Integration of MALDI-TOF MS and machine learning to classify enterococci: A comparative analysis of supervised learning algorithms for species prediction.

This research focused on distinguishing distinct matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral signatures of three Enterococcus species. We evaluated and compared the predictive performance of four supervised machine learning algorithms, K-nearest neighbor (KNN), support vector machine (SVM), and random forest (RF), to accurately classify Enterococcus species. This study involved a comprehensive dataset of 410 strains, generating 1640 individual spectra through on-plate and off-plate protein extraction methods. Although the commercial database correctly identified 76.9% of the strains, machine learning classifiers demonstrated superior performance (accuracy 0.991). In the RF model, top informative peaks played a significant role in the classification. Whole-genome sequencing showed that the most informative peaks are biomarkers connected to proteins, which are essential for understanding bacterial classification and evolution. The integration of MALDI-TOF MS and machine learning provides a rapid and accurate method for identifying Enterococcus species, improving healthcare and food safety.

Integrating MALDI-TOF Mass Spectrometry with Machine Learning Techniques for Rapid Antimicrobial Resistance Screening of Foodborne Bacterial Pathogens.

Although MALDI-TOF mass spectrometry (MS) is considered as the gold standard for rapid and cost-effective identification of microorganisms in routine laboratory practices, its capability for antimicrobial resistance (AMR) detection has received limited focus. Nevertheless, recent studies explored the predictive performance of MALDI-TOF MS for detecting AMR in clinical pathogens when machine learning techniques are applied. This chapter describes a routine MALDI-TOF MS workflow for the rapid screening of AMR in foodborne pathogens, with Campylobacter spp. as a study model.

Diversity of culturable bacterial isolates and their potential as antimicrobial against human pathogens from Afar region, Ethiopia.

Antimicrobial resistance is a growing global concern exacerbated by the scarcity of new medications and resistance to current antibiotics. Microbes from unexplored habitats are promising sources of natural products to combat this challenge. This study aimed to isolate bacteria producing secondary metabolites and assess their antimicrobial efficacy against human pathogens. Soil and liquid samples were collected from Afar region, Ethiopia. Bacterial isolates were obtained using standard serial dilution techniques. Antimicrobial activity was evaluated using agar plug and well diffusion methods. matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF MS) and whole-genome sequencing (WGS) were conducted for the isolate exhibiting the highest antimicrobial activity. Secondary metabolites were extracted and analyzed using gas chromatography-mass spectra (GC-MS). In this study, 301 bacteria isolates were identified, of which 68 (22.6%) demonstrated antagonistic activity against at least one reference pathogen. Whole-genome sequencing revealed that Sl00103 belongs to the genus Bacillus, designated as Bacillus sp. Sl00103. The extract of Sl00103 showed zones of inhibition ranging between 17.17 ± 0.43 and 26.2 ± 0.4 mm against bacterial pathogens and 19.5 ± 0.44 to 21.0 ± 1.01 mm against Candida albicans. GC-MS analysis of ethyl acetate and n-hexane extracts identified major compounds including (R,R)-butane-2,3-diol; 3-isobutylhexahydropyrrolo[1,2a] pyrazine-1,4-dione; cyclo(L-prolyl-L-valine); and tetradecanoic acid, 12-methyl-, methyl ester; hexadecanoic acid, methyl ester among other. In conclusion, this study isolated several promising bacterial strains from the Afar region in Ethiopia, with strain Sl00103 (Bacillus sp. Sl00103) demonstrating notable antimicrobial and antioxidant activities and warranting further studies. IMPORTANCE: Antimicrobial resistance (AMR) is an escalating global health threat affecting humans, animals, and the environment, underscoring the urgent need for alternative pathogen control methods. Natural products, particularly secondary metabolites from bacteria, continue to be a vital source of antibiotics. However, microbial habitats and metabolites in Africa remain largely unexplored. In this study, we isolated and screened bacteria from Ethiopia's Afar region, characterized by extreme conditions like high temperatures, volcanic activity, high salinity, and hot springs to identify potential bioactive compounds. We discovered diverse bacterial isolates with antimicrobial activity against various pathogens, including strain Sl00103 (Bacillus sp. Sl00103), which demonstrated significant antimicrobial and antioxidant activities. GC-MS analysis identified several antimicrobial compounds, highlighting strain Sl00103 as a promising source of secondary metabolites with potential pharmaceutical applications and warranting further investigation.

Utilization of MALDI-TOF MS in the etiological diagnosis of deep-seated anaerobic bacterial infections.

PURPOSE: Deep-seated abscesses can be caused by a wide array of bacteria in various anatomical sites, the precise identification of which is crucial for implementing organism-specific treatments which can reduce morbidity and mortality. MALDI-TOF MS is a powerful proteomic method for the swift and accurate identification of anaerobic organisms. The aim of this study was to investigate deep-seated infections by MALDI-TOF MS (in comparison to VITEK®2 ANC ID card and phenotypic biochemical tests) and to determine the susceptibility pattern of identified microorganisms. MATERIALS AND METHODS: A total of 104 samples from patients suspected of deep-seated infections were aseptically collected and subjected to microscopy, aerobic/anaerobic cultures and subsequent identification via MALDI-TOF MS followed by antimicrobial susceptibility testing. Anaerobic bacteria were also identified using the VITEK-2 system and phenotypic biochemical tests. RESULTS: Out of the 104 samples tested, 41.3 % (43/104) showed positive results, predominantly in pus specimens (88 %). Mixed infections were found in 21 % of the positive cases. Of the 52 organisms identified from positive specimens, 19.2 % (10/52) were obligate anaerobes, with Bacteroides fragilis group being the most prevalent, followed by both Clostridium perfringens and Clostridium sporogenes respectively. Escherichia coli was observed to be the most common facultative anaerobic isolate. All obligate anaerobes were successfully identified to the species level via MALDI-TOF MS. In contrast, the VITEK®2 ANC ID card identified only 40 % (4/10) anaerobic bacteria to the species level. All obligate anaerobic organisms showed 100 % susceptibility to metronidazole, vancomycin and ertapenem. 25 % of the Bacteroides spp. and 50 % of Clostridium perfringens isolates were found to be resistant to clindamycin. CONCLUSION: MALDI-TOF MS proves as a beneficial diagnostic tool for bacterial identification, eliminating the labour-intensive and time consuming conventional microbiological methods. Its accuracy of bacterial detection further helps in combating antibiotic resistance and improving patient outcomes in deep-seated infections.

Comparison of Nucleotide MALDI-TOF MS with Xpert MTB/RIF for Rifampicin Susceptibility Identification and Associated Risk Factors of Rifampicin Resistance Among Drug Resistant Mycobacterium tuberculosis.

Purpose: Nucleotide-based matrix-assisted laser desorption ionization time-of-flight mass spectrometry (nucleotide MALDI-TOF MS) is an emerging molecular technology used for the diagnosis of tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB)and its drug resistance. This study aimed to compare the ability of nucleotide MALDI-TOF MS to detect rifampicin (RIF) resistance in drug-resistant TB (DR-TB) patients with Xpert MTB/RIF and to analyze the disparate results individually. Additionally, potential factors associated with rifampicin resistance among DR-TB patients in Qingdao were investigated. Patients and Methods: A retrospective study was conducted at Qingdao Chest Hospital, and patients with DR-TB were enrolled. Corresponding frozen isolates were recovered and subjected to nucleotide MALDI-TOF MS, Xpert MTB/RIF, and phenotypic drug susceptibility testing (pDST). Sanger sequencing was performed for the discordant results of nucleotide MALDI-TOF MS and Xpert MTB/RIF. Univariate and multivariate logistic regression analyses were used to identify potential factors associated with rifampicin resistance among patients with DR-TB. Results: A total of 125 patients with DR-TB (18.8%, 125/668) were enrolled in this study from May 1 to July 31, 2023. Rifampicin-resistant (DR-TB/RR, 29) and rifampicin-sensitive (DR-TB/RS, 96) groups were divided according to the pDST results. Nucleotide MALDI-TOF MS performed better than Xpert MTB/RIF in terms of sensitivity, specificity, accuracy, and agreement with pDST. Only six cases had inconsistent results, and the sequencing results of five cases were identical to nucleotide MALDI-TOF MS. Furthermore, chest pain (aOR=12.84, 95% CI, 2.29-91.97, p=0.005), isoniazid sensitivity (aOR=0.14, 0.02-0.59, p=0.013), and ethambutol sensitivity (aOR=0.02, 0.00-0.10, p=0.000) were potential factors associated with rifampicin resistance among DR-TB patients in Qingdao. Conclusion: The overall concordance between nucleotide MALDI-TOF MS and Xpert MTB/RIF was 95.2%, with the former performing better in determining rifampicin susceptibility among DR-TB cases in Qingdao. Chest pain, isoniazid, and ethambutol resistance might be factors associated with RIF resistance among patients with DR-TB in Qingdao.

Evaluation of protein extraction protocols for MALDI-TOF Biotyper analysis of mycobacteria.

Infections caused by Mycobacterium tuberculosis and nontuberculous mycobacteria represent a significant global threat and medical concern. Therefore, accurate and reliable methods must be employed to identify mycobacteria rapidly. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique that compares the cellular protein profiles of unknown isolates with reference mass spectra in a database to identify microorganisms. However, the thick and waxy lipid layer, which is rich in mycolic acids and is present in mycobacterial cells, makes protein extraction challenging. To identify the optimal protocol for correctly identifying bacilli using MALDI-TOF mass spectrometry, this study compared four different cellular protein extraction methods. Four strains of M. bovis BCG were selected as representatives of slow-growing mycobacteria, while three strains of fast-growing mycobacteria were also included: M. peregrinum, M. smegmatis, and M. farcinogenes. The extraction method that proved most effective was the extraction of inactivated cells with chloroform and methanol, which partially delipidates the cells. These cells were then extracted with formic acid, as is standard practice for protein extraction. The advantage of this method is that it allows the parallel analysis of cellular lipids and proteins from a single sample. It is therefore important to optimize mycobacterial protein extraction for MALDI-TOF MS analysis in clinical microbiology laboratories.

Collision cross sections of large positive fullerene molecular ions and their use as ion mobility calibrants in trapped ion mobility mass spectrometry.

Positive-ion laser desorption/ionization (LDI) of fullerenes contained in soot as produced by the Krätschmer-Huffman process delivers a wide range of fullerene molecular ions from C56+• to above C300+•. Here, the collision cross section (CCS) values of those fullerene molecular ions are determined using a trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) instrument. While CCS values in the range from C60+• to C96+• are already known with high accuracy, those of ions from C98+• onward had yet to be determined. The fullerene molecular ions covered in this work have CCS values from about 200 to 440 Å2. The fullerene molecular ion series is evenly spaced at C2 differences in composition, and thus, small CCS differences of just 2.2-3.5 Å2 were determined across the entire range. Fullerene M+• ions may be employed as mobility calibrants, in particular, when very narrow 1/K0 ranges are being analyzed to achieve high TIMS resolving power. In addition, due to the simple elemental composition, M+• ions of fullerenes could also serve for mass calibration. This study describes the determination of CCS values of fullerene molecular ions from C56+• to C240+• and the application of ions from C56+• to C220+• to calibrate the ion mobility scale of a Bruker timsTOFflex instrument in any combination of LDI, matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI) modes in the CCS range from about 200 to 420 Å2. This use was exemplified along with ions from Agilent Tune Mix, leucine-enkephalin, angiotensin I, angiotensin II, and substance P.

Lipid imaging mass spectrometry: Towards a new molecular histology.

Lipid research is attracting greater attention, as these molecules are key components to understand cell metabolism and the connection between genotype and phenotype. The study of lipids has also been fueled by the development of new and powerful technologies, able to identify an increasing number of species in a single run and at decreasing concentrations. One of such key developments has been the image techniques that enable the visualization of lipid distribution over a tissue with cell resolution. Thanks to the spatial information reported by such techniques, it is possible to associate a lipidome trait to individual cells, in fixed metabolic stages, which greatly facilitates understanding the metabolic changes associated to diverse pathological conditions, such as cancer. Furthermore, the image of lipids is becoming a kind of new molecular histology that has great chances to make an impact in the diagnostic units of the hospitals. Here, we examine the current state of the technology and analyze what the next steps to bring it into the diagnosis units should be. To illustrate the potential and challenges of this technology, we present a case study on clear cell renal cell carcinoma, a good model for analyzing malignant tumors due to their significant cellular and molecular heterogeneity.

Bacterial Pesticides: Mechanism of Action, Possibility of Food Contamination, and Residue Analysis Using MS.

As Sustainable Development Goals (SDGs) and the realities of climate change become widely accepted around the world, the next-generation of integrated pest management will become even more important for establishing a sustainable food production system. To meet the current challenge of food security and climate change, biological control has been developed as one sustainable crop protection technology. However, most registered bacteria are ubiquitous soil-borne bacteria that are closely related to food poisoning and spoilage bacteria. Therefore, this review outlined (1) the mechanism of action of bacterial pesticides, (2) potential concerns about secondary contamination sources associated with past food contamination, and, as a prospective solution, focused on (3) principles and methods of bacterial identification, and (4) the possibility of identifying residual bacteria based on mass spectrometry.

Facile fabrication of the immuno-MALDI-MS chip for the enrichment of abused drug in human urine integrated with MALDI-MS analysis.

BACKGROUND: Drug abuse can result in both physical and mental health issues for individuals, and can also contribute to broader societal problems. The number of drug abuse cases rose to 296 million in 2021. The sample pretreatment methods commonly employed typically require longer processing times and occasionally necessitate derivatization. Furthermore, with the increase in sample sizes, traditional chromatography-mass spectrometry methods for analyzing abused drugs were no longer sufficient to handle such numerous samples. In this study, immuno-MALDI-MS chip were fabricated for specific enrichment of illicit drugs, integrating with the rapid and accurate capabilities of MALDI-MS for high-throughput analysis of drug abuse. RESULTS: The immuno-MALDI-MS chip was successfully prepared by coating an aluminum chip with antibody-conjugated boronic acid-modified gold nanoparticles. Ketamine, a frequently abused illicit drug, served as the proof of concept for this study. The immuno-MALDI-MS chip was employed to selectively enrich ketamine in human urine samples, facilitating direct MALDI-MS analysis with the addition of α-CHCA matrix solution. The challenge of detecting abused drugs, exacerbated by interfering peaks in the low m/z region from salts and small molecules in human urine samples, was successfully overcome. The developed method exhibited a wide linear range of 10-5000 ng/mL with a limit of detection of 3.3 ng/mL for ketamine. Notably, the proposed method enabled high-throughput screening and accurate confirmation of ketamine concentrations in suspects' urine samples within few minutes, requiring a minimal sample volume of 1 µL. The obtained data were in complete agreement with the previous GC/MS analysis. SIGNIFICANCE: A straightforward, cost-effective and sensitive method for the selective enrichment and absolute quantification of abused drugs was developed using a homemade immuno-MALDI-MS chip integrated with MALDI-MS analysis. This method combines the advantages of immunoassay and mass spectrometry, offering both speed and accuracy. The reported method for the quantification of ketamine in human urine offers a practical approach and has the potential to analyze emerging new psychoactive substances in the future.

Development of a rapid mass spectrometric method for the analysis of ten-eleven translocation enzymes.

In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.

Early organism identification by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) decreases the time to appropriate antibiotic modifications for common bacterial infections.

Objective: MALDI-TOF-MS facilitates the identification of microorganisms from positive cultures in a timely and accurate manner. It eliminates the necessity for the application of biochemicals and operates on the principle of proteomics. It decreases the time required to report culture results. Prompt detection and notification of the pathogen, prior to the disclosure of antimicrobial susceptibilities, could potentially shorten the duration until the initial antibiotic adjustment is necessary, thereby influencing patients' clinical prognoses. Methodology: Fifty patients in the conventional arm and one hundred patients in the interventional arm were compared in a pre and post quasi-experimental study conducted at a tertiary care centre in North India. Patients with positive cultures from medical wards and intensive care units were included. Comparing the time to first antibiotic modification after culture positivity, MALDI-TOF-MS-based identification, and clinical outcomes in both arms was the primary objective. Antibiotic modifications, escalation, and de-escalation were all recorded. Results: The intervention arm exhibited a substantially shorter median time to first antibiotic modification (2010 mins vs 2905 mins, p=0.002) than the conventional arm. In the interventional group, a total of 44 out of 100 antibiotic modifications were implemented. Of these, 19 (43.3%) were determined solely by the MALDI report, without the anticipation of susceptibility assessments. De-escalation of antibiotics constituted the pre-dominant form of modification (47.4%). The difference between the 27% and 32% mortality rates in the intervention arm and the conventional arm was not statistically significant (p=0.52). Conclusion: MALDI-TOF-MS facilitates the modification of antibiotics early on. The primary benefit lies in the reduction of superfluous antibiotic usage. Early organism identification and reporting prior to the availability of susceptibility results did not result in any mortality benefit. This strategy, when combined with a strong antimicrobial stewardship programme, can aid in the reduction of antibiotic use.

Proteomic analysis of two novel peptides from the Odontobuthus doriae scorpion venom.

The venom of the Odontobuthus doriae scorpion, prevalent in East Asia and Iran, has not been fully characterized. This study provides the first proteomic profile of O. doriae venom to explore its potential as a medical. 2D-PAGE analysis revealed 96 protein spots with isoelectric points from 3 to 9 and molecular weights between 6.6 to 205 kDa. Fourteen toxin fractions were isolated via HPLC, and SDS-PAGE showed seven protein bands ranging from 3.8 to 182 kDa. MALDI-TOF MS identified Peptide 1 and Peptide 2, resembling Hemoglobin beta-2 chain and Chaperonin HSP60 and suggest potential therapeutic applications for P1 and P2.

Xenophilus aerolatus isolate misidentified as Brucella spp. by MALDI-TOF MS (VITEK-MS) system.

We report the characterization of Xenophilus aerolatus strain identified from a screening rectal swab specimen in human, initially misidentified as Brucella spp. by MALDI-TOF MS (VITEK-MS system). The strain is able to growth on ColorexTM mSuperCARBA plate, suggesting carbapenem resistance. Whole genome sequencing identified several potential antimicrobial resistance mechanisms.

Exploring the versatility of mass spectrometry: Applications across diverse scientific disciplines.

Mass spectrometry (MS) has become a pivotal analytical tool across various scientific disciplines due to its ability to provide detailed molecular information with high sensitivity and specificity. MS plays a crucial role in various fields, including drug discovery and development, proteomics, metabolomics, environmental analysis, and clinical diagnostics and Forensic science. In this article we are discussing the application of MS across the diverse scientific disciplines by focusing on some classical examples from each field of application. As the technology continues to evolve, it promises to unlock new possibilities in scientific research and practical applications, cementing its position as an essential tool in modern analytical science.

Single-colony MALDI mass spectrometry imaging reveals spatial differences in metabolite abundance between natural and cultured Trichodesmium morphotypes.

Trichodesmium, a globally significant N2-fixing marine cyanobacterium, forms extensive surface blooms in nutrient-poor ocean regions. These blooms consist of a dynamic assemblage of Trichodesmium species that form distinct colony morphotypes and are inhabited by diverse microorganisms. Trichodesmium colony morphotypes vary in ecological niche, nutrient uptake, and organic molecule release, differentially impacting ocean carbon and nitrogen biogeochemical cycles. Here, we assessed the poorly studied spatial abundance of metabolites within and between three morphologically distinct Trichodesmium colonies collected from the Red Sea. We also compared these results with two morphotypes of the cultivable Trichodesmium strain IMS101. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) coupled with liquid extraction surface analysis (LESA) tandem mass spectrometry (MS2), we identified and localized a wide range of small metabolites associated with single-colony Trichodesmium morphotypes. Our untargeted MALDI-MSI approach revealed 80 unique features (metabolites) shared between Trichodesmium morphotypes. Discrimination analysis showed spatial variations in 57 shared metabolites, accounting for 62% of the observed variation between morphotypes. The greatest variations in metabolite abundance were observed between the cultured morphotypes compared to the natural colony morphotypes, suggesting substantial differences in metabolite production between the cultivable strain IMS101 and the naturally occurring colony morphotypes that the cultivable strain is meant to represent. This study highlights the variations in metabolite abundance between natural and cultured Trichodesmium morphotypes and provides valuable insights into metabolites common to morphologically distinct Trichodesmium colonies, offering a foundation for future targeted metabolomic investigations.IMPORTANCEThis work demonstrates that the application of spatial mass spectrometry imaging at single-colony resolution can successfully resolve metabolite differences between natural and cultured Trichodesmium morphotypes, shedding light on their distinct biochemical profiles. Understanding the morphological differences between Trichodesmium colonies is crucial because they impact nutrient uptake, organic molecule production, and carbon and nitrogen export, and subsequently influence ocean biogeochemical cycles. As such, our study serves as an important initial assessment of metabolite differences between distinct Trichodesmium colony types, identifying features that can serve as ideal candidates for future targeted metabolomic studies.

Preformulation evaluation of selumetinib for topical application: skin distribution and photodegradation analysis using MALDI imaging and LC-MS/MS.

Understanding drug behavior within the skin, especially for photosensitive compounds, is crucial for developing effective and safe topical therapies. This study employs Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to investigate the skin permeation and photostability of selumetinib, a MEK inhibitor used in treating type 1 neurofibromatosis (NF1). The highest amounts of selumetinib in the skin sections were obtained when using the gel formulation, suggesting that it is to be preferred to cream formulations to achieve higher permeation of the drug. Our study also revealed that selumetinib is amenable to photodegradation in ex vivo skin explants, and yields one main degradation product, whose degradation is likely triggered by hydrogen abstraction. MALDI-MSI results showed selumetinib and its degradation product concentrate in skin appendages, indicating these structures might serve as drug reservoirs, potentially prolonging retention and efficacy. This study demonstrates that combining MALDI-MSI with LC/MS-MS can highly contribute to the characterization of the fate of photosensitive compounds in the skin, an essential prerequisite to the development of compound-specific photoprotective measures. It will also pave the way for innovative topical delivery strategies for NF1 treatment.

Label-free assessment of complement-dependent cytotoxicity of therapeutic antibodies via a whole-cell MALDI mass spectrometry bioassay.

Potency assessment of monoclonal antibodies or corresponding biosimilars in cell-based assays is an essential prerequisite in biopharmaceutical research and development. However, cellular bioassays are still subject to limitations in sample throughput, speed, and often need costly reagents or labels as they are based on an indirect readout by luminescence or fluorescence. In contrast, whole-cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has emerged as a direct, fast and label-free technology for functional drug screening being able to unravel the molecular complexity of cellular response to pharmaceutical reagents. However, this approach has not yet been used for cellular testing of biologicals. In this study, we have conceived, developed and benchmarked a label-free MALDI-MS based cell bioassay workflow for the functional assessment of complement-dependent cytotoxicity (CDC) of Rituximab antibody. By computational evaluation of response profiles followed by subsequent m/z feature annotation via fragmentation analysis and trapped ion mobility MS, we identified adenosine triphosphate and glutathione as readily MS-assessable metabolite markers for CDC and demonstrate that robust concentration-response characteristics can be obtained by MALDI-TOF MS. Statistical assay performance indicators suggest that whole-cell MALDI-TOF MS could complement the toolbox for functional cellular testing of biopharmaceuticals.

Identification of N-linked glycans recognized by WGA in saliva from patients with non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths. Saliva diagnosis is an essential approach for clinical applications owing to its noninvasive and material-rich features. The purpose of this study was to investigate differences in wheat germ agglutinin (WGA)-based recognition of salivary protein N-linked glycan profiles to distinguish non-small cell lung cancer (NSCLC) patients from controls. We used WGA-magnetic particle conjugates to isolate glycoproteins in the pooled saliva of healthy volunteers (HV, n = 35), patients with benign pulmonary disease (BPD, n = 35), lung adenocarcinoma (ADC, n = 35), and squamous cell carcinoma (SCC, n = 35), following to release the N-linked glycans from the isolated proteins with PNGase F, and further identified and annotated the released glycans by MALDI-TOF/TOF-MS, respectively. The results showed that 34, 35, 39, and 44 N-glycans recognized by WGA were identified and annotated from pooled saliva samples of HV, BPD, ADC, and SCC, respectively. Furthermore, the proportion of N-glycans recognized by WGA in BPD (81.2 %), ADC (90.1 %), and SCC (88.7 %), increased compared to HV (71.9 %). Two N-glycan peaks (m/z 2286.799, and 3399.211) specifically recognized by WGA were present only in NSCLC. These findings suggest that altered salivary glycopatterns such as sialic acids and GlcNAc containing N-glycans recognized by WGA might serve as potential personalized biomarkers for the diagnosis of NSCLC patients.

Structural and spectroscopic characterization of N1,N2-diphenylacenapthylene-1,2-diimines and the influence of substituents on the viscosity of alkyl magnesium solutions.

Butyl octyl magnesium solutions are important raw materials in various chemical processes but suffer from their high reactivity with even traces of water, protic solvents or oxygen and an increased viscosity in hydrocarbon solution due to the formation of polymeric structures. N1,N2-diphenylacenaphthylene-1,2-diimines (BIANs) have already been identified as potential candidates to reduce the viscosity of alkyl magnesium solutions and this study provides a systematic insight into the dependence of this ability on the position and structures of substituents on the BIAN. Besides the various BIANs, ZnCl2 complexes and hydrogenated derivatives were characterized and tested for their ability to reduce the viscosity. HPLC-high resolution mass spectrometry, MALDI-ToF mass spectrometry, but most important FTIR and NMR experiments under inert conditions have been used to shine light on the interaction of the different BIAN derivatives with alkyl magnesium solutions. Hydrogenated BIANs, especially those with bulky alkyl groups in the ortho position(s) have been identified as the most promising candidates. An additional benefit of the hydrogenated species is that in contrast to BIANs and BIAN-Zn complexes they do not undergo permanent chemical modification and can be reused after extraction.