A Workflow for Meaningful Interpretation of Classification Results from Handheld Ambient Mass Spectrometry Analysis Probes.

While untargeted analysis of biological tissues with ambient mass spectrometry analysis probes has been widely reported in the literature, there are currently no guidelines to standardize the workflows for the experimental design, creation, and validation of molecular models that are utilized in these methods to perform class predictions. By drawing parallels with hurdles that are faced in the field of food fraud detection with untargeted mass spectrometry, we provide a stepwise workflow for the creation, refinement, evaluation, and assessment of the robustness of molecular models, aimed at meaningful interpretation of mass spectrometry-based tissue classification results. We propose strategies to obtain a sufficient number of samples for the creation of molecular models and discuss the potential overfitting of data, emphasizing both the need for model validation using an independent cohort of test samples, as well as the use of a fully characterized feature-based approach that verifies the biological relevance of the features that are used to avoid false discoveries. We additionally highlight the need to treat molecular models as "dynamic" and "living" entities and to further refine them as new knowledge concerning disease pathways and classifier feature noise becomes apparent in large(r) population studies. Where appropriate, we have provided a discussion of the challenges that we faced in our development of a 10 s cancer classification method using picosecond infrared laser mass spectrometry (PIRL-MS) to facilitate clinical decision-making at the bedside.

Metabolic Lipids in Melanoma Enable Rapid Determination of Actionable BRAF-V600E Mutation with Picosecond Infrared Laser Mass Spectrometry in 10 s.

Rapid molecular profiling of biological tissues with picosecond infrared laser mass spectrometry (PIRL-MS) has enabled the detection of clinically important histologic types and molecular subtypes of human cancers in as little as 10 s of data collection and analysis time. Utilizing an engineered cell line model of actionable BRAF-V600E mutation, we observed statistically significant differences in 10 s PIRL-MS molecular profiles between BRAF-V600E and BRAF-wt cells. Multivariate statistical analyses revealed a list of mass-to-charge (m/z) values most significantly responsible for the identification of BRAF-V600E mutation status in this engineered cell line that provided a highly controlled testbed for this observation. These metabolites predicted BRAF-V600E expression in human melanoma cell lines with greater than 98% accuracy. Through chromatography and tandem mass spectrometry analysis of cell line extracts, a 30-member "metabolite array" was characterized for determination of BRAF-V600E expression levels in subcutaneous melanoma xenografts with an average sensitivity and specificity of 95.6% with 10 s PIRL-MS analysis. This proof-of-principle work warrants a future large-scale study to identify a metabolite array for 10 s determination of actionable BRAF-V600E mutation in human tissue to guide patient care.

Reflection mode polarimetry guides laser mass spectrometry to diagnostically important regions of human breast cancer tissue.

To enhance the clinical utility of mass spectrometry (MS), lengthy dwell times on less informative regions of patient specimens (e.g., adipose tissue in breast) must be minimized. Additionally, a promising variant of MS known as picosecond infrared laser MS (PIRL-MS) faces further challenges, namely, lipid contamination when probing adipose tissue. Here we demonstrate on several thick non-sectioned resected human breast specimens (healthy and malignant) that reflection-mode polarimetric imaging can robustly guide PIRL-MS toward regions devoid of significant fat content to (1) avoid signal contamination and (2) shorten overall MS analysis times. Through polarimetric targeting of non-fat regions, PIRL-MS sampling revealed feature-rich spectral signatures including several known breast cancer markers. Polarimetric guidance mapping was enabled by circular degree-of-polarization (DOP) imaging via both Stokes and Mueller matrix polarimetry. These results suggest a potential synergistic hybrid approach employing polarimetry as a wide-field-imaging guidance tool to optimize efficient probing of tissue molecular content using MS.