matrix assisted laser desorptionionization Research Articles (Page 1)

This study investigated the temporal impact of mask wearing on cultivable conjunctival bacteria among healthcare workers. Conjunctival samples were collected from 48 participants, who were randomly assigned to wear surgical masks or KN95 respirators. Sampling occurred at baseline (T0, no mask), after 8h (T1), and after one week of daily wear (T2, ≥ 8h/day). Microbial cultures were analyzed using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry. The viable bacteria were of low diversity, dominated by Firmicutes, Actinobacteria, and Proteobacteria, encompassing seven families. Staphylococcaceae, Bacillaceae, and Corynebacteriaceae were most prevalent. In the KN95 group, Staphylococcaceae relative abundance was significantly higher at T2 than at T1 (P = 0.009). In the surgical mask group, Corynebacteriaceae abundance was significantly higher at T2 versus T0 and T1, while Bacillaceae was lower (all P < 0.01). Significant differences in Bacillaceae across all timepoints were unique to the KN95 group (all P < 0.01). At T1, Staphylococcaceae abundance was higher in the surgical mask group, while Bacillaceae was lower compared to the KN95 group (P = 0.025 and P = 0.023, respectively). At T2, Corynebacteriaceae abundance was significantly higher in the surgical mask group (P = 0.010). These findings raise the possibility that mask wearing may induce subtle, mask-type-dependent alterations in the cultivable conjunctival bacteria. Further study with a control group is warranted.

Low density polyethylene (LDPE) plastic films, which are widely used in greenhouses and mulch areas in the agricultural sector, can remain in the environment for a long time due to their resistance to decomposition structures and create large amounts of plastic pollution. In this study, it was aimed to isolate and identify bacteria which are capable of polyethylene (PE) degradation from greenhouse and mulch film soils containing plastic wastes in Izmir, one of the cities where greenhouse farming is most common in Türkiye, and to determine the degradation potentials of these bacteria. Bacteria were screened in soil samples taken from different greenhouses, mulched film areas and garden areas containing plastic waste, which have been covered with PE film for 10-30years. Eight bacterial isolates were identified for the first time from these areas using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. Plastic degradation properties of bacterial isolates were demonstrated after 30-days degradation study by reduction in PE film weight, FTIR-ATR analysis to determine structural changes on these films and SEM imaging to visualize changes in surface morphology. It was determined that the reduction rates in PE film weights of individual bacterial isolates after 30days varied between 25.39 and 7.08%, and the highest weight loss was recorded in Acinetobacter pittii HAT01. Also, the bacterium was observed to have biofilm formation capacity. FTIR-ATR analysis and SEM imaging findings of PE films supported the weight loss findings. This study provides new candidate strains adapted to long-term LDPE-contaminated agricultural soils and contributes to the development of sustainable biodegradation strategies.

Trichophyton rubrum and the Trichophyton mentagrophytes complex are the leading etiologic agents of human dermatophytosis. Terbinafine remains the first-line therapy for most dermatophyte infections; however, resistant strains have been increasingly reported worldwide, and the first cases of T. indotineae in Latin America have recently been reported. To update the limited regional information on Trichophyton spp. terbinafine resistance, we conducted a prospective laboratory study in Montevideo, Uruguay, from July 2023 to July 2024, using samples obtained through routine dermatological practice. Isolates were identified by morphological analysis and Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF). Terbinafine-resistant (TerR) clinical isolates were screened with the terbinafine-containing agar medium (TCAM) method, and their minimal inhibitory concentrations (MICs) for terbinafine and itraconazole were determined using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. The squalene epoxidase gene (SQLE) of TerR isolates was sequenced to detect resistance-associated mutations. Of 1252 processed samples, 543 were positive by microscopic examination and 281 by culture. Dermatophytes were identified in 231 of 281 positive cultures. The predominant species were T. rubrum (74.9%) and the T. mentagrophytes complex (19.9%), together accounting for 94.8% of isolates. Terbinafine susceptibility screening was performed on 171 isolates; 4 (2.34%) were resistant, with MICs ≥ 1 mg/l. Sequenced SQLE revealing amino acid substitutions at positions 393 or 397 in three T. rubrum and one T. mentagrophytes complex isolates. This study identifies the terbinafine resistance-associated mutations in Uruguay and highlights the urgent need for regional surveillance of this emerging phenomenon.

Size-exclusion chromatography (SEC) equipped with refractive index (RI) and multi-angle light scattering (MALS) detectors was carried out on a polystyrene (PS) sample with the weight-average molecular weight 2.07 ×€104 along with the matrix-assisted laser desorption flight time mass spectrometry (MALDI-TOF-MS). The MALDI-TO-MS result was corrected by the relative sensitivity, which was found to decay exponentially with the molecular weight. Exchanging the elution volume for molecular weight by the calibration relation, the molecular-weight distribution (MWD) curves were obtained from the chromatograms. The MWD curve determined by the SEC chromatogram was much broader than that obtained by MALS and MALDI-TOF-MS because of the peak-broadening effect. Based on the MWD determined by MALDI-TOF-MS, the RI chromatogram was closely described by the calculated values using the Gaussian function. Based on the results of the PS sample, RI and MALS data of polyethylene oxide samples in water were analyzed using the exponentially modified Gaussian equation.

Identifying disease-causing microorganisms in patient samples is a cornerstone activity in clinical diagnostic microbiology laboratories. This process relies on traditional microbiological techniques alongside advanced technological methods, such as matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF). Despite its growing prevalence in diagnostic settings, access to MALDI-TOF equipment remains limited in smaller laboratories, in regional centers, or in third-world nations. Consequently, many students in microbiology and related academic programs often lack opportunities for hands-on experience with this technology during their education. We overcame these limitations by implementing a blended teaching approach into an Infectious Diseases course, combining face-to-face practical inquiry with an innovative MALDI-TOF online simulation. The simulation enabled students to process patient samples from various body sites (e.g., swabs, cerebrospinal fluid, etc.) by selecting appropriate culture media and performing key microbiological tests. Students then confirmed preliminary identification by loading an individual colony onto a virtual MALDI-TOF target plate for analysis. The simulation then generated data mirroring a typical MALDI-TOF report, which uses a color-coded system to indicate confidence scores. The impact of this intervention on student learning was assessed through a written laboratory report and a questionnaire featuring Likert-scale and open-ended questions. Students demonstrated significant improvements in self-reported understanding, with nearly all participants successfully passing the written assessment. The approach was found to be enjoyable, interactive, and enhanced authentic learning. This study underscores the value of simulations as a cost-effective and scalable alternative for teaching advanced diagnostic techniques when access to expensive equipment is limited.

Antibody-drug conjugates (ADCs), comprising monoclonal antibodies and cytotoxic payloads, present inherent complexities in molecular design. The drug-to-antibody ratio (DAR) represents a critical determinant of ADC behavior, influencing pharmacokinetics and tissue distribution. In vivo studies in tumor-bearing murine models demonstrated comparable efficacy between DAR4 and DAR8 ADCs (DAR4: 6mg/kg, DAR8: 3mg/kg), suggesting that DAR alone does not dictate therapeutic outcome. Immunohistochemical (IHC) staining of human IgG, used to map intact ADC localization, demonstrated superior tumor penetration by DAR4 ADCs relative to their DAR8 counterparts. However, this enhanced penetration did not translate to improved therapeutic efficacy. To elucidate the mechanistic underpinning of DAR optimization, we leveraged matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) to spatially resolve the free payload distribution in tumors. The optimized MALDI imaging method enabled sensitive detection of released payload, revealing similar intensity and distribution pattern for both DAR4 and DAR8 ADCs at 2 and 24h post-dosing. This spatiotemporal uniformity of payload correlated with consistent pharmacodynamic (PD) responses. Moreover, equivalent payload signal intensities across DAR variants corroborated quantitative analyses of tumor lysates and subsequent efficacy data, reinforcing the pivotal role of intratumoral payload in driving in vivo efficacy. These findings underscore that DAR optimization is both target-dependent and payload-specific, suggesting that ADC design should prioritize factors governing active payload release over passive distribution metrics. The MALDI-IMS methodology developed in this study provides unprecedented spatial resolution of bioactive payload dynamics, establishing a robust platform for dissecting efficacy determinants in preclinical ADC development.

Mass spectrometry imaging (MSI) is widely used in biomedical sciences, as well as in plant sciences or microbiological studies. However, it is much less prominent in the earth sciences, despite holding enormous potential. For example, marine and lake sediments are excellent archives of the past, as they store the remnants of organisms from the water column and from surrounding land. These organisms leave behind fossil molecules that can be employed to reconstruct the environmental conditions during their lifetime, and hence to study the climate evolution of Earth. Focusing on matrix-assisted laser desorption/ionization (MALDI) imaging, we herein show that it is possible to read these molecular signatures in micrometer-sized spots, and thus to characterize climate variability with unprecedented resolution. However, obtaining this information is not trivial, and challenges arise from the nature of geological samples, low analyte concentrations, strong matrix effects, and from the need to reference the micrometer-scale maps to the original, large samples. Here we present a detailed tutorial on (i) how to select and prepare suitable samples, (ii) how to analyze them while avoiding the most common pitfalls and (iii) how to process the resulting large and complex dataset. We hope that this tutorial might contribute to inspire the use of MALDI in paleoenvironmental reconstructions in particular and in the earth sciences in general, and at the same time be useful to imaging applications across research fields.

Rapidly growing mycobacteria (RGM) are emerging pathogens with considerable variation in epidemiology and antimicrobial susceptibility patterns. Accurate species identification and antimicrobial susceptibility testing (AST) are essential to guide appropriate therapy. This study evaluated the epidemiology of RGM and the antimicrobial profile of the Mycobacterium abscessus complex (MABC). A retrospective study was conducted from June 2013 to May 2025at a tertiary care hospital in Mumbai. Clinical specimens were cultured using standard methods. RGM were identified by sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). AST for MABC was performed by broth microdilution (BMD) according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Inducible clarithromycin resistance (ICR) was also assessed. Sixty-eight RGM isolates were identified, most from patients aged 60-80 years, with a female predominance (58.8%). Pus and tissue were the most frequent specimen types (41.2%). MABC was the predominant species (67.65%), followed by M. fortuitum (14.71%). MABC showed high susceptibility to amikacin (91.1%), moderate activity to linezolid (37.8% susceptible, 42.2% intermediate), and 80% intermediate susceptibility to imipenem and cefoxitin. Resistance was high to cotrimoxazole, ciprofloxacin, and doxycycline. ICR was detected in 26.6% of tested MABC isolates. MABC is the predominant RGM in this setting and demonstrates high susceptibility to amikacin, with moderate activity of linezolid. ICR, tested and found in a subset of isolates, underscores the need for subspecies identification to optimize treatment. AST performed using recommended methods will help to guide therapy and formulate antimicrobial policy for managing these difficult to treat pathogens.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is integral to routine microbial identification. This study examines the performance of the VITEK® MS PRIME (bioMérieux; "PRIME") system for identification of clinically relevant bacteria, yeasts, and molds. We evaluated PRIME using the in vitro diagnostic (IVD) knowledge base version 3.2 (KB3.2) database on 652 clinical isolates, with reference identifications by polymerase chain reaction (PCR) and DNA sequencing. We also compared PRIME results with those obtained using the MALDI Biotyper (Bruker Daltonics; "Biotyper") for bacteria and yeasts in our routine workflow. PRIME correctly identified 311/317 bacteria (98.1%), 185/196 yeasts (94.4%), and 112/130 molds (86.2%); non-identification for molds was 16/130 (12.3%), with 2/130 (1.5%) misidentifications. Biotyper achieved 308/317 (97.2%) correct identifications for bacteria and 120/196 (61.2%) for yeasts with the IVD database; using the research use only (RUO) database increased the identification rate to 184/196 (93.9%) and reduced non-identifications from 76/196 (38.8%) to 12/196 (6.1%) for yeasts. Misidentifications by PRIME were uncommon, whereas molds-assessed only with PRIME-showed more frequent non-identification and occasional cross-taxon assignments. For example, Fusarium solani was reported as Rhizopus arrhizus, reflecting persistent challenges in MALDI-TOF MS for filamentous fungi and the impact of database breadth. Notably, mold performance observed here lies near the upper range of recent PRIME evaluations using IVD KB3.2. Overall, PRIME provided reliable routine identification for bacteria, yeasts, and molds; further expansion of fungal reference spectra and optimization of extraction workflows should improve mold identification. Pending such improvements, possible non-identifications or discordant assignments should be adjudicated by conventional microbiology before reporting.

Oxidative stress factors such as light and inflammation are known to damage nucleic acids through various mechanisms. Guanine, among the four canonical nucleobases, is particularly susceptible to oxidation, leading to the formation of oxidatively generated lesions such as 8-oxo-7,8-dihydroguanine (8-oxoG), spiroiminodihydantoin (Sp), and guanidinohydantoin (Gh). In this study, we now report an abasic (AP) site generation from guanine residue oxidation based on photocatalytic reactions. The study used Dickerson–Drew dodecamer DNA as the model oligo DNA for photocatalytic reactions, and the reactions were analyzed using denaturing polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry measurements. It was observed that guanine residues are converted into AP sites by reacting with singlet oxygen generated from the photocatalyst. Guanine residues with high solvent accessibility were found to be particularly reactive with singlet oxygen. Mechanistic investigations revealed that the photocatalytic generation of AP sites proceeds mainly via an 8-oxoG-independent mechanism. These findings are important for the design and development of photocatalyst-modified functional oligo probes and the understanding of their reactions.

Early diagnosis of Congenital Hypothyroidism (CH) is critical to prevent irreversible neurodevelopmental damage. However, current TSH-based newborn screening using Dried Blood Spots (DBS) is limited by factors that lead to false-positive and false-negative results, necessitating the development of alternative methods. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a high-throughput solution was investigated. This study presents a novel diagnostic approach utilizing DBS peptide barcoding, which was supported by comprehensive LC-MS/MS and network analysis. Peptide profiles showed a marked and reproducible difference between groups. MALDI-TOF MS identified a unique signature, including six dominant peptides specific to the CH-positive group. Subsequent analysis identified 37 candidate peptides, with network analysis linking 12 key proteins to established CH-related agents (e.g., thyroxine, TSHR). The MALDI-TOF MS peptidomic approach is validated as a robust, rapid, and cost-effective alternative for CH screening. This methodology represents a significant advance toward overcoming the limitations of current TSH assays and establishing a clinically translatable biomarker panel for improved personalized diagnosis of CH.

Purpose To characterize lipidomic profiles in the VX2 rabbit tumor model and assess potential lipid plasticity across tumor microenvironments in the liver and flank. Materials and Methods VX2 tumors were implanted into the liver in four rabbits and both flanks in two rabbits. After 10-14 days, tumor-containing tissues were harvested, snap frozen, sectioned, and analyzed using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in both positive and negative ion modes. Tandem mass spectrometry was used for further characterization. Lipid identities were determined based on accurate mass, fragmentation patterns, and database matching. Molecular ion distributions were spatially correlated with hematoxylin-eosin-stained sections. Results MALDI-MSI revealed distinct lipidomic profiles between liver and flank tumors. Oleate (mass-to-charge ratio [m/z] 281.24) was consistently localized to viable tumor regions in both sites. Phosphatidic acid 18:0/18:1(m/z 701.51) was detected in only liver tumors, whereas phosphatidylinositol 18:2/18:0 (m/z 861.55) was prominent in flank tumors but absent from liver tumors, despite being present in surrounding healthy parenchyma. Phosphatidylcholine (PC) species, notably PC 34:1 (m/z 798.52) and PC 36:2 (m/z 808.56), and PC (P-36:2) (m/z 824.56), showed heterogeneous spatial distributions across both anatomic sites. Conclusion MALDI-MSI enabled spatial mapping of lipid distributions within VX2 tumors, revealing both shared and site-specific alterations. These findings reveal heterogeneous spatial lipid distributions in the VX2 model and may reflect microenvironment-driven lipid plasticity. Keywords: Mass Spectrometry Imaging, VX2 Rabbit Tumor Model, Lipidomic Profiles, Ablation Techniques © RSNA 2026.

Human infection with non-aeruginosa Pseudomonas (NAP) is not commonly observed but can be severe and associated with significant morbidity and mortality. The aim for the present study was to investigate the prevalence, patient characteristics, risk factors, and clinical presentation of patients with NAP bacteremia. Additionally, the antibiotic sensitivity patterns of NAP isolates were assessed. The current retrospective study was conducted in the microbiology laboratory of a tertiary care center from January 2022 to October 2024. Two sets of blood cultures were received in BACTEC bottles (Becton, Dickinson and Company, Franklin Lakes, NJ) and were incubated. The blood samples were cultured on blood and MacConkey agar, and microscopy was performed using Gram staining. Species identification was performed via matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Antibiotic sensitivity testing was performed using meropenem, colistin, levofloxacin, amikacin, piperacillin-tazobactam, imipenem, aztreonam, ceftazidime, and cefoperazone-sulbactam. During the 2-year study period, 42 blood cultures grew Pseudomonas species other than Pseudomonas aeruginosa. The majority of patients were <60 years of age (76.1%). The age range of the population was 4-75 years. The male-to-female ratio of the population was 1.1:1. In this study, most isolates belonged to Pseudomonas mosselii (n = 13; 31%), followed by Pseudomonas stutzeri (n = 11; 26.1%), Pseudomonas putida (n = 9; 21.4%), Pseudomonas fluorescens (n = 5; 11.9%), and Pseudomonasalcaligenes (n = 3; 7.1%). The most common risk factors were the presence of a central venous catheter (n = 34; 81%) and mechanical ventilation (n = 25; 59.5%). Among the Pseudomonas mosellii isolates, the highest resistance was observed to meropenem (70%), imipenem (70%), and ceftazidime (70%). Timely diagnosis and treatment can help in reducing mortality and morbidity among NAP bacteremia patients.

We determined the optimal method to culture Mucorales species from air samples. Subsequently, we investigated the diversity of Mucorales species in Dutch air and compared these species with those causing mucormycosis in patients. We optimized the Mucorales culturing protocol by testing different growth conditions with samples from a newly developed air sampling approach. We used 120 air samples taken throughout the Netherlands in the project called Schimmelradar (September-October 2023). These samples were supplemented with additional air samples taken in the Netherlands (February 2024). The Mucorales species cultured from these air samples were compared to 17 clinical isolates (2016-2023) from a Dutch university medical center. Mucormycosis infections were classified using the European Organisation for Research and Treatment of Cancer (EORTC) criteria. All Mucorales were identified at genus level by culture morphology, and a subset was analyzed at species level using matrix-assisted laser desorption ionization-time of flight mass spectrometry, microscopy, and Internal Transcribed Spacer Polymerase Chain Reaction (ITS PCR). A combination of Sabouraud dextrose agar, voriconazole and incubation at 30°C yielded the broadest variety of Mucorales species from air samples. Species found in Dutch air included Rhizomucor pusillus, Rhizopus microsporus, and Mucor circinelloides. Clinical data showed that Rhizop. microsporus and M. circinelloides were most frequently identified in mucormycosis infections. We validated a selective method for the culture of Mucorales species from air samples using a delta trap air sampling method. Three of the 10 species cultured from air samples were also detected in clinical isolates. Although inhalation is assumed as primary route of infection, this is the first study demonstrating the similarity of Mucorales species between air and clinical samples.

Accumulating evidence suggests that N-glycosylation plays a crucial role in modulating ischemic pathophysiology. However, the dynamic alterations of N-glycosylation patterns during the early phase of acute ischemic stroke (AIS) have not been systematically investigated. We employed matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to profile the glycome of murine cerebral tissues and identify differentially expressed glycans and glycosylation features during early AIS progression. To validate these findings, we further analyzed serum glycome profiles from human AIS patients in the early disease stage. Comprehensive statistical analyses were conducted to identify potential glycome biomarkers. Comprehensive glycomic profiling identified 42 distinct N-glycan structures in murine brain tissues and 31 in serum samples. Through integrated multivariate statistical analyses, we identified 9 ischemia-sensitive cerebral glycans and 6 serum biomarkers. While no individual glycan structures were conserved between species, we identified a conserved downregulation of fucosylation as a key feature (murine cohort AUC = 0.98; human cohort AUC = 0.78). This study presents the first comprehensive comparison of glycomic profiles in early AIS from mouse brain tissue and human serum, identifying a conserved fucosylation deficiency as a potential class of diagnostic indicators for clinical detection and therapeutic targeting. The observed deficiency was correlated with a potential mechanistic link to dysregulated glycan-mediated inflammatory responses and impaired cellular stress response pathways.

Introduction. Aspergillosis represents a significant global health threat, with increasing concerns about azole resistance.Hypothesis/Gap Statement. There is limited evidence on the prevalence and distribution of azole resistance among clinical Aspergillus isolates.Aim. This study investigated the prevalence of azole resistance in clinical Aspergillus isolates and evaluated different susceptibility testing methods.Methodology. A total of 125 Aspergillus spp. isolates were collected from clinical samples (abscess, corneal abscess, biopsy, tissue and respiratory samples). Species identification was performed using conventional morphological methods and Matrix assisted laser desorption Ionization time of flight massspectrometry (MALDI-TOF) MS. Azole susceptibility testing was conducted using the gradient test (E-test), the agar plate screening method and broth microdilution for voriconazole (VOR), itraconazole (ITR) and posaconazole (POS). Molecular analysis was performed to detect cyp51A gene mutations associated with resistance.Results. Among 125 isolates, species distribution was 44% Aspergillus fumigatus, 33.6% Aspergillus flavus, 5% Aspergillus terreus, 3% Aspergillus niger and 14% Aspergillus spp. Using gradient testing, A. fumigatus showed 1.8% resistance to VOR, 5.45% to ITR and 1.8% to POS, with one isolate resistant to all azoles. A. terreus showed 16.7% resistance to VOR, A. niger 25% resistance to ITR and Aspergillus spp. showed various resistance patterns. The agar plate method detected resistance with 100% susceptibility/specificity for non-fumigatus species but 33.3% susceptibility for A. fumigatus ITR resistance. CypA-L98H mutations were detected in six isolates and CypA-M220 mutations in seven isolates.Conclusion. This study confirms the presence of azole resistance in clinical Aspergillus isolates with species-specific variations. The agar plate screening method shows promise for non-fumigatus species but requires optimization for A. fumigatus.

Surface-capping ligands on quantum dots (QDs) modify the physical and chemical properties of QDs to enable diverse applications. Accurate qualitative and quantitative analyses of these ligands are therefore essential for the advancement of QD research; however, conventional methods (e.g., UV-vis, TEM) seldom provide direct stoichiometric insight. MS can offer a solution, but traditional MALDI induces an excessive ligand loss. Here, we employed a highly electronegative, fluorinated porphyrin (TPFP) as a soft MALDI matrix for CdSe QDs to preserve the ligands and yield high ion signals at reduced laser power. Comparative studies with electron-transfer matrixes (DCTB, 9-nitroanthracene, dithranol, and pentacene) indicate that the highly electronegative TPFP extracts electrons from photoexcited QDs, lowering ionization thresholds and preventing ligand detachment. Using TPFP-MALDI-MS, we determined QD core sizes and counted the number of surface ligands in carboxylate- and thiolate-capped QDs, and for thiolate-capped QDs in particular, we further evaluated ligand numbers and ligand densities with various core sizes. This approach provides a reliable strategy to determine QD core sizes, to count ligand numbers, and to estimate the surface coverage with improved resolution. These findings underscore the importance of electron affinity of a matrix for soft ionization and expand MS-based tools for probing QD core and surface chemistry, paving the way for more reliable characterization of nanocrystalline systems.

Background/Objectives: There is an increasing need for methods that provide improved insight into the molecular basis of colorectal cancer (CRC) and thus a better understanding of its morphological heterogeneity. The objectives of this study were to evaluate the use of imaging mass spectrometry (IMS) to examine tumor margins and gain insight into the molecular heterogeneity of CRC. Methods: An observational study involving 10 cases was conducted. Native tissue samples were collected during the subject's surgery, and consecutively taken tissue sections were immediately prepared for light microscopic and IMS analysis. IMS was performed across the 200-1000 Da mass range, divided into four sub-ranges, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) in both negative and positive modes of ionization. For tumor margin and tissue heterogeneity assessment, image segmentation was used. Segmented MS images were analyzed against the respective light microscopy images of hematoxylin-eosin-stained tissue sections. Results: Quantitative analysis of the sample collection indicated that IMS enabled correct recognition of tumor margin in the 800-1000 Da range using binary segmentation. Denary image segmentation depicted tissue heterogeneity in greater detail. The strongest m/z signals specific to tumor, peritumor, and margins were identified and tentatively annotated: aside from dCTP, all other compounds were patient-specific, indicating interindividual variations in the molecular composition of CRC. Conclusions: IMS provides new insights into the morphological and biochemical properties of CRC: binary segmented MS images can clearly depict the tumor margin in the 800-1000 Da range, while denary segmented MS images depict intra- and inter-individual molecular heterogeneity of CRC.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows species-level microbial identification quickly and cost-effectively. The aim of this study was to validate MALDI-TOF MS for the identification of Legionella anisa, Legionella longbeachae and Legionella pneumophila and to examine whether incubation time or sample preparation method affect identification results. Environmental Legionella strains and ATCC reference strains were used in the study. The strains underwent 16S rRNA sequencing and latex agglutination and fluorescence tests to which MALDI-TOF MS results were compared. The strains, incubated for 24 and 48h, were analysed with MALDI-TOF MS using direct transfer and extraction methods. Following performance characteristics were calculated from the results: sensitivity, specificity, relative accuracy, predictive values, repeatability and reproducibility. Species-level identification was achieved for 63 out of the 69 environmental and reference strains regardless of incubation time or preparation method. MALDI-TOF MS proved to be very specific (100%) and sensitive (94.6-100%) to the Legionella species analysed. With the extraction method, incubation time had a statistically significant, but species-dependent effect on the interpretative criteria, i.e. score values, in all species studied. No significant difference in any of the species' score values was found between the direct transfer and extraction methods with 24 h incubation. MALDI-TOF MS score values showed high repeatability (std 0.01-0.23) and reproducibility (std 0.02-0.18) for all Legionella species analysed. Based on the results, MALDI-TOF MS suits well for the analysis of environmental Legionella strains already after 24h of incubation and using direct transfer method.

Mass spectrometry imaging (MSI) is a versatile technique for spatially resolved multi-omics analysis in clinical research. However, its proteomic applications remain constrained by issues such as poor ionisation efficiency and signal interference from complex tissue matrices. On-tissue mass-tag labelling (OTMT) overcomes these challenges by employing affinity-based imaging agents that carry cleavable, highly ionisable reporter groups known as mass-tags (MTs). In this work, we introduce TATE-Ru(ii)MT, which comprises a somatostatin receptor subtype 2 (SSTR2)-targeting cyclic peptide and a photocleavable Ru(ii) polypyridine complex. We demonstrate that this MT enables multimodal visualisation of SSTR2 in tissue using matrix-assisted laser desorption/ionisation (MALDI) and desorption electrospray ionisation (DESI) MSI, following a staining workflow fully compatible with the widely used, commercially available Miralys™ MTs from AmberGen. Unlike commercial MTs, which use peptide sequences as photocleavable reporter groups, Ru(ii)-based MTs are not restricted to MALDI-MSI, offering a flexible platform with variable MT design and broad methodological applicability.