9th CONFERENCE OF THE CZECH SOCIETY FOR MASS SPECTROMETRY - BOOK OF ABSTRACTS

Significance of the Topic
Recent advances in mass spectrometry have expanded its role from qualitative studies to precise quantitative analyses in research, clinical diagnostics, and industrial quality control. Methods like LC–MS, CE–MS, and MALDI–MS allow the detection of low-abundance biomarkers, provide structural details in proteomics and metabolomics, and identify small molecules in complex matrices. These innovations improve disease diagnosis, environmental monitoring, and drug development.

Study Objectives and Overview
This collection summarizes analytical approaches presented at the Ninth Annual Conference of the Czech Society for Mass Spectrometry, highlighting: 1. Metabolomics and proteomics workflows for biomarker discovery. 2. Glycoprotein and lipid analyses by LC–MS/MS and HILIC separations. 3. Microbiological applications: detection of siderophores, metallophores, and bacterial virulence factors. 4. Clinical microsampling techniques for noninvasive biofluid analysis. 5. Structural MS methods (FPOP, HDX, top-down) for protein–DNA and protein–protein interactions. 6. Clinical diagnostics of infection (Aspergillus) and inherited metabolic disorders by targeted MS.

Methodologies and Instrumentation

• UHPLC–HRMS (Orbitrap Fusion, Q Exactive) and UHPLC–QqQ for metabolomics and proteomics:
  – Exploratory workflows combining XCMS Online detection, blank subtraction, retention time prediction (cLogP model) to reduce false positives by 88%.
  – Targeted quantification of 50 proteins per analysis in single cerebral organoids by SRM-MS.
  – Evaluation of peptide winged internal standards for absolute quantitation.
  
• Reversed-phase and HILIC LC–Q-TOF-MS for glycopeptide and glycoprotein profiling:
  – CSHTM columns with CID fragmentation produce oxonium ions for glycan assignment.
  – Charged surface hybrid columns allow low-formic acid mobile phases (0.01%) for 40–50% increased ESI sensitivity in proteomics.
  
• MALDI–FTICR, MALDI–TOF, qMSI for spatial metabolomics and lipidomics:
  – MALDI imaging of neurotransmitters (norepinephrine, epinephrine) and α-tocopherol in brain slices post-hypoxic-ischemia.
  – qMSI-uD combining in vivo neuropharmacokinetics and in vitro slice studies to quantify unbound drugs (risperidone, clozapine, olanzapine) across BBB in 20 µm resolution.
  
• CE–MS (CE–ICP-MS, CE–ESI–MS) for siderophore analysis:
  – Native and Ga-labeled forms of ferricrocin, TAFC, pyoverdines achieve 500× lower LODs via CE–ICP-MS with SDS BGE.
  
• Fast Photochemical Oxidation of Proteins (FPOP) & Top-down MS:
  – vT-nESI IMS–MS (timsTOF Pro) for protein–DNA complex melting transitions and footprinting.
  – EAD and Zeno trap on Q-TOF for complementary fragmentation of lipids & peptides.
  
• Sample preparation and microsampling:
  – QuEChERS extraction and UHPLC–Q-Orbitrap for pesticide profiling in soils.
  – SLIDE 3D-printed porous glass device for apocrine sweat lipidomics, PQN normalization.
  – Dry blood spots, saliva, tears as emerging microsamples.
  
• Clinical and environmental diagnostics:
  – MALDI affinity chips for procalcitonin detection in sepsis (10 ng/mL LOD).
  – LC–MS/MS panel of 147 urinary OAs, acylglycines, acylcarnitines for IMD diagnosis (90+ disorders).
  – Urinary screening of antibiotics in wastewaters by SPE–LC–MS/MS reveals hospital sources exceeding PNEC for resistance selection.
  

Main Results and Discussion
1. False positives in untargeted metabolomics can be drastically reduced using retention-time filtering, enhancing biomarker validity.
2. Glycopeptide separations on HILIC vs RP columns: each stationary phase resolves different glycoforms; HILIC-A and HILIC-N separate core/outer fucosylation.
3. Low-formic acid mobile phases on CSH columns improve proteomic sensitivity without peak distortion.
4. MALDI imaging uncovers region-specific metabolite changes in brain injury and drug distribution across the BBB at subregional levels.
5. CE–ICP-MS with Ga-labeling allows ultra-trace siderophore quantification for infection diagnostics.
6. FPOP top-down reveals protein–DNA interaction interfaces and dynamics faster than bottom-up.
7. Microsampling (dry blood spots, SLIDE sweat) and PQN normalization enable noninvasive lipidomic analyses.

Benefits and Practical Applications

• Biomarker discovery: robust untargeted workflows and retention–time models accelerate disease marker identification.
• Therapeutic monitoring: qMSI-uD informs drug dosing by mapping unbound drug distribution.
• Microbial infection: siderophore/metallophore detection guides rapid pathogen diagnosis, noninvasive aspergillosis monitoring by urine MS.
• Clinical analytics: multiplexed SRM panels improve newborn screening, IMD diagnosis, treatment monitoring.
• Environmental surveillance: UHPLC–MS screens soils and wastewaters for pesticide and antibiotic residues impacting resistance selection.
• Structural biology: FPOP-TD and IMS-MS allow mapping of macromolecular interactions and conformational changes.

Future Trends and Possibilities

• AI-driven integration of MS imaging and pharmacokinetics to predict drug efficacy and BBB permeability.
• Single-cell MS proteomics with ultra-high sensitivity for cellular heterogeneity in organoids or tumors.
• Microfluidic-based MS sampling for point-of-care diagnostics of infections and metabolic disorders.
• Complementary fragmentation (EAD, UVPD) with Zeno trap for comprehensive lipid and protein isomer characterization.
• High-throughput bioinformatics for large-scale untargeted MS data and metabolite annotation.
• Expanded panels of metalophores as universal infection biomarkers across pathogens.

Conclusion
Recent developments in mass spectrometry—from advanced LC, CE, and MALDI workflows to high-resolution imaging and structural MS—are transforming analytical chemistry. These methods offer higher sensitivity, specificity, and spatial resolution, enabling new insights into disease biomarkers, drug delivery, infection diagnostics, and environmental monitoring. Integrating robust sample preparation, innovative instrumentation, and data analysis tools paves the way for personalized medicine, point-of-care tests, and improved quality control across industries.

Instrumention Highlight
• UHPLC–Orbitrap (Fusion, Q Exactive) and Q-TOF (timsTOF, Synapt, Vion) for HRMS.
• CE–ICP-MS and CE–ESI-MS (Agilent 7100/6460, 7700x) for trace metal complexes.
• MALDI–FTICR, MALDI–TOF (timsTOF, Rapiflex) with imaging capabilities.
• EAD on SCIEX 6600+ with Zeno trap for enhanced MS/MS duty cycle.

References
1. Gadara et al., Anal. Chem. 93, 9103 (2021).
2. Skriba et al., Front Microbiol. 9, 1 (2018).
3. Luptáková et al., Mol Psychiatry (2021).
4. Švecová et al., Microchem J. (2021).
5. Kvasnička et al., Int J Mol Sci. 22, 8054 (2021).