15^th European Fourier Transform Mass Spectrometry Workshop

Over the past 20 years, advances in modern high resolution / ultrahigh-resolution mass spectrometry have forever changed the expectations of complex mixture analysis. Aided by advances in ionization methodologies that provide course chemical selectivity in ionization that facilitates the molecular-level analysis of polar (acidic / basic) and aromatic species, ultrahigh-resolution mass spectrometry routinely resolves and identifies tens-of-thousands (at the level of elemental composition assignment) components in complex mixtures. Furthermore, the highest magnetic field instruments enable on-line coupling of separation methods with ultrahigh resolution mass spectrometry that provides unprecedented insight into even the most complex mixtures. Here, we present the latest efforts to address complex organic mixture analyses by high field Fourier Transform ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) and highlight on-line separation strategies used to alleviate / minimize the impact of selective ionization and aggregation. Applications highlighted include biomass pyrolysates, petroleum, dissolved organic matter, and complex emerging contaminants. Recent advances in data reduction / visualization strategies and updates to the PyC2MC software platform that facilitate rapid, confidence-based data processing are also discussed.

Polyethylene terephthalates are one of the most abundant polymers in our daily life. These polyesters are formed of terephthalic acid and ethylene glycol units. In the environment, they degrade and release micro- and nano-PET, a major pollution source especially for marine ecosystems. Huge amounts of nano-PET were recently detected in water of PET bottles. Many analyses were performed on PET. Unfortunately, they were not satisfying for the study of PET structural modifications and their quantification. Our new methodology identifies and quantifies PET including their additives, allows the study of PET environmental modifications after ageing by UV, salinity, micro-organisms, waste-treatment, and permits the study of modifications’ degree between micro-, nano- and macro-PET which affects their toxicity.
Our methodology starts by PET transamidation using N,N′-dimethyl-1,3-propanediamine (DMAPA) at room temperature after a solubilization step by 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The resulting products were neutralized, extracted with ultrapure water and ethyl acetate, then analyzed using MALDI ionization on an ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) 9.4 Tesla and ESI ionization on an Orbitrap LC-MS. The step of PET pre-solubilization by HFIP allowed a transamidation at room temperature instead of 70°C and improved the extraction yield. To quantify PET, an internal standard was successfully synthesized. For better identifying polyols, we derivatized them with 4-N,N’-dimethylbenzoyl chloride prior to their analysis. This benzoylation allows the study of polyols modifications during PET manufacturing and their environmental modifications.
Many PET containers and bottles from France and US of different brands and contents (oils, vinegar, water, wine, beer and carbonated liquids) were analyzed. PETs were also studied after accelerated ageing; and marine environmental PET were analyzed. For each, a specific signature was determined. Indeed, PET were differentiated by their chemical composition and by the difference in intensities of the common peaks; dimers, oligomers, crosslinked and oxidized products. For example, cyclohexanedimethanol of PET-G was detected in the carbonated-containing bottles. This methodology allows, in addition to the identification of PET and their additives (plasticizers and antioxidants), the detection of products embedded in PET. The micro-PET analysis and the study of the new similar biodegradable generation (PBAT) will be also presented.

Recent years have seen significant growth in the methods of structural proteomics, which had a significant impact in the field of structural and molecular biology. These methods may unveil answers related to structure and dynamics of protein and protein complexes, making them favorable for studying protein-DNA interactions. One specific method, radical covalent labelling, has emerged as an effective analytical tool for characterization of biomolecules. Fast Photochemical Oxidation of Proteins (FPOP), the most common radical labelling method, is now exclusively employed only for mapping the structure and interaction of proteins. Nevertheless, during the early 80’s, when radical chemistry was only involving, it was primarily used for assessing biding motif between DNA and transcription factors by hydroxyl radical damage of the DNA.
We show in this study an approach to analyze FPOP-induced damage of DNA coupled to high-resolution mass spectrometry analysis. Our model system 17bp double-stranded DNA, Insulin Response Element (IRE), was fragmented in FPOP apparatus in the absence and in the presence of its cognate binding partner, FOXO4. Residual protein was digested using Proteinase K, and resulting DNA fragments were separated in LC and analyzed using high-resolution 15T-FT-ICR mass spectrometry operated in negative ion mode. The study emphasizes the fragmentation mechanism of nucleic acid in solution, and outlines both benefits and drawbacks associated with analyzing DNA fragments throughout the experimental process, including FPOP oxidation, LC-MS, and data analysis.
Analysis of separated IRE fragments revealed that hydroxyl radicals cleave the DNA nonspecifically, creating a complementary set of 3’OH, 3’P, 5’OH and 5’P terminal fragment ions. Combining MS/MS analysis, mass accuracy precision and high resolution obtained by FT instrument and in-silico modeling of isotopic envelopes confirmed the presence of generated DNA fragments. Complementary fragments were found in the LC-MS trace and quantified. Comparison of IRE fragment ions revealed a significant protective effect around the binding sequence in the major groove of DNA, also lower protection in the minor groove. Expanding the scope, our study delved into a complex involving two transcription factors, FOXO4 and TEAD1, interacting with one oligonucleotide to form a ternary complex. This investigation illustrates how FPOP can effectively monitor minor structural changes in DNA upon the binding of transcription factors, underscoring its full potential for studying dynamic complexes in solution.

Two-dimensional mass spectrometry (2DMS) allows acquisition of fragment information from all precursors simultaneously by modulation of a trapped ion packet position through a fragmentation zone. This fragmentation zone is best created using either an electron beam (ExD methods) or by using a laser (IRMPD or UVPD). The modulation is created using a classic Gaumann pulse sequence (a low amplitude frequency sweep pulse (P1), an iterated delay (T), and a second sweep pulse (P2=P1) or by using the SWIFT/SWIM pulse sequences from Ross and Marshall. In either case, all ions are modulated at their own cyclotron frequencies, through the fragmentation zone and fragments are born (or not) at the cyclotron frequency of the precursor ions, so a Fourier transform of the peak intensities of all peaks will extract the modulation frequencies of all ions, thereby correlating fragments with their precursor ions, even in complex mixtures.

The advantages of 2DMS are several. 2DMS does not require precursor ion isolation prior to fragmentation, so resolution in the precursor ion dimension is only limited by the number of scan lines chosen, and the ion beam stability. In 2DMS, accumulation of signal over a series of scans increases signal/noise as the square root of the number of scan lines (the Fellgett signal averaging advantage). Having all fragments from all precursors allows the user to avoid any ion selection biases (either by the user or by the DDA algorithm). Having all fragments from all precursors also allows interesting chemical correlations to be revealed in the spectra.

Over the past 10 years, our group has been working to implement this methodology and have done so using all the fragmentation methods above. We have also implemented an MS/2DMS methodology and a TIMS/2DMS methodology as well. Furthermore, we have shown the ability to differente fragments at greater than 4 sigma) from two different precursors which are only 19 mDa apart.

This presentation will discuss the current state of the art in 2DMS methodologies, and we will also attempt to project the longer-term utility of these techniques.

Energy transition is a major topic facing climate change. Besides hydrogen, methanol, and ammonia, renewable carbon-based biofuels, such as fatty acid methyl esters (FAME), hydrated vegetable oils (HVO), or Fischer-Tropsch synthesis products raise in importance. [1] During combustion in heating systems, fossil fuels tend to form deposits over time. In this project, we investigated the influence of different fossil fuels, biofuels and fuel mixtures (fresh/aged) on the amount of formed deposit and their chemical composition. As the deposits’ chemical composition was expected to be very complex, high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was applied for in-depth molecular characterization.

Fuels and fuel mixtures were aged with two different methods: 1) Storage at 40 °C for 6 month, 2) fast-aging with BigOxy (DGMK 763) at 105 °C, 4.8 bar and 32 h. Deposits were generated on a modified stationary heating with exchangeable evaporation unit.
Fresh/aged fuels were characterized by TAN, GC-MS and ESI/APPI-FT-ICR MS. Deposits were investigated by TGA, pyr-GC-MS and DIP-APPI-FT-ICR MS. [2,3]

Aging of fuels and fuels mixtures led to an increase of oxygen-containing compounds, especially when FAME was present. However, in the presence of octanol, no aging effects were observed.
The investigation of deposits revealed that they are composed of 70-85 % elemental carbon, 15-30 % pyrolyzable organic carbon, and less than 5% comprises remaining fuel. Pyr-GC-MS revealed the presence of alkyl-side chains with up to 20 carbons as well as the presence of alkylated benzenes and naphthalenes, which seem to be attached to higher molecular structures.
DIP-APPI-FT-ICR MS showed differences in the high complex molecular fingerprints of deposits from different fuels and fuel mixtures. Deposits are composed of high aromatic compounds, while alkylation differs between the investigated heating oil (higher alkylation) and diesel (lower alkylation). Main compound classes found for all types of deposits were the CH-class and O1-3-class. Furthermore, the addition of rapeseed oil methyl ester (RME) increased the amount of pyrolyzable organic carbon. Deposits of aged fuels showed compounds with slightly higher aromaticity compared to deposits from non-aged fuels.

Literature:
[1] A. Krutof, Renewable Sustainable Energy Rev., 2016, 59
[2] C.P. Rüger, Anal. Chem., 2015, 87
[3] C.P. Rüger, Energy Fuels, 2018, 32

The analysis of polymer by mass spectrometry offers information on the repeating unit(s), average molar mass, end-groups and additives. Electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI) ion sources are commonly used. However, they can require sample preparation steps, as the solubilization of the sample in a sufficiently polar and volatile solvent for ESI, and homogeneous mixing with a matrix in MALDI.

In some cases, solubilization and preparation steps are not only time consuming but are real limitations, as exemplified by poly(vinylidene fluoride), PVDF, a polymer bearing a C₂H₂F₂ repeating unit. It is well-known that PVDFs are poorly (or non-soluble) in common organic solvents. Desorption/ionization techniques are useful in that case, but PVDF does not absorb UV and cannot be investigated by UV laser desorption/ionization. Direct analysis in real time (DART) is most suited and generates ions by shooting the sample with a hot metastable He or N₂ stream. DART-MS was successfully employed for the differentiation of PVDFs according to their end-groups. (Pacholski et al. 2023)

For in-depth PVDF characterization, a thermodesorption/pyrolysis (TD/Py) device was added to the DART ion source. A temperature gradient from 35 °C to 600 °C ensured to observe both thermal desorption and pyrolysis products. Classically, pyrolysis leads to the formation of a huge number of compounds. After ionization, the resulting mass spectrum may be very complex and bear thousands of features. For this reason, the TDPy-DART ion source was coupled to a 7T 2XR Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Up to our knowledge, this study presents the first TD/Py-DART-FT-ICR MS coupling. The very high resolution and the high m/z measurement accuracy allowed the unambiguous ions formula attribution, leading to a deep understanding of the generated compounds. In one of the investigated PVDFs, an unexpected amount of fluorine (F/C greater than 1) revealed the presence of a perfluorinated comonomer, which was not evidenced by “simple”-DART analysis. This result was confirmed by ¹⁹F NMR, which demonstrated the presence of 5% of hexafluoropropylene (HFP) comonomer. This sample was compared to a VDF homopolymer and a poly(VDF-co-HFP) copolymer with mol.%HFP > 5. A correlation between the HFP content in the polymer and the number of CF₂ units measured in pyrolysis product was observed.

The findings highlighted in this study showed the potential of TD/Py-DART-FT-ICR MS to quickly reveal (the time for an analysis is shorter than 10 minutes) insights on polymers and differentiate copolymers according to their monomer composition.

Metabolomics offers the most direct readout of the phenotype, thus being of immense value to describe the current state of a living organism. In contrast to genomics or proteomics that rely on well established and virtually universal methods, the chemical diversity and dynamic range of the metabolome pose unique challenges. Thus, a plethora of analytical methods, straight from the analytical chemistry arsenal, has been applied to metabolomics, mainly CGMS and LCMS, that provide fractional and biased coverages of the metabolome. Moreover, all require extensive sample preparation and rely on the concept of sequential analysis through previous chromatographic or other separative techniques thus hindering throughput. Metabolomics calls for parallelization and simultaneous, unbiased high throughput analysis. Magnetic Resonance methods open that path, through nuclear magnetic resonance (NMR) or magnetic resonance mass spectrometry (MRMS). MRMS, correctly termed Fourier transform ion-cyclotron resonance mass spectrometry (FT-ICR-MS) offers a broad mass range coverage with extreme resolution and mass accuracy as well as a suitable dynamic range for the simultaneous analysis of all metabolites. Maximizing the information content in biological context of accurate mass measurements of whole metabolomes is the purpose of single-shot metabolomics. Here we show how mass spectra obtained from direct injection at extreme resolution and mass accuracy can be converted into biological information, addressing issues such as metabolite identification, quantification as well as future directions based on computational approaches.

The performances of lithium-ion batteries (LIBs) are closely related to the control of the electrolyte composition and solid-electrolytes interface (SEI) stability [1-2]. To decrease the capacity fading electrolyte formulation has been continuously optimized over years and much attention has been paid to identifying the SEI composition. However, deciphering the molecular diversity of both liquid electrolytes and solid interphase remains extremely challenging because few analytical techniques show the appropriate sensitivity and spatial resolution without modifying their composition. Here, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was used for the first time to characterize at the molecular level both parts of LIBs. The liquid electrolytes were characterized using atmospheric pressure chemical ionization to cover a broad polarity range. Surprisingly, obtained mass spectra revealed hundreds of signals even if the initial electrolytes cocktail was prepared using 10 species. Using mass defect molecular maps, it was possible to delimit regions of interest for each component and easily perform reverse engineering. Two anodes coming from LIBs with different performances were analyzed using laser desorption ionization operated in imaging mode. After the subtraction of species coming from the carbon reference electrode, the unique contribution of each distinct SEI was observed and known reaction products of electrolytes were researched and identified[3].

[1] Larcher, D.; Beattie, S.; Morcrette, M.; Edstroem, K.; Jumas, J.-C.; Tarascon, J.-M. J. J. o. M. C. Recent findings and prospects in the field of pure metals as negative electrodes for Li-ion batteries - Journal of Materials Chemistry - 2007, 17 (36), 3759-3772.
[2] Liu, Y.; Zhang, R.; Wang, J.; Wang, Y. J. I. Current and future lithium-ion battery manufacturing - iScience - 2021, 24 (4), 102332.
[3] Maillard, JF; Demeaux, J.; Mase, C.; Gajan, A.; Tessier, C.; Bernard, P.; Afonso, C.; Giusti, P. – Unambiguous molecular characterization of solid electrolyte interphase species on graphite negative electrodes - Journal of power sources - 2023, 582, 233516

Deposition of ions from a gas phase, a process often referred to as ion soft landing, allows patterning of protein molecules onto surfaces in the form of different microarrays that can form protein chips with many different functionalities. Successful protein ion soft landing with retention of activity was first demonstrated by Cooks (1), while electrospray deposition of protein ions at atmospheric pressure was developed by Morozov (2). Ion soft landing can be used to prepare protein chips compatible with MALDI ionization, which gives the MALDI experiment new possibilities to perform in-situ affinity chemistry on the MALDI plate (3). Different workflows can be used to perform enrichment and clean-up prior MALDI ionization, a workflow previously named “lab-on-plate” (4). We have developed a method to use ambient ion soft landing, or controlled electrospray deposition, to prepare MALDI chips of large variety of protein functionalities that can be applied to perform different affinity assays detected by MALDI mass spectrometry (5). While MALDI has been coupled with time-of-flight mass analyzer from its inception, and this coupling has been extremely successful, sometime the MALDI performance is limited by the insufficient resolution of the time-of-flight. Here we show examples of biochemically and clinically relevant MALDI-MS assays, where ultrahigh resolution of FTICR significantly expanded information that can be obtained from MALDI protein chip applications.

References
1/ Quyang et al. Science 2003, 301, 5638, 1351–1354.
2/ Morozov et al. Anal. Chem. 1999, 71, 7, 1415–1420.
3/ Blacken et al., Anl. Chem. 2007, 79, 14, 5449-5456.
4/ Urban et al. Mass Spectrometry Reviews, 2011, 30, 435– 478.
5/ Pompach et al. Anal. Chem. 2016, 88, 17, 8526–8534.

Trapped ion mobility spectrometry (TIMS) has advanced greatly in recent years, yet FT-ICR MS has only had limited exposure to the IMS separation technique. A new FT-ICR MS instrument has been designed, constructed, and characterised which overhauls the entire front end of the SolariX MRMS platform to benefit from various ion optic advancements, including a contemporary, dual-accumulation and analysis TIMS cartridge as used on commercial TIMS Tof systems. The energy landscape of the system was retuned for ultra-low energy and low activation transmission of delicate analytes. Extensive ion current measurements were used to optimise each transfer and isolation region, with energy measurements informing optimum transfer and detection characteristics.

The TIMS MRMS system was modified to operate under gated TIMS (gTIMS) conditions using a dual-lens and/or fast switching quadrupole as a gating element. gTIMS allowed time-binned analysis of TIMS-separated ions in order to accommodate the FT-ICR MS’s variable, and longer detection times required for ultra-high resolution MS analysis. This decoupling of the IMS separation (millisecond timescale) and MS (second timescale) allowed for ultra-high resolution analysis and the ability to accumulate large ion populations for subsequent analysis, enhancing dynamic range for complex mixture samples such as SRFA and alternative future fuels. Gated TIMS was optimised for a wide mobility range (e.g. 1/K0 0.01-2.3Vs/cm²) and was operated in both sweeping mode to study mobility of species, or filtering mode to pass individual species/isomers on for MSⁿ and/or high resolution detection of gas phase fractionated ions.

Ultra-high resolution MS performance of FT-ICR and UHV was maintained; mass resolving power of several million in broadband mode, and over 14 million in narrowband mode was readily achieved. Front-end CID and in-cell ExD (ECD, EID, EDD) are compatible with up front gTIMS separation. Calibration against known compounds showed that gTIMS elution voltage to mobility relationship was preserved to an R² of 0.9999. The inherent mass accuracy of FT-ICR was achieved without compromise; 95ppb standard deviation for Tunemix ions 622-2722m/z on a 7T magnet system.

To demonstrate the capabilities of the prototype system a range of complex mixtures were analysed, such as Swannee river fulvic acids (SRFA) and alternative (green) future fuels. Combining ultra-high MS resolution and gTIMS allowed m/z specific IMS analysis down to the milli-Dalton level, required for studying the isomeric complexity of such complex mixtures.

Initial MALDI imaging analysis of human brain and kidney tissue sections is also shown from the prototype instrument, showing 10um microprobe spatial resolution with high spatial fidelity and 500,000 to 1,000,000 MS resolving power (at 400m/z). Incorporation of a microgrid MALDI stage to achieve 5um spatial resolution is underway.

The raw signal from FT-ICR or Orbitrap MS is constituted by sum of damped sine in the time-domain called transient. From this transient a spectrum in the frequency domain is obtained Discrete Fourier Transform (DFT). Then the spectrum in frequency is transformed in the desired m/z scale. In mass spectrometry DFT has a major limitation: the peak with in the spectrum is proportional to the transient duration in the time domain. Obtaining peaks in the spectrum sharper than those obtained by DFT is called super-resolution. We will show that by searching a sum of damped sines in the time domain by a genetice algorithm we peak sharpening by a factor almost 4 which is close from the theoretically predicted limit.
The sinus_it algorithm runs on Nvidia RTX 2080 Ti Graphics Processing Units (GPU) hosted by a 2 × 16 cores 2.1 Ghz Intel Xeon Dell Precision 7920 server. Nvidia RTX 2080 Ti GPU is a gaming CPU which is inexpensive (approximately 1500 USD). Sinus_it is written in C++ and CUDA the specific language for Nvidia GPU. We used the open source EASNA platform from ICube laboratory in Strasbourg (France) to implement it. Sinus_it was applied on simulated as well as experimental signals acquired on a Bruker SolariX 9.4 Tesla FTICR mass spectrometer.
Sinus_it searches a of sine sum that fits the transient using a genetic evolutionary algorithm. In a genetic algorithm, like in Nature, a population of solutions is evolved toward better solutions by mutation and mixing solutions. Sinus_it determines for each sine the intensity, frequency and damping factor. Preliminary results which showed that Sinus_it running time is proportional to data points in the transient and sines to be searched. So, Sinus_it was initially implemented with 2 search modes: coarse and fine. In coarse mode Sinus_it searches for the full set of isotopes of a substance whereas on fine mode Sinus_it searches for the fine structure of a given isotope. Transients with limited band width around relevant frequencies are obtained by classical Butterworth filtering. In all cases a super-resolution near the theoretical limit of 4 was obtained. In other words, we can use only the first quarter of the transient where the signal is more intense affording more accurate isotopic ratios. Genetic evolutionary algorithms are by nature stochastic, which mean that the results of a run depend on the random numbers drawn. By running sinus_it about thirty times we will show that the standard deviation on sine parameters is low and inversely proportional to the signal to noise. The high accuracy of sinus_it is proved by the perfect linear dependence of the phases with the starting point in the gradient. Furthermore, we will present the possibility of performing information driven harmonic inversion when the expected m/z are known for example when performing isotope ratios measurements.

The fast pyrolysis of synthetic and natural polymers is a promising source of renewable molecules and fuels. The parameters employed during the pyrolysis process (raw material, heating rate(1), gas flow) and the use of a catalytic treatment(2) have a significant impact on the physical properties of the resulting oil as well as its molecular composition. In addition, these oils contain tens of thousands of different compounds, whose chemical properties (weight, polarity, stability) can vary significantly. Optimizing pyrolysis parameters to obtain oils with the desired properties and yields for the targeted compounds is a critical step. This optimization process is complex and time-consuming and often require repeated production and characterization according to a petroleomic approach of the different oils in respect with the used pyrolysis parameters.

To shorten the time required to optimize the pyrolysis parameters, the on-line mass spectrometry analysis of the pyrolysis products is well suited. In this frame, a modification of the direct insertion probe (DIP) coupled to a FT-ICR Mass Spectrometer (FT-ICR MS) was developed. Indeed, the DIP device ensures to quickly heat a solid at temperatures reaching hundreds of Celsius. Adding a flow of inert gas inside the probe allowed to mimic the thermal degradation of raw materials occurring in pyrolysis reactors. To investigate the resulting species, an atmospheric pressure chemical ionization source (APCI) was used prior to the analysis of the resulting ions by FT-ICR MS. The proposed device allows the investigation in a few minutes of the thermal degradation products of natural and synthetic polymers.

The influence of the employed gas flow as well as the nature of the polymer’s conversion products have been investigated. For cellulose, the pyrolysis produced well-known markers(3) such as cellobiosan and levoglucosan which have been unambiguously identified by tandem mass spectrometry. The dependence of the distribution and nature of the pyrolysis products of polystyrene with respect to its average molecular mass was also studied.

References
(1) Dement’ev K. et al. Polymers 2023, 15 (2), 290.
(2) Hertzog, J. et al. ACS Sustainable Chem. Eng. 2018, 6 (4), 4717–4728.
(3) Zhu, X. et al. Biomass; 2010.

Keywords: DIP, FT-ICR MS, polymers, pyrolysis, analytical chemistry

Plants and their environment, including microorganisms such as bacteria and fungi, are surprisingly complex at the molecular level. This is not only due to their nature, but also to their interactions, which can be symbiotic, neutral or pathogenic.
MALDI mass spectrometry is a powerful technique for probing and imaging this molecular diversity on the surface of a sample. In addition, the use of FT-ICR mass spectrometry with high mass resolution and mass measurement accuracy increases the confidence and selectivity of the spatial distribution of many compounds. However, application to plant and/or microbial samples remains an analytical challenge. Firstly, the topology and heterogeneity of the sample surface can vary greatly and influence the MALDI MSI response. Secondly, the molecular composition of these samples can be so extensive and complex that no relevant chemical variation from one region to another may be highlighted if no compounds are targeted first.
In this presentation we will discuss two examples of such MSI problems with plants and microorganisms and how they can be overcome. First, we will look at MALDI MSI of soil bacterial colonies on agar media, which introduces the problem of sample heterogeneity and topology. Secondly, we will look at the molecular complexity of a grape leaf and its response to biotic stress. In this last case, the large number of mass features led us to employ a new data analysis approach to treat non-targeted MSI data.
These two works highlight the ability of MSI to probe the dynamics and spatial variations of metabolic profiles of plants and their microbiome.

Bio-oils produced from the pyrolysis of lignocellulosic biomass have proven to be a promising renewable energy source. These bio-oils are highly complex organic mixtures consisting of thousands of compounds covering a wide range of mass and polarity. However, the liquid obtained after pyrolysis is rich in oxygen, which causes instability and corrosion problems. Therefore the use of processes such as hydrotreatment is required to increase the H/C ratio of the products to be used as fuels or chemicals. Advanced molecular level characterization of these samples is necessary to understand the efficiency of these processes. However, this requires the use of ultra-high resolution instruments such as the Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer. This instrument offers the best performance in terms of resolution, dynamic range, and mass accuracy. It allows unique molecular formulas to be unambiguously assigned to all compounds in the sample. To extend the range of molecules that can be characterized, the use of separation method on line or off line can be used to separate different families of molecules including isomers.
In this work high performance thin layer chromatography (HPTLC), flash chromatography (FC) an trapped ion mobility spectrometry (TIMS) coupled to FTICR MS were used to provide a more accurate chemical description of biooil composition [1]. In addition, the use of different ionization methods including ESI, APCI, APPI and MALDI was used to obtain different ionization selectivities [2, 3]. In particular, it was shown that APCI was particularly useful for the detection of aliphatic compounds, while MALDI was very effective for the detection of a wide range of molecules present in the biooils.

Literature:
[1] Mase, C., et al., J. Anal. Appl. Pyrolysis, 2024, 177. [2] Mase, C., et al., J. Anal. Appl. Pyrolysis, 2022. 167. [3] Mase, C., et al., J. Am. Soc. Mass Spectrom., 2023. 34(8): p. 1789-1797.

While the introduction of electron capture dissociation (ECD) in 1998 ^[1] was a catalyst for transformative radical-driven tandem mass spectrometry (MS/MS) of multiply-charged biomolecular cations, the idea of electron bombardment as an MS/MS activation method in FT-ICR MS had been previously introduced for singly-charged analytes under the names ‘electron impact excitation of ions from organics’ (EIEIO) ^[2] and ‘electron induced dissociation’ (EID) ^[3]. Furthermore, the term electron collision dissociation was used for related experiments in magnetic sector mass spectrometers ^[4]. Following the introduction of ECD, related techniques such as “hot” ECD ^[5] and electron ionization dissociation ^[6] (EIoD ^[7]) have been proposed to occur at higher electron energies than “true” ECD. Here, we show that phenomena previously attributed to “hot” ECD and EIoD can occur at low electron energy, similar to that in “true” ECD. These phenomena include secondary fragmentation and tandem ionization. We show that electron flux rather than energy is the main determinant for these processes, analogous to previously published findings from smaller cluster ions, demonstrating that multiple collisions with 5 eV electrons can raise analyte internal energy to ~15 eV ^[3].

1. Zubarev et al. J. Am. Chem. Soc. 1998, 120, 3265-3266.

2. Cody and Fraiser. Anal. Chem. 1979, 51, 547-551.

3. Gord et al. J. Am. Soc. Mass Spectrom. 1993, 4, 145-151.

4. Aberth and Burlingame. Proc. 2nd Int. Symp. Mass Spectrometry in the Health and Life Sciences. 1990, pp 217-221.

5. Kjeldsen et al. Chem. Phys. Lett. 2002, 356, 201-206.

6. Fung et al. J. Am. Chem. Soc. 1998, 131, 9977-9985.

7. Baba et al. J. Am. Soc. Mass Spectrom. 2022, 33, 1723-1732.

Heat shock cognate 71 kDa protein (Hsc70) is a representative of heat shock protein 70 family encoded by a HSPA8 gene in human genome. It fulfills several important tasks in cells, including proper folding of newly translated and misfolded proteins, as well as stabilize or degrade mutant proteins; it also participates in other biological processes including signal transduction, apoptosis, autophagy, protein homeostasis, cell growth and differentiation. HSC70 cellular pool regulation is thus tremendously important; one of the current hypotheses of HSC70 describes the inactivation through oligomerization, which was already proven by analytical size-exclusion chromatography. This work pursues the exploration of oligomerization properties of Hsc70 WT and its mutants by chemical cross-linking and mass spectrometry including stable isotope labeling of recombinant proteins by 15N. The last technique demands the production of 15N-versions of each studied protein.

The reconstruction of past environmental conditions from sedimentary archives via the extraction of organic biomarker molecules is a proven and well-established approach referred to as molecular stratigraphy (Brassel et al., 1986). Linked to biological precursor compounds, such as microbial membrane lipids, plant waxes, pigments, sterols, or hopanoids, these geochemical fossils are witnesses of past climate conditions and thus can provide key insights into understanding current and future effects of climate change.

Classical biomarker analysis requires the extraction of organic compounds from typically cubic-centimeter-sized samples. Considering that marine or lacustrine sediment deposits typically build up by a less than a few millimeters per year, a sample spanning one centimeter of depth averages the information deposited during decades, centuries, or more time into one signal.

Recently, the Organic Geochemistry working group at MARUM – Center for marine environmental sciences at the University of Bremen revolutionized the approach to molecular stratigraphy by using Matrix-Assisted Laser Desorption/Ionization coupled to a 7T solariX XR MRMS system to obtain molecular information directly from intact sediment samples, without previous sub-sampling or extraction. This approach offers unmatched spatial and thus temporal resolution for biomarker-based paleoclimate studies and further enables exploring the two-dimensional spatial distribution of the molecular information in a sample.

Here we will present some of the advances facilitated by this unique and dedicated facility for high-resolution paleoclimatology. For instance, on sediments deposited in the last century that can be compared with instrumental and historical climate data (Alfken et al., 2020; Alfken et al., 2021; Napier et al., 2022), to high-frequency fluctuations and abrupt environmental responses during the Younger Dryas (~12 ka) (Obreht et al., 2020, Wörmer et al., 2022) and the last warm climate period of the Last Interglacial (~129 - 116 ka) (Obreht et al., 2022), and even new findings into the drivers of the end-Permian mass extinction (~250 Ma) (Saito et a., 2023).

References:

Alfken, S., Wörmer, L., Lipp, J.S., et al., 2020. Mechanistic insights into molecular proxies through comparison of subannually resolved sedimentary records with instrumental water column data in the Santa Barbara Basin, Southern California. Paleoceanography and Paleoclimatology 35 (10). https://doi.org/10.1029/2020PA004076

Alfken, S., Wörmer, L., Lipp, J.S., et al., 2021 Disrupted coherence between upwelling strength and redox conditions reflects source water change in Santa Barbara Basin during the 20th century. Paleoceanography and Paleoclimatology 36 (12), https://doi.org/10.1029/2021PA004354

Brassell, S. C., Eglinton, G., Marlowe, I. T., et al., 1986 et al. Molecular stratigraphy: a new tool for climatic assessment. Nature 320, 129–133. https://doi.org/10.1038/320129a0

Napier, T.J., Wörmer, L., Wendt, et al., 2022. Sub-annual to interannual Arabian Sea upwelling, sea surface temperature, and Indian monsoon rainfall reconstructed using congruent micrometer-scale climate proxies. Paleoceanography and Paleoclimatology 37 (3). https://doi.org/10.1029/2021PA004355

Obreht, I., Wörmer, L., Brauer, A., et al., 2020. An annually resolved record of Western European vegetation response to Younger Dryas cooling. Quaternary Science Reviews 231, https://doi.org/10.1016/j.quascirev.2020.106198

Obreht, I., De Vleeschouwer, D., Wörmer, L. et al., 2022 Last Interglacial decadal sea surface temperature variability in the eastern Mediterranean. Nat. Geosci. 15, 812–818. https://doi.org/10.1038/s41561-022-01016-y

Wörmer, L., Wendt, J., Boehman, B. et al., 2022 Deglacial increase of seasonal temperature variability in the tropical ocean. Nature 612, 88–91. https://doi.org/10.1038/s41586-022-05350-4

Saito, R., Wörmer, L., Taubner, H. et al., 2023 Centennial scale sequences of environmental deterioration preceded the end-Permian mass extinction. Nat Commun 14, 2113. https://doi.org/10.1038/s41467-023-37717-0

Methylenation reactions in the gas phase are of great significance in interstellar chemistry involved in carbon chain elongation of small molecules. Electron ionization of appropriate volatile precursors, like ketene, oxirane, and ethers, has been employed to simulate these processes. Preliminary experiments in the cell of an FT-ICR mass spectrometer were carried out, where ionized ethylene oxide was allowed to react with CH₃CHO. Interestingly, a net CH₂^+· transfer occurred forming [C₃H₆O]^+· (m/z 58) ions, whose structure was probed by ion-molecule reactions (IMR), collision induced dissociation (CID) and mid-infrared vibrational spectroscopy. In particular, candidate isomers obtained by ionization of suitable neutral precursors, including 4-methyl-1,3-dioxolane and acetone, were assayed and compared to characterize different isomers possibly participating in the sampled ion population. [1, 2]
The same multimethodological approach was applied to the study of a bare prototypical perfluoroalkyl anion, C₂F₅^-, to reveal the possible existence of isomeric structures, either a covalently bound species or a loosely bound complex, and the operation of negative hyperconjugation effects. This species is relevant in fluorocarbon plasmas broadly used for the deposition and etching in semiconductor industry. [3] The structure and bonding features of C₂F₅^- conforms to the behavior of a covalently bound ion, according to the evidence gained by the reactivity pattern, the IRMPD spectrum and the molecular geometries optimized at MP2/cc-pVTZ level. [4]
[1] P. Gerbaux, R. Flammang, M. T. Nguyen, J.-Y. Salpin, G. Bouchoux, J. Phys. Chem. A 1998, 102, 861.
[2] E. P. Burrows. J. Mass Spectrom. 2005, 30, 312.
[3] I. Reid, G. Hughes, Semiconductor Sci. Technol. 2006, 21, 1354.
[4] M. E. Crestoni, B. Chiavarino, J. Lemaire, P. Maitre, S.Fornarini, Chem. Phys. 2012, 398, 118.

Hardware architectures of contemporary built-in data acquisition and processing (DAQ/P) systems in the FTMS instruments often remain sub-optimal in performance, including phase distortions requiring post-acquisition phase correction, reduced resolution and duty cycle, and compromised S/N and sensitivity. A direct consequence of these limitations is restricted access to the absorption mode FT (aFT) mass spectra. Furthermore, the current generation of built-in DAQ/P systems, accompanied by rigid data acquisition control software, may inhibit experimental flexibility, for instance, by prohibiting the stable acquisition of ultra-short or ultra-long time-domain transients.

Previously, we demonstrated that the state-of-the-art field-programmable-gate-array (FPGA) technology enables a new-generation DAQ/P architecture with powerful in-line digital signal processing helping to overcome the said disadvantages. The new-generation high-performance DAQ/P systems, e.g., the FTMS Boosters, can directly yield the in-hardware phased transients, which can be readily converted into equally informative aFT mass spectra. The flexibility of the external and in-parallel interfaced high-performance DAQ/P systems to commercial and custom FTMS instruments provides additional benefits.

We will overview recent advances in high-performance external phased transient acquisition and processing technology and its use in selected applications. The recent examples include advancing Orbitrap-, ICR-, and custom FTMS-based charge detection mass spectrometry (CDMS), mass spectrometry imaging and bottom-up/top-down proteomics applications [1-3].

[1] A. Kozhinov, A. Johnson, K. Nagornov et al., Anal. Chem., 95, 7, 3712–3719 (2023)
[2] A. Grgic, K. Nagornov, A. Kozhinov, et al., Anal. Chem. 96, 2, 794–801(2024)
[3] E. Deslignière et al., Nature Methods, DOI 10.1038/s41592-024-02207-8 (2024)

Efficient and informative fragmentation is key to the success of many MS-based experiments studying both small molecules as well as large multi-protein assemblies. Especially so, in the case of proteins, since the realization of both structural biologists and mass spectrometrists that there exists yet another level of complexity involved in the regulation and controlling of protein structure and functions beyond pure single gene = single protein equation. Through RNA splicing, mutations as well as post- and co-translational modifications and involvement in larger non-covalent assemblies, individual proteins generate complex landscapes of proteoforms with huge implications on their functioning in organisms. [1]

Therefore, both proteomics as well as structural MS have been often dependent on various means of fragmentation for which diverse MS dissociation techniques and their combinations are often needed. This contribution will report on the successful implementation of both 10.6 µm CO2 and 193 nm ArF lasers for infrared multi-photon dissociation (IRMPD) and ultraviolet photodissociation (UVPD), respectively, for in-cell dissociation inside the 15T SolariX Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer at BIOCEV in Prague. It will also highlight a particular success story, where the molecular structure of nostatin A – a novel polypeptidic highly bioactive compound synthesized and heavily co-translationally modified in algae has been solved by top-down MS/MS after years of resisting efforts to crystallize it or determine it using nuclear magnetic resonance alone.

References:
[1] Smith L.M. & Kelleher N.L. Nat. Methods 10, 186–187 (2013).

Two-dimensional mass spectrometry (2D MS) is a tandem mass spectrometry method where precursor and fragment ions are correlated without isolation. A multifaceted data-independent acquisition technique, 2D MS has found applications from agrochemicals to top-down proteomics with both laser-based and electron-based fragmentation [1]. Developments in signal processing have shaped 2D MS into a fully-fledged analytical method. For narrowband 2D mass spectra with limited precursor m/z ranges, phase-corrected absorption mode, which improves resolving power and signal-to-noise ratios, has enabled the distinction between fragments from histone peptides with m/z 0.006 difference [2]. This study seeks to extract analytical information from all 2D mass spectra with maximum signal-to-noise ratio and resolving power by expanding phase-corrected absorption mode to broadband 2D MS.
2D mass spectra of diverse samples (intact proteins, peptide isoform mixtures, protein digests, plant extracts) were recorded on FT-ICR mass spectrometers with magnetic fields between 7-15 T (Bruker Daltonics, Germany) with Gäumann’s pulse sequence and both laser-based and electron-based fragmentation [1]. The frequency range for precursor ions in the vertical dimension ranged from 10 to 1000 kHz (i.e. precursor m/z 5-2000 ranges). All data processing and visualization was performed with the open-source Spectrometry Processing Innovative Kernel (SPIKE) software, developed in 64-bit python language [3]. For all 2D mass spectra, parameter optimization was performed in python notebooks before data processing in batch processing to eliminate limitations in dataset size due to RAM.
Phase progression in 2D mass spectra is linear with frequency in the vertical precursor ion dimension and quadratic in the horizontal fragment dimension [4]. For accurate phase correction, different parameters need to be optimized: 3 coefficients for horizontal phase correction, 2 coefficients for vertical phase correction, and the modulation frequency. All parameters are dependent on the experimental conditions determined in the pulse sequence. Phase-corrected absorption mode narrowband 2D mass spectra show that both signal-to-noise ratio and resolving power can be doubled compared to magnitude mode. In addition, phase correction acts as a filter for artefacts from harmonic peaks in 2D mass spectra. Data processing for absorption mode is performed in batch processing to eliminate caps on dataset size imposed by available RAM. We discuss the stability of empirically optimized parameters (modulation frequency, all phase correction coefficients) for automated processing of multiple 2D mass spectra acquired with identical experimental conditions. We establish routines in SPIKE for front-end data processing to produce internally calibrated peaklists for input in data analysis software (e.g. MASCOT for bottom-up proteomics, FAST-MS for top-down proteomics, PyC2MC for petroleomics). We examine disturbances introduced by factors like frequency range and speed of phase accretion as a function of cyclotron frequency (for fragment ion m/z) or of modulation frequency (for precursor ion m/z). We discuss solutions to these issues: asymmetrical apodisation, reconstruction of initial datapoints through autoregressive models, non-uniform sampling, and one-dimensional phase correction.
[1] https://doi.org/10.1007/s00249-019-01348-5
[2] https://doi.org/10.1021/jasms.2c00319
[3] https://arxiv.org/abs/1608.06777
[4] https://doi.org/10.3390/molecules26113388

The integration of Ion Mobility (IM) devices with mass spectrometers has facilitated the precise determination of peptide collisional cross sections (CCS), introducing an additional dimension to Mass Spectrometry (MS)-based analysis. Utilization of transient decay rate in Fourier Transform Mass Spectrometry (FTMS) for assessing CCS values has been reported for Orbitrap and Ion Cyclotron Resonance (ICR) MS systems, eliminating the need for a dedicated IM device. This study presents a new method that enables accurate and consistent measurement of CCS using FTMS in high-throughput LC/MS analyses on the proteome scale.

All the experiments were conducted on a modified Thermo Scientific™ Orbitrap Exploris™ 480 MS. HeLa cells were harvested in boiling SDS lysis buffer. Protein aggregation capture (PAC) was induced by the addition of organic solvent and magnetic microbeads before overnight on-bead digestion with Lys-C and Trypsin. Offline High pH Reversed-Phase HPLC Fractionation was performed using a Waters XBridge BEH130 C18 3.5 μm 4.6 × 250 mm column on an Ultimate 3000 high-pressure liquid chromatography (HPLC) system. Briefly, phosphopeptide enrichment was performed using TilMAC-HP beads on a KingFisher™ Flex robot. The ionic CCS values of peptides were evaluated based on the reported resolution values in MS1 scans. The processing was done using an in-house developed computational pipeline.

The inferred CCS values showed strong correlation with the IM-MS values from the literature and demonstrated comparable separation of different peptide charge states in the m/z vs CCS space. The reproducibility of the FTMS CCS measurements in the shotgun proteomics experiments is on the same scale as the reported CCS values obtained with the dedicated IM devices.
Moreover, our findings illustrate the utility of the CCS in the analysis of the peptide secondary structures and suggest that the conformation of peptides is likely determined by the balance between hydrophobic interactions driving more compressed forms and charge repulsion promoting extended conformations. Additionally, in the analysis of Post Translational Modifications (PTM), the CCS showed that phosphopeptides generally exhibit smaller CCS values compared to their unmodified counterparts; this trend particularly affects peptides with higher charge states. Conversely, methylated and acetylated peptides have larger CCS compared to their unmodified versions, likely due to increased hydrophobicity and loss of charge. Finally, we envision that this strategy can benefit the understanding and utility of CCS in proteomics workflows, contributing to increased confidence in peptide, phosphorylation site, and protein identification.