User-friendly platform for analysis of high mass intact proteins and glycopeptides by laser desorption/ionization-mass spectrometry based on copper oxide particles.

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) based on micro/nanostructured materials with different natures has received increasing attention for the analysis of a wide variety of analytes. However, up to now, only a few studies have shown the application of simple platforms in MALDI-MS for the identification of intact proteins. The present work reports on the application of copper oxide particles (Cu2O PS), obtained by a greener route, in combination with low amounts of 2,5-dihydroxybenzoic acid (DHB) as a novel hybrid platform. The combined Cu2O PS@DHB matrix, containing only 2.5 mg mL-1 of particles and 10 mg mL-1 of DHB, was easily applicable in MALDI-MS without surface modification of target plates. Under optimal conditions, the analysis of intact proteins up to 150,000 Da was possible, including immunoglobulin G, bovine serum albumin, and cytochrome C with adequate spot-to-spot signal reproducibility (RSD < 10%). In addition, the analysis of glycopeptides from IgG digests was carried out to prove the multipurpose application of the Cu2O PS@DHB platform in the low m/z range (2500-3000 Da). From the obtained results, it can be concluded that the optical and surface properties of as-synthesized Cu2O PS are likely to be responsible for the superior performance of Cu2O PS@DHB in comparison with conventional matrices. In this sense, the proposed user-friendly methodology opens up the prospect for possible implementation in bioanalysis and diagnostic research.

Lignin Structure and Reactivity in the Organosolv Process Studied by NMR Spectroscopy, Mass Spectrometry, and Density Functional Theory.

There is need for well-defined lignin macromolecules for research related to their use in biomaterial and biochemical applications. Lignin biorefining efforts are therefore under investigation to meet these needs. The detailed knowledge of the molecular structure of the native lignin and of the biorefinery lignins is essential for understanding the extraction mechanisms as well as chemical properties of the molecules. The objective of this work was to study the reactivity of lignin during a cyclic organosolv extraction process adopting physical protection strategies. As references, synthetic lignins obtained by mimicking the chemistry of lignin polymerization were used. State-of-the-art nuclear magnetic resonance (NMR) analysis, a powerful tool for the elucidation of lignin inter-unit linkages and functionalities, is complemented with matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF MS), to gain insights into linkage sequences and structural populations. The study unraveled interesting fundamental aspects on lignin polymerization processes, such as identifications of molecular populations with high degrees of structural homogeneity and the emergence of branching points in lignin structure. Furthermore, a previously proposed intramolecular condensation reaction is substantiated and new insights into the selectivity of this reaction are introduced and supported by density functional theory (DFT) calculations, where the important role of intramolecular π-π stacking is emphasized. The combined NMR and MALDI-TOF MS analytical approach, together with computational modeling, is important for deeper fundamental lignin studies and will be further exploited.

The reaction of organic peroxy radicals with unsaturated compounds controlled by a non-epoxide pathway under atmospheric conditions.

Today, the reactions of gas-phase organic peroxy radicals (RO2) with unsaturated Volatile Organic Compounds (VOC) are expected to be negligible at room temperature and ignored in atmospheric chemistry. This assumption is based on combustion studies (T ≥ 360 K), which were the only experimental data available for these reactions until recently. These studies also reported epoxide formation as the only reaction channel. In this work, the products of the reactions of 1-pentylperoxy (C5H11O2) and methylperoxy (CH3O2) with 2,3-dimethyl-2-butene ("2,3DM2B") and isoprene were investigated at T = 300 ± 5 K with Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) and Gas Chromatography/Electron Impact Mass Spectrometry. Unlike what was expected, the experiments showed no measurable formation of epoxide. However, RO2 + alkene was found to produce compounds retaining the alkene structure, such as 3-hydroxy-3-methyl-2-butanone (C5H10O2) with 2,3DM2B and 2-hydroxy-2-methyl-3-butenal (C5H8O2) and methyl vinyl ketone with isoprene, suggesting that these reactions proceed through another reaction pathway under atmospheric conditions. We propose that, instead of forming an epoxide, the alkyl radical produced by the addtion of RO2 onto the alkene reacts with oxygen, producing a peroxy radical. The corresponding mechanisms are consistent with the products observed in the experiments. This alternative pathway implies that, under atmospheric conditions, RO2 + alkene reactions are kinetically limited by the initial addition step and not by the epoxide formation proposed until now for combustion systems. Extrapolating the combustion data to room temperature thus underestimates the rate coefficients, which is consistent with those recently reported for these reactions at room temperature. While slow for many classes of RO2, these reactions could be non-negligible at room temperature for some functionalized RO2. They might thus need to be considered in laboratory studies using large alkene concentrations and in biogenically-dominated regions of the atmosphere.

Thiol-ene-based microfluidic chips for glycopeptide enrichment and online digestion of inflammation-related proteins osteopontin and immunoglobulin G.

Proteins, and more specifically glycoproteins, have been widely used as biomarkers, e.g., to monitor disease states. Bottom-up approaches based on mass spectrometry (MS) are techniques commonly utilized in glycoproteomics, involving protein digestion and glycopeptide enrichment. Here, a dual function polymeric thiol-ene-based microfluidic chip (TE microchip) was applied for the analysis of the proteins osteopontin (OPN) and immunoglobulin G (IgG), which have important roles in autoimmune diseases, in inflammatory diseases, and in coronavirus disease 2019 (COVID-19). TE microchips with larger internal surface features immobilized with trypsin were successfully utilized for OPN digestion, providing rapid and efficient digestion with a residence time of a few seconds. Furthermore, TE microchips surface-modified with ascorbic acid linker (TEA microchip) have been successfully utilized for IgG glycopeptide enrichment. To illustrate the use of the chips for more complex samples, they were applied to enrich IgG glycopeptides from human serum samples with antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The dual functional TE microchips could provide high throughput for online protein digestion and glycopeptide enrichment, showing great promise for future extended applications in proteomics and the study of related diseases.

Chemical mass shifts of cluster ions and adduct ions in quadrupolar ion traps revisited and extended.

RATIONALE: Chemical mass shifts in quadrupolar ion traps have been studied previously but only for a limited number of analytes and mass ranges. Here, mass shifts of cluster ions, commonly used as calibrants, and other analytes are qualitatively evaluated on the Bruker amaZon spherical ion trap (QIT) and the Finnigan LXQ linear ion trap (LIT). To extend the mass range from previous experiments m/z up to 4000 are investigated. METHODS: Chemical mass shifts of CsI, Y(HCOO)3 , and NaCF3 COO cluster ions, CF3 COO- , Na+ , and Cs+ adduct ions, protonated commercial calibration solutions and peptides, and deprotonated peptides were investigated on the Bruker amaZon speed QIT and some of these were also investigated on the Finnigan LXQ LIT. RESULTS: On both instruments, peak distortions and mass shifts toward lower m/z became apparent as m/z approached 1000. To some extent, the issues were more severe at slower scans. Peak distortions included loss of resolution, tailing, or fronting and were different between the amaZon QIT and the LXQ LIT. The noncluster and nonadduct ions analyzed showed no obvious mass shifts or peak distortions under the same analysis conditions. CONCLUSIONS: As expected, the ion traps investigated here showed mass shift and peak distortion issues, and such issues persisted at m/z up to 4000 on both instruments. Peak distortions were different between the amaZon QIT and the LXQ LIT, and were not always visible despite mass shifts. Both mass shifts and peak distortions make cluster ions and some adduct ions unsuitable for ion trap calibration.

Capillary and microchip electrophoresis method development for amino acid monitoring during biopharmaceutical cultivation.

The increased use of biopharmaceuticals calls for improved means of bioprocess monitoring. In this work, capillary electrophoresis (CE) and microchip electrophoresis (MCE) methods were developed and applied for the analysis of amino acids (AAs) in cell culture supernatant. In samples from different days of a Chinese hamster ovary cell cultivation process, all 19 proteinogenic AAs containing primary amine groups could be detected using CE, and 17 out of 19 AAs using MCE. The relative concentration changes in different samples agreed well with those measured by high-performance liquid chromatography (HPLC). Compared to the more commonly employed HPLC analysis, the CE and MCE methods resulted in faster analysis, while significantly lowering both the sample and reagent consumption, and the cost per analysis.

Method development for mono- and disaccharides monitoring in cell culture medium by capillary and microchip electrophoresis.

The rapidly growing, competitive biopharmaceutical market requires tight bioprocess monitoring. An integrated, automated platform for the routine online/at-line monitoring of key factors in the cell culture medium could greatly improve process monitoring. Mono- and disaccharides, as the main energy and carbon source, are one of these key factors. A CE-LIF method was developed for the analysis of several mono- and disaccharides, considering requirements and restrictions for analysis in an integrated, automated monitoring platform, such as the possibility for miniaturization to microchip electrophoresis. Analysis was performed after fluorescent derivatization with 8-aminopyrene-1,3,6-trisulfonic acid. The derivatisation reaction and the separation BGE were optimized using design of experiments. The developed method is applicable to the complex matrix of cell culture medium and proved transferable to microchip electrophoresis.

Chemical Diversity between Three Graminoid Plants Found in Western Kenya Analyzed by Headspace Solid-Phase Microextraction Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS).

In recent work, it was shown that the graminoid plants Cynodon dactylon (Poaceae), Cyperus exaltatus (Cyperaceae), and Panicum repens (Poaceae) have an ovipositional effect on the malaria vector Anopheles gambiae in olfactometric bioassays. In order to get a view of the diversity of semiochemicals present in the environment of the vector during olfactometric trials, in the present work, the volatile profiles of these graminoid plants were analyzed using headspace solid-phase microextraction (HS-SPME) together with gas chromatography-mass spectrometry (GC-MS). In addition, one-way ANOVA comparison of compounds detected in two or more headspace samples are presented in order to provide a basis for comparison of compounds that could constitute a starting point for novel blends of volatile organic compounds to be tested as oviposition attractants.

Grass-like plants release general volatile cues attractive for gravid Anopheles gambiae sensu stricto mosquitoes.

BACKGROUND: Understanding the ecology and behaviour of disease vectors, including the olfactory cues used to orient and select hosts and egg-laying sites, are essential for the development of novel, insecticide-free control tools. Selected graminoid plants have been shown to release volatile chemicals attracting malaria vectors; however, whether the attraction is selective to individual plants or more general across genera and families is still unclear. METHODS: To contribute to the current evidence, we implemented bioassays in two-port airflow olfactometers and in large field cages with four live graminoid plant species commonly found associated with malaria vector breeding sites in western Kenya: Cyperus rotundus and C. exaltatus of the Cyperaceae family, and Panicum repens and Cynodon dactylon of the Poaceae family. Additionally, we tested one Poaceae species, Cenchrus setaceus, not usually associated with water. The volatile compounds released in the headspace of the plants were identified using gas chromatography/mass spectrometry. RESULTS: All five plants attracted gravid vectors, with the odds of a mosquito orienting towards the choice-chamber with the plant in an olfactometer being 2-5 times higher than when no plant was present. This attraction was maintained when tested with free-flying mosquitoes over a longer distance in large field cages, though at lower strength, with the odds of attracting a female 1.5-2.5 times higher when live plants were present than when only water was present in the trap. Cyperus rotundus, previously implicated in connection with an oviposition attractant, consistently elicited the strongest response from gravid vectors. Volatiles regularly detected were limonene, ß-pinene, ß-elemene and ß-caryophyllene, among other common plant compounds previously described in association with odour-orientation of gravid and unfed malaria vectors. CONCLUSIONS: The present study confirms that gravid Anopheles gambiae sensu stricto use chemical cues released from graminoid plants to orientate. These cues are released from a variety of graminoid plant species in both the Cyperaceae and Poaceae family. Given the general nature of these cues, it appears unlikely that they are exclusively used for the location of suitable oviposition sites. The utilization of these chemical cues for attract-and-kill trapping strategies must be explored under natural conditions to investigate their efficiency when in competition with complex interacting natural cues.

Analysis of amino acids in cell culture supernatant using capillary electrophoresis with contactless conductivity detection.

CE-C4 D methods for the analysis of amino acids (AAs) are presented. Combining the results from two methods with acetic acid and cyclodextrin-based BGEs, 20 proteinogenic AAs could be analyzed using CE. CE-C4 D was also, for the first time, applied to analyze free AAs in samples of mammalian cell culture supernatant. After dilution as only sample preparation, combining the results of the two CE methods allowed monitoring the concentration changes of 17 AAs in samples taken during the cultivation of CHO cells.

Analytical survey of tattoo inks-A chemical and legal perspective with focus on sensitizing substances.

BACKGROUND: Tattoo inks have been reported to elicit allergic contact dermatitis. OBJECTIVES: To investigate the labels and the contents of metals and pigments in tattoo inks, considering restrictions within the European Union. METHODS: Seventy-three tattoo inks currently available on the market, either bought or donated (already used), were investigated for trace metals and pigments by inductively coupled plasma mass spectrometry and by matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry. RESULTS: Ninety-three percent of the bought tattoo inks violated European, legal requirements on labeling. Fifty percent of the tattoo inks declared at least one pigment ingredient incorrectly. Sixty-one percent of the inks contained pigments of concern, especially red inks. Iron, aluminium, titanium, and copper (most in green/blue inks) were the main metals detected in the inks. The level of metal impurities exceeded current restriction limits in only a few cases. Total chromium (0.35-139 µg/g) and nickel (0.1-41 µg/g) were found in almost all samples. The levels of iron, chromium, manganese, cobalt, nickel, zinc, lead, and arsenic were found to covary significantly. CONCLUSIONS: To prevent contact allergy and toxic reactions among users it is important for tattoo ink manufacturers to follow the regulations and decrease nickel and chromium impurities.

SpheriCal® -ESI: A dendrimer-based nine-point calibration solution ranging from m/z 273 to 1716 for electrospray ionization mass spectrometry peptide analysis.

RATIONALE: A calibration solution for mass spectrometry needs to cover the range of interest with intense and sufficiently narrowly spaced peaks. Limited options fulfilling this may lead to compromises between performance and ease of use. SpheriCal® -ESI was designed to combine high calibration performance for electrospray ionization (ESI) mass spectrometric analysis of peptides in positive mode with quick and easy use. METHODS: The developed calibration solution was tested using three mass spectrometers: two ion traps and one tandem quadrupole. The m/z errors of SpheriCal® -ESI itself and of a tryptic digest of cytochrome C were measured after calibration. The results were compared with those achieved with ESI Tuning Mix. The memory effects of the dendrimers, and contamination from Na+ in the calibration solution, were evaluated. RESULTS: SpheriCal® -ESI showed good shelf life as powder and was quickly reconstituted for use. Achieving intense and stable signals was straightforward. The accuracies and precisions were as expected for the instruments. SpheriCal® -ESI was more precise and at least as accurate as ESI Tuning Mix. The memory effects and Na+ contamination were found to be negligible in typical peptide solvents. In addition, the dendrimers showed predictable dissociations with product ions common to collision-induced dissociation in both ion trap and tandem quadrupole mass spectrometers. CONCLUSIONS: SpheriCal® -ESI provided easily accessible calibration by showing intense signals at low infusion rates and at source settings equal or similar to those used in peptide analysis. Nine calibration points in the range of interest gave precise and accurate results. Memory effects and contamination were negligible even without rinsing.

Implementation of a ultraviolet area imaging detector for analysis of polyvinyl alcohol microbubbles by capillary electrophoresis.

Contrast agents are widely used to enhance the image quality in clinical imaging using e.g. ultrasound. The contrast agents used for ultrasound imaging are mainly microbubbles (MBs) with a soft or hard shell encapsulating a core of gas. In the present study, MBs with a hard shell of polyvinyl alcohol (PVA), and a core of air were analysed in a capillary electrophoretic system using a UV area imaging detector. The detector was operating at 3 wavelengths; 214 nm, 255 nm and 525 nm, and the highest absorbance for individual PVA-MBs were obtained at 214 nm. Two detection windows and a vertical loop capillary position enabled tracking of the PVA-MBs both in an upward and a downward flow direction, where PVA-MBs had different flow distributions and slightly higher average flow velocity upwards, attributed to temperature differences in the capillary that was part within the instrument and part outside. The tracking also allowed counting and quantification of the PVA-MBs. Separation of PVA-MBs from proteins present in human blood plasma was achieved, with multi-wavelength imaging showing best contrast at 525 nm. The PVA-MBs absolute values of negative zeta potential and anionic mobility when injected from plasma in the pH 12 background electrolyte are higher than those obtained for MBs injected from buffer, consistent with their increased negative charge due to a protein corona coating of the PVA-MBs.

Amino Acid-Functionalized Two-Dimensional Hollow Cobalt Sulfide Nanoleaves for the Highly Selective Enrichment of N-Linked Glycopeptides.

Leaf-like hollow cobalt sulfides with a sulfur-gold-cysteine (S-Au-Cys) structure on the surface have been synthesized for efficient N-glycopeptide enrichment. A two-dimensional (2D) zeolitic imidazolate framework with cobalt (ZIF-L-Co, L for leaf) was used as a self-sacrificed template. After sulfidation, the S-Au-Cys architecture was created on the surface of the leaf-like hollow cobalt sulfide to obtain a material denoted ZIF-L-Co-S-Au-Cys. Enrichment of glycopeptides from trypsin digests of immunoglobulin G (IgG) standard samples and of IgG isolated from real human plasma samples was accomplished via hydrophilic interaction liquid chromatography processes using ZIF-L-Co-S-Au-Cys. The good sensitivity and selectivity ensure the effectiveness and robustness of ZIF-L-Co-S-Au-Cys for sample preconcentration, which is comparable to a commercial HILIC product. This work provides an efficient way to produce transition metal sulfides with a low-dimensional morphology and provides a novel concept for material design for exploitation in sample preparation, especially in glycoproteomics.

An antibody-free sample pretreatment method for osteopontin combined with MALDI-TOF MS/MS analysis.

Osteopontin is an osteoblast-secreted protein with an aspartic acid-rich, highly phosphorylated, and glycosylated structure. Osteopontin can easily bind to integrins, tumor cells, extracellular matrix and calcium, and is related to bone diseases, various cancers, inflammation etc. Here, DEAE-Cibacron blue 3GA was used to extract recombinant osteopontin from human plasma, and to deplete abundant plasma proteins with an antibody-free method. Using selected buffer systems, osteopontin and human serum albumin could be bound to DEAE-Cibacron blue 3GA, while immunoglobulin G was excluded. The bound osteopontin could then be separated from albumin by using different sequential elution buffers. By this method, 1 µg/mL recombinant osteopontin could be separated from the major part of the most abundant proteins in human plasma. After trypsin digestion, the extracted osteopontin could be successfully detected and identified by MALDI-TOF MS/MS using the m/z 1854.898 peptide and its fragments.

An automated system for CE-MALDI and on-target digestion under a fluorocarbon lid applied on spermatophore proteins from Pieris napi.

A method for off-line CE-MALDI-TOF-MS and MS2, and on-target digestion under a fluorocarbon lid was developed and applied for the analysis of proteins in the spermatophore of the butterfly Pieris napi. Fractionation revealed many peptides otherwise not detected or resolved. Automated fractionation was performed with an in-lab developed robotic system, and automated on-target tryptic digestion under a fluorocarbon lid was demonstrated with the same system. Fractionation onto a pre-structured MALDI-concentration plate facilitated aligned deposition of trypsin and MALDI-matrix with the deposited sample, also under the fluorocarbon lid. Some indications of indigenous proteolysis of spermatophore proteins were seen, and searching MS2 spectra suggested three tentative sequence homologies to P. rapae. The study demonstrates the functionality of the lab-made robot. Detailed manufacturing instructions and code are provided. The feasibility of automated on-target digestion under a fluorocarbon lid, and the usefulness of a structured concentration plate in CE-MALDI fractionation was shown. Further, it constitutes a preliminary study of P. napi spermatophore proteins.

Analysis of butterfly reproductive proteins using capillary electrophoresis and mass spectrometry.

A method for analysis of proteins from spermatophores transferred from male to female Pieris napi butterflies during mating has been developed. The proteins were solubilized from the dissected spermatophores using different solubilization agents (water, methanol, acetonitrile and hexafluoroisopropanol). Capillary electrophoresis (CE) analysis was performed using an acidic background electrolyte containing a fluorosurfactant to avoid protein-wall adsorption, and to increase separation performance. The samples were also analyzed with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), in a lower m/z range (1000-6000) and a higher m/z range (6000-12000). Solubilization with different solvents and the use of alternative matrices gave partly complementary profiles.

Structural basis for the formation of soy protein nanofibrils.

Amyloid-like protein nanofibrils (PNFs) can assemble from a range of different proteins including disease-associated proteins, functional amyloid proteins and several proteins for which the PNFs are neither related to disease nor function. We here examined the core building blocks of PNFs formed by soy proteins. Fibril formation at pH 2 and 90 °C is coupled to peptide hydrolysis which allows isolation of the PNF-forming peptides and identification of them by mass spectrometry. We found five peptides that constitute the main building blocks in soy PNFs, three of them from the protein ß-conglycinin and two from the protein glycinin. The abilities of these peptides to form PNFs were addressed by amyloid prediction software and by PNF formation of the corresponding synthetic peptides. Analysis of the structural context in the native soy proteins revealed two structural motifs for the PNF-forming peptides: (i) so-called ß-arches and (ii) helical segments involved in quaternary structure contacts. However, the results suggest that neither the native structural motifs nor the protein of origin defines the morphology of the PNFs formed from soy protein isolate.