Untargeted (Non-Targeted) HRMS Assay
Beyond the pre-defined toxin panel: a full-spectrum, open-ended approach that measures every detectable intracellular compound — known and unknown — using data-independent acquisition HRMS and computational exposome mapping.
What Is an Untargeted Assay?
The Cell Biology Lab targeted toxin panel — like all targeted assays — works by instructing the mass spectrometer to look for a specific, pre-defined list of compounds. This approach delivers the highest sensitivity and clinical reliability for those compounds, but it has a fundamental limitation: it can only find what it is looking for.
An untargeted assay (also called a non-targeted assay, suspect screening, or exposome-wide analysis) takes the opposite approach. The instrument records every detectable ion across the full mass range in a single acquisition — a technique called full-scan data-independent acquisition (DIA). Post-acquisition software then searches this data against comprehensive spectral libraries to identify what is present, rather than confirming what was expected.
This approach is the analytical equivalent of sequencing an entire genome rather than testing for specific known mutations. It is more complex, more data-intensive, and currently less clinically standardized — but it is capable of detecting novel, emerging, and unexpected intracellular contaminants that no targeted panel would ever find.
The Terminology
Targeted vs. Untargeted: A Direct Comparison
| Aspect | Targeted Panel (Current) | Untargeted Assay (Future) |
|---|---|---|
| What is measured | A pre-defined list of specific compounds (e.g., lead, OTA, PFOS, atrazine) | Every detectable compound above the reporting threshold — known and unknown |
| Analytical approach | Selected Reaction Monitoring (SRM) or targeted MS/MS — instrument programmed for specific masses | Full-scan data-independent acquisition (DIA) or data-dependent acquisition (DDA) — entire mass range recorded |
| Sensitivity | Highest sensitivity for the specific compounds on the panel | Slightly lower per-compound sensitivity; compensated by broad coverage |
| Compound identification | Confirmed against certified reference standards (CRM) | Matched against spectral libraries (mzCloud, HMDB, EPA DSSTox, MassBank) — confidence levels I–IV |
| Novel / unexpected toxins | Cannot detect — if it is not on the panel, it is not reported | Can detect and flag novel, emerging, or unexpected compounds |
| Data complexity | Straightforward — pass/fail against reference ranges | Highly complex — thousands of features per sample; requires bioinformatics pipeline |
| Clinical readiness | Clinically validated; reference ranges established | Research-grade; clinical reference ranges not yet established for most features |
| Best use case | Routine clinical screening; monitoring known exposures; treatment response | Unexplained illness; novel exposure investigation; research; exposome mapping |
How the Untargeted PBMC-HRMS Workflow Works
The PBMC isolation step is identical to the targeted assay — the same blood draw and cell preparation supports both analyses. The key difference is in the mass spectrometer acquisition method and the downstream data processing pipeline.
PBMC Isolation
Identical to targeted assay — density gradient centrifugation, viability confirmation, protein normalization. The same PBMC lysate can be split for both targeted and untargeted analysis from a single blood draw.
Full-Scan HRMS Acquisition
Orbitrap HRMS operated in full-scan DIA mode (resolution ≥120,000). The instrument records every detectable ion across the full mass range (m/z 50–1,200) in both positive and negative ionization modes. No pre-selection of target compounds.
Feature Detection & Alignment
Raw data processed by XCMS or MZmine software. Thousands of chromatographic features (unique m/z × retention time combinations) are detected, deconvoluted, and aligned across samples. Isotope patterns and adducts are resolved.
Library Matching & Suspect Screening
Each feature is searched against curated spectral libraries: mzCloud (>650,000 spectra), HMDB (Human Metabolome Database), EPA DSSTox (>875,000 chemicals), and MassBank. Identification confidence is assigned per the Schymanski scale (Level 1–5).
Bioinformatics & Pathway Analysis
Identified compounds are mapped to toxicological pathways, endocrine disruption networks, and disease-associated signatures. Statistical comparison against reference population data flags compounds with anomalous intracellular accumulation.
Clinical Report Generation
Confirmed Level 1–2 identifications are reported with quantitative estimates. Level 3–4 suspects are flagged for follow-up targeted confirmation. Novel features of interest are archived for longitudinal tracking and research collaboration.
Spectral Libraries Used for Compound Identification
Untargeted identification confidence is graded using the Schymanski scale (Levels 1–5): Level 1 = confirmed by reference standard; Level 2 = probable structure; Level 3 = tentative candidate; Level 4 = unequivocal molecular formula; Level 5 = exact mass only. Only Levels 1–2 are reported in clinical contexts.
When Would an Untargeted Assay Be Used?
Unexplained Chronic Illness
A patient with CIRS, chronic fatigue, or multi-system inflammatory syndrome whose targeted panel returns normal results. The untargeted assay may detect novel mycotoxin metabolites, transformation products, or emerging contaminants not yet on any targeted panel.
Novel or Emerging Contaminant Exposure
Occupational exposures (semiconductor workers, firefighters, agricultural workers) where the specific chemical mixture is unknown. Full-scan DIA captures the entire intracellular chemical fingerprint without prior knowledge of the exposure.
Exposome Research
Population-level studies mapping the full environmental chemical burden in specific disease cohorts — autism, Parkinson's, breast cancer, autoimmune disease. Untargeted PBMC-HRMS can identify novel disease-associated chemical signatures for future targeted panel development.
Treatment Monitoring & Detox Assessment
Tracking the mobilization and clearance of intracellular toxins during chelation, binder therapy, or sauna protocols. Untargeted profiling can detect mobilized compounds and transformation products that targeted panels miss.
Phase 1 — Current
- ▸Targeted HRMS panel (31 toxins)
- ▸EIS intracellular electrical capacity
- ▸Cell biology damage markers
- ▸Clinical reference range development
Phase 2 — Near Term
- ▸Full-scan DIA acquisition protocol
- ▸XCMS/MZmine bioinformatics pipeline
- ▸Library integration (mzCloud, EPA DSSTox)
- ▸Suspect screening validation
Phase 3 — Future
- ▸Clinical-grade untargeted reporting
- ▸Exposome reference population database
- ▸Disease-signature library (CIRS, ASD, Parkinson's)
- ▸Combined targeted + untargeted report
Interested in Untargeted Assay Development?
We are actively seeking research collaborators, bioinformatics partners, and funding to develop the untargeted PBMC-HRMS pipeline into a clinically validated assay. If you are a researcher, clinician, or investor interested in this work, we would like to hear from you.
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