Matrix-assisted laser desorption/ionization matrix incorporation evaluation algorithm for improved peak coverage and signal-to-noise ratio in mass spectrometry imaging.

RATIONALE: Despite decades of implementation, the selection of optimal sample preparation conditions for matrix-assisted laser desorption/ionization (MALDI) imaging is still ambiguous due to the lack of a universal and comprehensive evaluation methodology. Thus, numerous experiments with different matrix application conditions accompany a translation of the method to novel sample types and matrices. METHODS: Mouse brain tissues were covered with 9-aminoacridine through sublimation, followed by recrystallization in vapors of 5% (v/v) methanol solution in water. The samples were analyzed by MALDI time-of-flight mass spectrometry, and the efficiency of lipid and small-molecule ionization was evaluated with different metrics. RESULTS: We first investigate the dependency of matrix density and recrystallization conditions on the thickness of an analyte-empty matrix layer to roughly evaluate the laser shot number required to obtain an intense signal with minimal noise. Then, we introduce metrics for the analysis of small imaging datasets (small sample regions) of model samples based on median quantity of peaks in spectra (medQP) and weighted median signal-to-noise ratio (wmSNR). The evaluation of small regions and taking median values for metrics help overcome the sample heterogeneity and allow for the simultaneous comparison of different acquisition parameters. CONCLUSIONS: Here, we propose a methodology based on gradual laser ablation of small regions of sample and further implementation of weighted signal-to-noise ratio to assess various matrix application conditions. The proposed approach helps reduce the number of test samples required to determine optimal sample preparation conditions and improve the overall quality of images.

Determination of Brain Tissue Samples Storage Conditions for Reproducible Intraoperative Lipid Profiling.

Ex-vivo molecular profiling has recently emerged as a promising method for intraoperative tissue identification, especially in neurosurgery. The short-term storage of resected samples at room temperature is proposed to have negligible influence on the lipid molecular profiles. However, a detailed investigation of short-term molecular profile stability is required to implement molecular profiling in a clinic. This study evaluates the effect of storage media, temperature, and washing solution to determine conditions that provide stable and reproducible molecular profiles, with the help of ambient ionization mass spectrometry using rat cerebral cortex as model brain tissue samples. Utilizing normal saline for sample storage and washing media shows a positive effect on the reproducibility of the spectra; however, the refrigeration shows a negligible effect on the spectral similarity. Thus, it was demonstrated that up to hour-long storage in normal saline, even at room temperature, ensures the acquisition of representative molecular profiles using ambient ionization mass spectrometry.

Spherical Sampler Probes Enhance the Robustness of Ambient Ionization Mass Spectrometry for Rapid Drugs Screening.

Ambient ionization mass spectrometry has become one of the most promising approaches for rapid and high-throughput screening of small molecules in complex biological matrices for emergency medicine, forensics, and food and agriculture applications. The simple procedures for sample collection and ionization without additional pretreatment are vital in these fields. Many efforts have been devoted to modifying various ambient ionization techniques to simplify the procedures and improve the robustness and sensitivity of the methods. Here, we demonstrate the implementation of rigid spherical sampler probes to improve the robustness of touch spray ionization mass spectrometry. The sphericity of the probes increases the stability of the cone-jet mode of electrospray, reduces the requirements for fine positioning of a sampler in the ion source, and decreases the possibility of corona discharge occurrence. The utilization of spherical sampler probes allows fast, non-invasive sampling, followed by rapid analysis for various drugs of different chemical classes in complex biological matrices, such as the whole blood or sebum collected from the skin surface. The linearity of the analytical signal response from drug concentration confirms the possibility of creating a simple semiquantitative method for small molecules monitoring using spherical sampler probes.

Mass spectrometry based proteome profiling of the exhaled breath condensate for lung cancer biomarkers search.

INTRODUCTION: Lung cancer remains the most prevalent cause of cancer mortality worldwide mainly due to insufficient availability of early screening methods for wide-scale application. Exhaled breath condensate (EBC) is currently considered as one of the promising targets for early screening and is particularly attractive due to its absolutely noninvasive collection and possibility for long-term frozen storage. EBC proteome analysis can provide valuable information about the (patho)physiological changes in the respiratory system and may help to identify in time a high risk of lung cancer. Mass spectrometry (MS) profiling of EBC proteome seems to have no alternative in obtaining the most extensive data and characteristic marker panels for screening. AREAS COVERED: This special report summarizes the data of several proteomic studies of EBC in normal and lung cancer (from 2012 to 2021, PubMed), focuses on the possible reasons for the significant discrepancy in the results, and discusses some aspects for special attention in further studies. EXPERT OPINION: The significant discrepancy in the results of various studies primarily highlights the need to create standardized protocols for the collection and preparation of EBC for proteomic analysis. The application of quantitative and targeted LC-MS/MS based approaches seems to be the most promising in further EBC proteomic studies.

Normalization methods for reducing interbatch effect without quality control samples in liquid chromatography-mass spectrometry-based studies.

Data normalization is an essential part of a large-scale untargeted mass spectrometry metabolomics analysis. Autoscaling, Pareto scaling, range scaling, and level scaling methods for liquid chromatography-mass spectrometry data processing were compared with the most common normalization methods, including quantile normalization, probabilistic quotient normalization, and variance stabilizing normalization. These methods were tested on eight datasets from various clinical studies. The efficiency of the data normalization was assessed by the distance between clusters corresponding to batches and the distance between clusters corresponding to clinical groups in the space of principal components, as well as by the number of features with a pairwise statistically significant difference between the batches and the number of features with a pairwise statistically significant difference between clinical groups. Autoscaling demonstrated the most effective reduction in interbatch variation and can be preferable to probabilistic quotient or quantile normalization in liquid chromatography-mass spectrometry data.

Rapid estimation of tumor cell percentage in brain tissue biopsy samples using inline cartridge extraction mass spectrometry.

Tumor cell percentage (TCP) is an essential characteristic of biopsy samples that directly affects the sensitivity of molecular testing in clinical practice. Apart from clarifying diagnoses, rapid evaluation of TCP combined with various neuronavigation systems can be used to support decision making in neurosurgery. It is known that ambient mass spectrometry makes it possible to rapidly distinguish healthy from malignant tissues. In connection with this, here we demonstrate the possibility of using non-imaging ambient mass spectrometry to evaluate TCP in glial tumor tissues with a high degree of confidence. Molecular profiles of histologically annotated human glioblastoma tissue samples were obtained using the inline cartridge extraction ambient mass spectrometry approach. XGBoost regressors were trained to evaluate tumor cell percentage. Using cross-validation, it was estimated that the TCP was determined by the regressors with a precision of approximately 90% using only low-resolution data. This result demonstrates that ambient mass spectrometry provides an accurate method todetermine TCP in dissected tissues even without implementing mass spectrometry imaging. The application of such techniques offers the possibility to automate routine tissue screening and TCP evaluation to boost the throughput of pathology laboratories. Rapid estimation of tumor cell percentage during neurosurgery.

Probabilistic model applied to ion abundances in product-ion spectra: quantitative analysis of aspartic acid isomerization in peptides.

Evaluation of post-translational modifications of protein molecules is important for both basic and applied biomedical research. Mass spectrometric quantitative studies of modifications, which do not change the mass of the protein, such as isomerization of aspartic acid, do not necessarily require the use of isotope-labelled standards. However, the accurate solution of this problem requires a deep understanding of the relationship between the mole fractions of the isomers and the peak intensities in the mass spectra. In previous studies on the isomerization of aspartic acid in short beta-amyloid fragments, it has been shown that calibration curves used for such quantitative studies often have a non-linear form. The reason for the deviation in the shape of the calibration curves from linearity has not yet been established. Here, we propose an explanation for this phenomenon based on a probabilistic model of the fragmentation process and present a general approach for the selection of fragments that can be used for quantitative studies of the degree of isomerization. Graphical Abstract.

Dissection of the deep-blue autofluorescence changes accompanying amyloid fibrillation.

Pathogenesis of numerous diseases is associated with the formation of amyloid fibrils. Extrinsic fluorescent dyes, including Thioflavin T (ThT), are used to follow the fibrillation kinetics. It has recently been reported that the so-called deep-blue autofluorescence (dbAF) is changing during the aggregation process. However, the origin of dbAF and the reasons for its change remain debatable. Here, the kinetics of fibril formation in model proteins were comprehensively analyzed using fluorescence lifetime and intensity of ThT, intrinsic fluorescence of proteinaceous fluorophores, and dbAF. For all systems, intensity enhancement of the dbAF band with similar spectral parameters (∼350 nm excitation; ∼450 nm emission) was observed. Although the time course of ThT lifetime (indicative of protofibrils formation) coincided with that of tyrosine residues in insulin, and the kinetic changes in the ThT fluorescence intensity (reflecting formation of mature fibrils) coincided with changes in ThT absorption spectrum, the dbAF band started to increase from the beginning of the incubation process without a lag-phase. Our mass-spectrometry data and model experiments suggested that dbAF could be at least partially related to oxidation of amino acids. This study scrutinizes the dbAF features in the context of the existing hypotheses about the origin of this spectral band.

Fourier transform ion cyclotron resonance (FT ICR) mass spectrometry: Theory and simulations.

Fourier transform ion cyclotron resonance (FT ICR) mass spectrometer offers highest resolving power and mass accuracy among all types of mass spectrometers. Its unique analytical characteristics made FT ICR important tool for proteomics, metabolomics, petroleomics, and investigation of complex mixtures. Signal acquisition in FT ICR MS takes long time (up to minutes). During this time ion-ion interaction considerably affects ion motion and result in decreasing of the resolving power. Understanding of those effects required complicated theory and supercomputer simulations but culminated in the invention of the ion trap with dynamic harmonization which demonstrated the highest resolving power ever achieved. In this review we summarize latest achievements in theory and simulation of FT ICR mass spectrometers.

Atmospheric pressure thermal ionization ion source for peptide analysis.

A novel atmospheric pressure thermal ionization (APTI) ion source was developed for the analysis of liquid samples. The feasibility of the ion source was demonstrated on peptides using two configurations-assisted by hot wire or hot surface. Microalloyed molybdenum, used as a thermal ion- emitter, notably facilitated the formation of multiply-charged ions, but fragmentation products were still observed. Peptide fragmentation accompanying thermal ionization can be used for peptide identification. The described method is promising for studies of biological samples with minimal pre-treatment.

Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo.

The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD(50) of S-(2-(diethylamino)ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.

Synthesis of model humic substances: a mechanistic study using controllable H/D exchange and Fourier transform ion cyclotron resonance mass spectrometry.

The products of the oxidative coupling of phenols are frequently used as synthetic analogues to natural humic substances (HS) for biomedical research. However, their molecular compositions and exact structures remain largely unknown. The objective of this study was to develop a novel approach for the molecular-level analysis of phenolic polymerisates that is capable of inventorying molecular constituents and resolving their distinct structural formulas. For this purpose, we have synthesized the model HS using the oxidative coupling of a specifically designed phenylpropanoic monomer, 3-(4-hydroxy-3-methoxyphenyl)-3-oxopropionic acid, to hydroquinone. We have characterized the synthesized model HS using high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS), (1)H NMR spectroscopy, and controllable hydrogen/deuterium (H/D) exchange. We succeeded in the molecular inventory of the model HS. The assigned molecular formulas occupied the substantial space of CHO compositions in the Van Krevelen diagram with a maximum density found in the regions of tannins and lignins, resembling those of natural HS. To identify the exact structural formulas of the individual constituents in the model HS, we have applied selective H/D exchange of non-labile backbone protons by a choice of basic or acidic catalytic conditions followed by FTICR MS. The determined formulas allowed us to verify the proposed pathways of hydroxylation and carboxylation in the course of the phenolic coupling and to identify the acetylation of aromatic rings as an important side reaction. We conclude that the proposed analytical approach may be used to identify the molecular carriers of biological activity within the phenolic polymerisates and eventually within natural HS.

Application of de novo sequencing tools to study abiogenic peptide formations by tandem mass spectrometry. The case of homo-peptides from glutamic acid complicated by substitutions of hydrogen by sodium or potassium atoms.

RATIONALE: Peptides and proteins are among the most important components of living systems. Different attempts have been made to experimentally model the formation of peptides from amino acid monomers in investigation of the origin of life. Detailed characterization of peptides formed under various conditions in such reactions is very important for understanding processes of abiogenic peptide formation. METHODS: We used liquid chromatography coupled with tandem mass spectrometry (MS/MS) for an accurate study of homo-peptides formed in a model reaction: glutamic acid oligomerization catalyzed by 1,1'-carbonyldiimidazole in aqueous solution with 1 M of sodium or potassium chloride and without any salts. We used de novo sequencing software for peptide identification. In addition we propose an approach that uses more spectral information for de novo sequencing then standard methods. RESULTS: Peptides up to 9 amino acids long were found in the experiments with KCl, while in experiments with NaCl and without salts only peptides of up to 7 amino acids were detected. Due to high salt concentrations in samples a high number of singly charged peptide ions with up to 4 substitutions of hydrogen atoms by sodium or potassium atoms were observed. De novo sequencing software provided correct identifications even for peptide ions with substitutions. CONCLUSIONS: Multiple substitutions of hydrogen by alkali metal atoms in peptide ions strongly change their fragmentation patterns. Proposed approach for de novo sequencing was found very effective, even for ions with substitutions. So, it may be useful in more complicated cases like sequencing abiogenic peptides consisting of different amino acids.

Molecular mapping of sorbent selectivities with respect to isolation of Arctic dissolved organic matter as measured by Fourier transform mass spectrometry.

The objectives of this study were to identify molecular features characteristic to arctic DOM from the Kolyma River basin and to elucidate structural imprints induced by a choice of the sorption technique. To achieve this goal, DOM was isolated from the Kolyma River basin with a use of three nonionic sorbents: Amberlite XAD-8 resin, PPL- and C18 - SPE cartridges, and one anion exchanging resin-diethylaminoethyl (DEAE) -cellulose. The structural studies were conducted with a use of electrospray ionization Fourier Transform Ion Cyclotron Resonance (ESI FT-ICR) mass spectrometry and liquid state (1)H NMR spectroscopy. The DOM isolates obtained with a use of PPL and C18 cartridges were characterized with higher content of aliphatic compounds as compared to XAD-8 and DEAE-isolates. In total, for all arctic DOM isolates we observed predominance of hydrogen saturated compounds with high H/C values of identified formulas from FT-ICR MS data. (1)H NMR spectroscopy studies have confirmed this trend and revealed high contribution of alkyl-chain protons into the spectral density of the arctic DOM reaching 43% for PPL isolates.

Potassium ions are more effective than sodium ions in salt induced peptide formation.

Prebiotic peptide formation under aqueous conditions in the presence of metal ions is one of the plausible triggers of the emergence of life. The salt-induced peptide formation reaction has been suggested as being prebiotically relevant and was examined for the formation of peptides in NaCl solutions. In previous work we have argued that the first protocell could have emerged in KCl solution. Using HPLC-MS/MS analysis, we found that K(+) is more than an order of magnitude more effective in the L-glutamic acid oligomerization with 1,1'-carbonyldiimidazole in aqueous solutions than the same concentration of Na(+), which is consistent with the diffusion theory calculations. We anticipate that prebiotic peptides could have formed with K(+) as the driving force, not Na(+), as commonly believed.

Incorporation of a Disposable ESI Emitter into Inline Cartridge Extraction Mass Spectrometry Improves Throughput and Spectra Stability.

Rapid and reliable methods for detecting tumor margins are crucial for neuro-oncology. Several mass spectrometry-based methods have been recently proposed to address this problem. Inline Cartridge Extraction (ICE) demonstrates the potential for clinical application, based on ex-vivo analysis of dissected tissues, but requires time-consuming steps to avoid cross-contamination. In this work, a method of incorporating a disposable electrospray emitter into the ICE cartridge by PEEK sleeves melting is developed. It reduces total analysis time and improves throughput. The proposed setup also improves the robustness of the ICE molecular profiling as demonstrated with human glial tumor samples in that stability and reproducibility of the spectra were increased.

Fine structure in isotopic peak distributions measured using a dynamically harmonized Fourier transform ion cyclotron resonance cell at 7 T.

The fine structure of isotopic peak clusters in mass spectra of reserpine and substance P are measured using Fourier transform ion cyclotron resonance mass spectrometry at a 7 T magnetic field. The resolved peaks in the fine structure consist of (13)C, (15)N, (17)O, (18)O, (2)H, (33)S, (34)S, and combinations of them. A recently introduced high-resolution ion cyclotron resonance cell (Nikolaev, E. N.; Boldin, I. A.; Jertz, R.; Baykut, G. J. Am. Soc. Mass Spectrom. 2011, 22, 1125-1133) is used in these experiments. The positions of the experimentally obtained fine structure peaks on the mass scale agree with the isotopic distribution simulations with ≤200 ppb error. Some deviation from the theoretical isotopic distribution is observed, less abundant peaks in the fine structure patterns are a little suppressed compared to the larger ones. We present a method for atomic composition determination using accurate mass data and fine isotopic structure of the mass spectrum. Our method combines the traditional atomic composition determination from accurate mass data by enumeration of all possible formulas within constraints defined a priori with the estimation of element coefficients from resolved isotopic structures. These estimated values allow one to narrow the search space for the composition and therefore to reduce the number of candidate formulas.

Absorption-mode spectra on the dynamically harmonized Fourier transform ion cyclotron resonance cell.

RATIONALE: The recently designed dynamically harmonized Fourier transform ion cyclotron resonance (FT-ICR) cell creates a more harmonized electric field for the detection of the cyclotron motion of ions and prolongs the ion transient from seconds to minutes. In order to achieve its best performance, phase correction was applied in the spectra, and new advantages of the absorption-mode were revealed. METHODS: Spectra were acquired from both simple standard and complex mixtures using either narrowband or broadband mode, and the data were processed to compare the performance of the spectra in magnitude and absorption-mode. RESULTS: The research shows that phase correction works well with data from Nikolaev's new cell, which produces the maximum improvement in resolving power (2×), and improves the match with the theoretical intensities of the isotopic peaks. In addition, the harmonic peaks can be diagnosed immediately in the absorption-mode. CONCLUSIONS: The manuscript demonstrates absorption-mode spectra from Nikolaev's ICR cell, which will be of interest to the community. The improved relative peak intensities and immediate identification of harmonic peaks will facilitate data interpretation.

Phosphorylation and nitration levels of photosynthetic proteins are conversely regulated by light stress.

Using a label-free mass spectrometric approach, we investigated light-induced changes in the distribution of phosphorylated and nitrated proteins within subpopulations of native photosynthetic complexes in the thylakoid membrane of Arabidopsis thaliana leaves adapted to growth light (GL) and subsequently exposed to high light (HL). Eight protein phosphorylation sites were identified in photosystem II (PSII) and the phosphorylation level of seven was regulated by HL as determined based on peak areas from ion chromatograms of phosphorylated and non-phosphorylated peptides. Although the phosphorylation of PSII proteins was reported in the past, we demonstrated for the first time that two minor antenna LHCB4 isoforms are alternately phosphorylated under GL and HL conditions in PSII monomers, dimers and supercomplexes. A role of LHCB4 phosphorylation in state transition and monomerization of PSII under HL conditions is proposed. We determined changes in the nitration level of 23 tyrosine residues in five photosystem I (PSI) and nine PSII proteins and demonstrated for the majority of them a lower nitration level in PSI and PSII complexes and supercomplexes under HL conditions, as compared to GL. In contrast, the nitration level significantly increased in assembled/disassembled PSI and PSII subcomplexes under HL conditions. A possible role of nitration in (1) monomerization of LHCB1-3 trimers under HL conditions (2) binding properties of ferredoxin-NADP+ oxidoreductase to photosystem I, and (3) PSII photodamage and repair cycle, is discussed. Based on these data, we propose that the conversely regulated phosphorylation and nitration levels regulate the stability and turnover of photosynthetic complexes under HL conditions.