) or electron-capture dissociation (ECD). The resulting 2D mass spectra retain information equivalent to a set of TDP MS3 experiments on the selected protein. Up to n - 1 fragmentation steps can be added to the process, as long as an ion of interest can be unambiguously fragmented before the ICR cell, leading to an MS n/2DMS experiment whose output is a 2D mass spectrum retaining information equivalent to MS n. MS/2DMS and MS/MS/2DMS are used in this work for the structural analysis of ubiquitin (Ubi), noting several unique features which aid fragment identification. The use of CAD-MS/IRMPD-2DMS, CAD-MS/ECD-2DMS, and MS2/2DMS using, respectively, in-source dissociation (ISD), CAD, and ECD-2DMS led to 97% cleavage coverage for Ubi.

Water-Soluble Organic Composition of the Arctic Sea Surface Microlayer and Association with Ice Nucleation Ability.

Organic matter in the sea surface microlayer (SML) may be transferred to the atmosphere as sea spray and hence influence the composition and properties of marine aerosol. Recent work has demonstrated that the SML contains material capable of heterogeneously nucleating ice, but the nature of this material remains largely unknown. Water-soluble organic matter was extracted from SML and underlying seawater from the Arctic and analyzed using a combination of mass spectrometric approaches. High performance liquid chromatography-ion trap mass spectrometry (LC-IT-MS), and Fourier transform ion cyclotron resonance MS (FT-ICR-MS), showed seawater extracts to be compositionally similar across all stations, whereas microlayer extracts had a different and more variable composition. LC-IT-MS demonstrated the enrichment of particular ions in the microlayer. Ice nucleation ability (defined as the median droplet freezing temperature) appeared to be related to the relative abundances of some ions, although the extracts themselves did not retain this property. Molecular formulas were assigned using LC-quadrupole time-of-flight MS (LC-TOF-MS2) and FT-ICR-MS. The ice nucleation tracer ions were associated with elevated biogenic trace gases, and were also observed in atmospheric aerosol collected during the summer, but not early spring suggesting a biogenic source of ice nuclei in the Arctic microlayer.

Mass spectrometric revival of an l-rhamnose– and d-galactose–specific lectin from a lost strain of Streptomyces

Blood type B-specific Streptomyces sp. 27S5 hemagglutinin (SHA) was discovered and characterized in the 1970s. Although strain 27S5 has been lost, the purified SHA protein survived intact under frozen conditions and retained its activity. Using modern techniques, here we further characterized SHA. Fourier-transform ion cyclotron resonance MS analysis determined the average molecular mass of SHA as 13,314.67 Da. MS of digested SHA peptides, Streptomyces genomic database matching, and N-terminal sequencing solved the 131-residue amino acid sequence of SHA. We found that SHA is homologous to N-terminally truncated hypothetical proteins encoded by the genomes of Streptomyces lavendulae, Streptomyces sp. Mg1, and others. The gene of the closest homologue in S. lavendulae, a putative polysaccharide deacetylase (PDSL), encodes 68 additional N-terminal amino acids, and its C terminus perfectly matched the SHA sequence, except for a single Ala-to-Glu amino acid difference. We expressed recombinant SHA(PDSL-A108E) (rSHA) as an enzymatically cleavable fusion protein in Escherichia coli, and glycan microarray analyses indicated that refolded rSHA exhibits the blood type B- and l-rhamnose-specific characteristics of authentic SHA, confirming that rSHA is essentially identical with SHA produced by Streptomyces sp. 27S5. We noted that SHA comprises three similar domains, representing 70% of the protein, and that these SHA domains partially overlap with annotated clostridial hydrophobic with conserved W domains. Furthermore, examination of GFP-tagged SHA revealed binding to microbial surfaces. rSHA may be useful both for studying the role of SHA/clostridial hydrophobic with conserved W domains in carbohydrate binding and for developing novel diagnostics and therapeutics for l-rhamnose-containing microorganisms.

Characterization of fast pyrolysis oil from short-rotation willow by high-resolution Fourier transform ion cyclotron resonance mass spectrometry

Fast pyrolysis oil (FPO) produced from short-rotation willow was characterized by using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry and various bulk chemical analyses. The pyrolysis experiments were carried out on a bench-scale bubbling fluidized bed pyrolysis reactor operating at the temperature of 500°C. The resulting pyrolysis oil was a single-phase liquid with relatively low oxygen content and had pH and total acid number values of 3.0 and 129mg/g, respectively. A wealth of compositional information of the willow FPO was obtained using FT-ICR MS in combination with electrospray ionization (ESI) and atmospheric-pressure photoionization (APPI) techniques. While ESI preferentially ionized acidic extractives, small phenolics and anhydrosugars, APPI ionized clearly larger compounds, including neutral lipophilic extractives, triterpenoids and high molecular mass pyrolytic lignin, most of which are beyond conventional gas chromatography–mass spectrometry analyses.

Structural characterization of major soyasaponins in traditional cultivars of Fagioli di Sarconi beans investigated by high-resolution tandem mass spectrometry.

Major soyasaponins, i.e., soyasaponins I, V, βg, and αg from traditional Fagioli di Sarconi beans (Phaseolus vulgaris L., ecotype Tabacchino), were analyzed by reversed-phase liquid chromatography-mass spectrometry (MS) using high-resolution Fourier transform ion cyclotron resonance (FTICR) MS on electrospray ionization in positive-ion mode. Fagioli di Sarconi beans are protected by the European Union [Commission Regulation (EC) No 1263/96] with the mark PGI (for "Protected Geographical Indication"), and are cultivated in Basilicata (southern Italy). Protonated adducts of soyasaponins I, V, βg, and αg were observed at m/z 943.5262, 959.5213, 1069.5583, and 1085.5534, respectively. Gas-phase dissociation of soyasaponins by infrared multiphoton dissociation FTICR MS was performed using a CO2 laser source at a wavelength of 10.6 μm. Most of the fragment ions were identified unambiguously by using the high-resolution and accurate mass value provided by the FTICR mass spectrometer. All soyasaponins exhibit a sequential and neutral loss of sugar moieties at relatively short irradiation times (i.e., less than 50 ms). When the pulse length was increased, a more pronounced fragmentation occurred, with several signals in the lower part of the mass spectrum. In the case of soyasaponins βg and αg, the occurrence of the conjugated product ion at m/z 127.0389 ([C6H6O3 + H](+), 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one) was evidenced. Coupling reversed-phase liquid chromatography with high-performance FTICR MS in combination with infrared multiphoton dissociation tandem MS proved to be very promising for the structural characterization of soyasaponins, and is also suitable for the rapid and accurate structural investigation of other saponins. Graphical Abstract Representative Infrared Multiphoton Dissociation (IRMPD)-FTICR MS spectra of main group B saponins in Fagioli di Sarconi beans.

CuO-oxidized dissolved organic matter (DOM) investigated with comprehensive two dimensional gas chromatography-time of flight-mass spectrometry (GC × GC-TOF-MS)

GC×GC-TOF-MS was applied to investigate the molecular diversity of CuO-oxidized, trimethylsilyl (TMS) derivatized DOM isolated from Lake Tahoe’s main tributary, the Upper Truckee River (UTR), California. Many (4789) chromatographic peaks were resolved in a single sample at a signal/noise (S/N) ratio of 10 or greater and>300 (>6%) were assigned discrete structures. Mapping the two dimensional elution time space revealed 8 homologous series defined by successive CH2 additions. Series of other compounds related to one another by OCH3 and OH substitutions were also assigned. Elucidation of the retention time (RT) displacements affected by these molecular transformations guided the discovery of novel compounds as well as those that had previously escaped detection within DOM, including a suite of lignin-derived cyclobutane photodimers. Analysis of RT shifts among sets of benzene polycarboxylic acid (BPCA) isomers revealed the second chromatographic dimension to strongly retain sterically strained isomers, providing a basis for assigning groups of isomers that were otherwise unassignable. The heightened chromatographic resolution also revealed a substantial and hitherto analytically inaccessible diversity of isomers, which appear to be uniquely resolved with the method, but which would fall outside the analytical window of other high resolution MS methods, such as Fourier transform ion cyclotron resonance MS (FT-ICR-MS). The study likely represents the most comprehensive compound-specific elucidation of any natural OM (NOM) sample to date.