Chapter Four · failure evidence
What Mass Spectrometry Imaging got wrong, from 23 dissertations
Mass spectrometry imaging workflows frequently encounter technical bottlenecks across sample preparation, analyte ionization, and downstream computational analysis. Physical degradation of tissue, in-source fragmentation of fragile functional groups, severe ion suppression, and software compatibility constraints consistently impede reliable spatial profiling. These records come from PhD theses at 10 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Tissue preparation, embedding, and matrix application methods introduce artifacts or degrade spectral quality
Non-aromatic deparaffinization solvents and cryoprotectant additives in embedding matrices caused tissue section degradation, altered adduct profiles, and signal loss. Matrix sublimation under high vacuum led to premature matrix loss, matrix spraying dispersed stain dyes across membranes, and matrix deposition introduced adduct formation and throughput bottlenecks.
Tried and failed
applying MALDI matrices for laser desorption mass spectrometry applied to ambient laser desorption mass spectrometry. Outcome: worse than baseline. Reason: caused severe chemical selectivity, abundant matrix adduct formation, and poor reproducibility
Towards single cell analysis using ambient mass spectrometry techniques · Imperial
Tried and failed
matrix sublimation for MALDI mass spectrometry imaging applied to tissue sections under high vacuum. Outcome: unstable. Reason: sample chamber heating under high vacuum caused premature matrix loss off slides via reverse sublimation
Tried and failed
cryoprotectant additives in tissue embedding matrix applied to desorption electrospray ionization mass spectrometry. Outcome: worse than baseline. Reason: caused lower signal intensity, altered adduct profiles, and tissue ablation under electrospray
Considered and rejected
Considered and rejected: Rejected MALDI-based mass spectrometry imaging (MSI) as a primary screening method for large candidate probiotic libraries due to multi-hour sample prep per slide, matrix optimization challenges, and low throughput.
Metabolomic Analysis of Chemical Interactions between Burkholderia thailandensis and Host to Inform Alternative Treatments · Georgia Tech
Considered and rejected
Considered and rejected: Rejected using neutral red cell staining for AP-MALDI-MS imaging because matrix spraying dispersed the dye across the insert membrane and obscured metabolic signals.
Investigating anti-tumour effects and associated metabolomic impacts of repurposed therapeutics for glioblastoma · University of Nottingham Repository
Tried and failed
non-aromatic solvent deparaffinization applied to FFPE tissue mass spectrometry imaging. Outcome: worse than baseline. Reason: caused tissue section degradation and yielded inferior spectral information compared to xylene
On-tissue ion suppression and low spatial precursor abundance hinder analyte and standard detection
Adding or spotting internal standards onto tissue sections caused severe ion suppression of endogenous species or resulted in suppression of the standards themselves. Complex tissue matrices also generated severe suppression during ambient desorption ionization, while low precursor abundance and signal instability prevented direct on-tissue tandem mass spectrometry.
Tried and failed
spiking internal standards into biological matrix applied to mass spectrometry imaging quality control. Reason: added isotope-labeled compound caused ion suppression of endogenous metabolites and lipids
Tried and failed
on-tissue internal standard deposition for mass spectrometry imaging applied to metabolite detection in tissue sections. Outcome: no signal. Reason: matrix-assisted ionization suffered severe on-tissue ion suppression of the spotted metabolite standard
Tried and failed
flow focusing ambient desorption ionization mass spectrometry applied to drug detection in biological tissue. Outcome: no signal. Reason: severe ion suppression and low ionization sensitivity in tissue matrices
Instrumental development of DESI imaging MS and its applications · Imperial
Tried and failed
direct on-tissue tandem mass spectrometry applied to spatial tissue sections. Outcome: no signal. Reason: unstable signal and low spatial abundance of precursor ions on tissue
Software and computational pipelines face acquisition constraints and limited analytical resolution
Meandering acquisition raster patterns were rejected because downstream imaging software required coordinate coordination with fly-back lines. Meanwhile, spatial segmentation and pathway enrichment were hindered by low isotope signal intensities and sparse metabolite annotations, and cross-validation accuracy suffered from small cohort sizes.
Tried and failed
semi-supervised clustering spatial segmentation applied to mass spectrometry imaging data. Outcome: data insufficient. Reason: low signal intensities from isotope labeling prevented detailed feature resolution across segments
Advanced analyses for mass spectrometry: From atomic to molecular · Iowa State
Considered and rejected
Considered and rejected: Rejected meandering scanning patterns for DESI FT-ICR MSI because MALDI FlexImaging sequence processing required fly-back line coordination.
Method Development and Applications: Exploration of Biological Specimens Through Both Ambient and Direct Forms of Fourier Transform Mass Spectrometry · DSpace at SUNY Buffalo
Tried and failed
mass spectrometry imaging classification with patient-level cross-validation applied to tissue spatial lipidomics profiles. Outcome: did not generalise. Reason: small sample size led to poor clustering and reduced predictive accuracy under cross-validation
A next-generation -omics analysis of the radiotherapy response in rectal cancer · Imperial
Tried and failed
metabolite pathway enrichment analysis applied to mass spectrometry imaging data. Outcome: no signal. Reason: low annotation coverage of small polar metabolites precluded statistical pathway significance
Machine Learning Enabled Stem Cell Lipidomics · Georgia Tech
In-source fragmentation and low ionization efficiency limit spatial detection of fragile or intact molecules
Nucleotide di- and triphosphates suffered in-source phosphate loss, while glycosaminoglycan sulfate substituents readily dissociated without stabilization during ionization. Furthermore, intact protein imaging exhibited poor molecular coverage because large intact proteins had low ionization efficiency compared to digested peptide workflows.
Tried and failed
DESI mass spectrometry imaging applied to nucleotide di- and triphosphates detection. Outcome: no signal. Reason: in-source phosphate loss during ionization and acquisition
Considered and rejected
Considered and rejected: Rejected MALDI mass spectrometry imaging for glycosaminoglycan analysis because fragile sulfate substituents dissociate without stabilization.
Considered and rejected
Considered and rejected: Decided against utilizing intact protein imaging alone for endometriosis tissue profiling due to poor molecular coverage from low ionization efficiency of large intact proteins, replacing it with sequential N-glycan and on-tissue tryptic peptide imaging.
Left open by the authors
Problems the authors named and did not get to.
Left open
Develop and validate a mass spectrometry acquisition workflow combining SureQuant trigger peptides with isobaric multiplex labeling for sample multiplexing. Blocker: Requires wet lab sample preparation, isobaric mass tags (TMTpro), and a high-resolution mass spectrometer (Orbitrap Exploris 480).
Left open
Refine mass spectrometry quantification methods and evaluate lectin microarrays for high-throughput glycome profiling. Blocker: Requires mass spectrometry instruments, wet-lab facilities, and clinical samples
Mass spectrometric investigation into glycomes implicated in human reproductive biology · Imperial
Left open
Compare the quantification of monoclonal proteins using the Hevylite immunoassay against liquid chromatography-tandem mass spectrometry (LC-MS/MS). Blocker: Requires patient serum samples and wet lab LC-MS/MS equipment.
Left open
Perform large-scale glycopeptide enrichment using DVA magnetic beads and systematically compare performance against standard enrichment methods using mass spectrometry. Blocker: Requires a wet lab, synthesis of DVA magnetic beads, biological samples, and mass spectrometry instrumentation.
Developing novel mass spectrometry-based approaches to globally investigate protein glycosylation · Georgia Tech
Left open
Validate DESI-MSI lipid findings using positive ionisation mode UPLC-MS on cellular extracts. Blocker: Requires wet lab facilities, tissue samples/cellular extracts, and mass spectrometry instruments
A next-generation -omics analysis of the radiotherapy response in rectal cancer · Imperial
Left open
Apply ion accumulation methods such as CASI and SIM-stitching to enhance relative isotopic abundance accuracy in DI-FT-ICR mass spectrometry. Blocker: Requires direct access to a high-field FT-ICR mass spectrometer and sample introduction apparatus.
Left open
Perform MS/MS tandem mass spectrometry experiments on selected tissue features against known chemical standards to confirm molecular identifications. Blocker: Requires a mass spectrometer, tissue samples, and physical chemical reference standards in a wet lab.
Molecular Diagnosis of Glial Cell Brain Cancers Using Handheld Mass Spectrometry Device · UT Austin
Left open
Quantify relative levels of free nucleotides in Alzheimer's disease patient and control samples using liquid chromatography-mass spectrometry. Blocker: Requires wet lab facilities, patient tissue/blood samples, and mass spectrometry apparatus.
Mitochondrial One-Carbon Metabolism and Alzheimer's Disease. · Cambridge
Left open
Perform MS/MS fragmentation to confirm putative DESI-MSI and OrbiSIMS ion annotations and resolve isobaric species in lung metastatic tissue. Blocker: Requires mass spectrometry hardware (MS/MS instrumentation, DESI-MSI, OrbiSIMS) and physical tissue samples.
Left open
Perform proteomic quantification comparing One Pot PURE and commercial PURE systems to optimize translation factor ratios. Blocker: Requires wet lab access, mass spectrometry instrumentation, and cell-free protein synthesis system samples
Checking a claim in this area?
We can run the same search on any method or claim. If nothing turns up, we will say so, and that proves nothing on its own.