Lipidomic-Based Approach to 10 s Classification of Major Pediatric Brain Cancer Types with Picosecond Infrared Laser Mass Spectrometry.

Picosecond infrared laser mass spectrometry (PIRL-MS) is shown, through a retrospective patient tissue study, to differentiate medulloblastoma cancers from pilocytic astrocytoma and two molecular subtypes of ependymoma (PF-EPN-A, ST-EPN-RELA) using laser-extracted lipids profiled with PIRL-MS in 10 s of sampling and analysis time. The average sensitivity and specificity values for this classification, taking genomic profiling data as standard, were 96.41 and 99.54%, and this classification used many molecular features resolvable in 10 s PIRL-MS spectra. Data analysis and liquid chromatography coupled with tandem high-resolution mass spectrometry (LC-MS/MS) further allowed us to reduce the molecular feature list to only 18 metabolic lipid markers most strongly involved in this classification. The identified 'metabolite array' was comprised of a variety of phosphatidic and fatty acids, ceramides, and phosphatidylcholine/ethanolamine and could mediate the above-mentioned classification with average sensitivity and specificity values of 94.39 and 98.78%, respectively, at a 95% confidence in prediction probability threshold. Therefore, a rapid and accurate pathology classification of select pediatric brain cancer types from 10 s PIRL-MS analysis using known metabolic biomarkers can now be available to the neurosurgeon. Based on retrospective mining of 'survival' versus 'extent-of-resection' data, we further identified pediatric cancer types that may benefit from actionable 10 s PIRL-MS pathology feedback. In such cases, aggressiveness of the surgical resection can be optimized in a manner that is expected to benefit the patient's overall or progression-free survival. PIRL-MS is a promising tool to drive such personalized decision-making in the operating theater.

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.

Picosecond Infrared Laser Mass Spectrometry Identifies a Metabolite Array for 10 s Diagnosis of Select Skin Cancer Types: A Proof-of-Concept Feasibility Study.

Currently, a large number of skin biopsies are taken for each true skin cancer case detected, creating a need for a rapid, high sensitivity, and specificity skin cancer detection tool to reduce the number of unnecessary biopsies taken from benign tissue. Picosecond infrared laser mass spectrometry (PIRL-MS) using a hand-held sampling probe is reported to detect and classify melanoma, squamous cell carcinoma, and normal skin with average sensitivity and specificity values of 86-95% and 91-98%, respectively (at a 95% confidence level) solely requiring 10 s or less of total data collection and analysis time. Classifications are not adversely affected by specimen's quantity of melanin pigments and are mediated by a number of metabolic lipids, further identified herein as potential biomarkers for skin cancer-type differentiation, 19 of which were sufficient here (as a fully characterized metabolite array) to provide high specificity and sensitivity classification of skin cancer types. In situ detection was demonstrated in an intradermal melanoma mouse model wherein in vivo sampling did not cause significant discomfort. PIRL-MS sampling is further shown to be compatible with downstream gross histopathologic evaluations despite loss of tissue from the immediate laser sampling site(s) and can be configured using selective laser pulses to avoid thermal damage to normal skin. Therefore, PIRL-MS may be employed as a decision-support tool to reduce both the subjectivity of clinical diagnosis and the number of unnecessary biopsies currently required for skin cancer screening.

Mass Spectrometry Imaging Reveals a Gradient of Cancer-like Metabolic States in the Vicinity of Cancer Not Seen in Morphometric Margins from Microscopy.

Spatially resolved ambient mass spectrometry imaging methods have gained popularity to characterize cancer sites and their borders using molecular changes in the lipidome. This utility, however, is predicated on metabolic homogeneity at the border, which would create a sharp molecular transition at the morphometric borders. We subjected murine models of human medulloblastoma brain cancer to mass spectrometry imaging, a technique that provides a direct readout of tissue molecular content in a spatially resolved manner. We discovered a distance-dependent gradient of cancer-like lipid molecule profiles in the brain tissue within 1.2 mm of the cancer border, suggesting that a cancer-like state progresses beyond the histologic border, into the healthy tissue. The results were further corroborated using orthogonal liquid chromatography and mass spectrometry (LC-MS) analysis of selected tissue regions subjected to laser capture microdissection. LC-MS/MS analysis for robust identification of the affected molecules implied changes in a number of different lipid classes, some of which are metabolized from the essential docosahexaenoic fatty acid (DHA) present in the interstitial fluid. Metabolic molecular borders are thus not as sharp as morphometric borders, and mass spectrometry imaging can reveal molecular nuances not observed with microscopy. Caution must be exercised in interpreting multimodal imaging results stipulated on a coincidental relationship between metabolic and morphometric borders of cancer, at least within animal models used in preclinical research.

Potential impact of tissue molecular heterogeneity on ambient mass spectrometry profiles: a note of caution in choosing the right disease model.

This review provides a summary of known molecular alterations in commonly used cancer models and strives to stipulate how they may affect ambient mass spectrometry profiles. Immortalized cell lines are known to accumulate mutations, and xenografts derived from cell lines are known to contain tumour microenvironment elements from the host animal. While the use of human specimens for mass spectrometry profiling studies is highly encouraged, patient-derived xenografts with low passage numbers could provide an alternative means of amplifying material for ambient MS research when needed. Similarly, genetic preservation of patient tissue seen in some organoid models, further verified by qualitative proteomic and transcriptomic analyses, may argue in favor of organoid suitability for certain ambient profiling studies. However, to choose the appropriate model, pre-evaluation of the model's molecular characteristics in the context of the research question(s) being asked will likely provide the most appropriate strategy to move research forward. This can be achieved by performing comparative ambient MS analysis of the disease model of choice against a small amount of patient tissue to verify concordance. Disease models, however, will continue to be useful tools to orthogonally validate metabolic states of patient tissues through controlled genetic alterations that are not possible with patient specimens.

Dual Laser and Desorption Electrospray Ionization Mass Spectrometry Imaging Using the Same Interface.

For a more comprehensive characterization of molecular heterogeneities of matter, multimodal mass spectrometry imaging must be developed to take advantage of the complementarity of information available through different ionization mechanisms. We report the design, implementation, and performance validation of a laser desorption imaging interface composed of add-on components that adapt a commercial Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging interface for dual imaging of Picosecond Infrared Laser Mass Spectrometry (PIRL-MS) with DESI-MS. The interface utilizes hardware elements and data analysis pipelines already established for DESI-MS imaging, and was further validated in cancer margin assessments using human medulloblastoma cancers. The PIRL-MS images were robust and reproducible across multiple experimental runs on independently prepared xenograft tumors, and could be segmented into cancer and healthy regions in concordance with pathology using a variety of supervised and unsupervised clustering methods. The spectral quality and complexity obtained with this interface were examined with infiltrating and noninfiltrating tumors, and were comparable to other mass spectrometry analysis interfaces. The average PIRL-MS spectra from spatially resolved images could be used for robust cancer m/z model building to classify medulloblastoma cancer from healthy tissue without any misclassifications, an observation that held true over close to 70 sampling data points. While the unsupervised spectral analysis methods suggested a slight suppression of signal in the phospholipid range compared to the hand-held configuration, these changes were insufficient to hamper utility in cancer margin assessment with spatially resolved data obtained with our interface. Dual PIRL-MS and DESI-MS imaging of consecutive sections, as suggested by multivariate loading plots, revealed highly complementary molecular information with m/z values identifiable with one desorption method sufficient to reveal cancer regions being absent in another, further emphasizing the need for effective hardware and software interfaces for dual mass spectrometry imaging.

Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells.

Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function.

Platforms for rapid cancer characterization by ambient mass spectrometry: advancements, challenges and opportunities for improvement towards intrasurgical use.

Ambient Mass Spectrometry (MS) analysis is widely used to characterize biological and non-biological samples. Advancements that allow rapid analysis of samples by ambient methods such as Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) and Rapid Evaporative Ionization Mass Spectrometry (REIMS) are discussed. A short, non-comprehensive overview of ambient MS is provided that only contains example applications due to space limitations. A spatially encoded mass spectrometry analysis concept to plan cancer resection is introduced. The application of minimally destructive tissue ablation probes to survey the surgical field for sites of pathology using on-line analysis methods is discussed. The technological challenges that must be overcome for ambient MS to become a robust method for intrasurgical pathology assessments are reviewed.

Rapid determination of the tumour stroma ratio in squamous cell carcinomas with desorption electrospray ionization mass spectrometry (DESI-MS): a proof-of-concept demonstration.

Squamous cell carcinomas constitute a major class of head & neck cancers, where the tumour stroma ratio (TSR) carries prognostic information. Patients affected by stroma-rich tumours exhibit a poor prognosis and a higher chance of relapse. As such, there is a need for a technology platform that allows rapid determination of the tumour stroma ratio. In this work, we provide a proof-of-principle demonstration that Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) can be used to determine tumour stroma ratios. Slices from three independent mouse xenograft tumours from the human FaDu cell line were subjected to DESI-MS imaging, staining and detailed analysis using digital pathology methods. Using multivariate statistical methods we compared the MS profiles with those of isolated stromal cells. We found that m/z 773.53 [PG(18:1)(18:1) - H]-, m/z 835.53 [PI(34:1) - H]- and m/z 863.56 [PI(18:1)(18:0) - H]- are biomarker ions that can distinguish FaDu cancer from cancer associated fibroblast (CAF) cells. A comparison with DESI-MS analysis of controlled mixtures of the CAF and FaDu cells showed that the abundance of the biomarker ions above can be used to determine, with an error margin of close to 5% compared with quantitative pathology estimates, TSR values. This proof-of-principle demonstration is encouraging and must be further validated using human samples and a larger sample base. At maturity, DESI-MS thus may become a stand-alone molecular pathology tool providing an alternative rapid cancer assessment without the need for time-consuming staining and microscopy methods, potentially further conserving human resources.

Correction: Rapid determination of the tumour stroma ratio in squamous cell carcinomas with desorption electrospray ionization mass spectrometry (DESI-MS): a proof-of-concept demonstration.

Correction for 'Rapid determination of the tumour stroma ratio in squamous cell carcinomas with desorption electrospray ionization mass spectrometry (DESI-MS): a proof-of-concept demonstration' by Michael Woolman et al., Analyst, 2017, 142, 3250-3260.

Variations in the Abundance of Lipid Biomarker Ions in Mass Spectrometry Images Correlate to Tissue Density.

While mass spectrometry (MS) imaging is widely used to investigate the molecular composition of ex vivo slices of cancerous tumors, little is known about how variations in the cellular properties of cancer tissue can influence cancer biomarker ion images. To better understand the basis for variations in the abundances of cancer biomarker ions seen in MS images of relatively homogeneous ex vivo tumor samples, sections of snap frozen human breast cancer murine xenografts were subjected to desorption electrospray ionization mass spectrometry (DESI-MS) imaging. Serial sections were then stained with hematoxylin and eosin (H&E) and subjected to detailed morphometric cellular analysis, using a commercial digital pathology platform augmented with custom-tailored image analysis algorithms developed in-house. Gross morphological heterogeneities due to stroma, vasculature, and noncancer cells were mapped in the tumor and found to not correlate with the areas of suppressed cancer biomarker abundance. Instead, the ion abundances of major breast cancer biomarkers were found to correlate with the cytoplasmic area of cancer cells that comprised the tumor tissue. Therefore, detailed cellular analyses can be used to rationalize subtle heterogeneities in ion abundance in MS images, not explained by the presence of gross morphological heterogeneities such as stroma.

Contrast Agent Mass Spectrometry Imaging Reveals Tumor Heterogeneity.

Mapping intratumoral heterogeneity such as vasculature and margins is important during intraoperative applications. Desorption electrospray ionization mass spectrometry (DESI-MS) has demonstrated potential for intraoperative tumor imaging using validated MS profiles. The clinical translation of DESI-MS into a universal label-free imaging technique thus requires access to MS profiles characteristic to tumors and healthy tissues. Here, we developed contrast agent mass spectrometry imaging (CA-MSI) that utilizes a magnetic resonance imaging (MRI) contrast agent targeted to disease sites, as a label, to reveal tumor heterogeneity in the absence of known MS profiles. Human breast cancer tumors grown in mice were subjected to CA-MSI using Gadoteridol revealing tumor margins and vasculature from the localization of [Gadoteridol+K](+) and [Gadoteridol+Na](+) adducts, respectively. The localization of the [Gadoteridol+K](+) adduct as revealed through DESI-MS complements the in vivo MRI results. DESI-MS imaging is therefore possible for tumors for which no characteristic MS profiles are established. Further DESI-MS imaging of the flux of the contrast agent through mouse kidneys was performed indicating secretion of the intact label.

Gas-Phase FRET Efficiency Measurements To Probe the Conformation of Mass-Selected Proteins.

Electrospray ionization and mass spectrometry have revolutionized the chemical analysis of biological molecules, including proteins. However, the correspondence between a protein's native structure and its structure in the mass spectrometer (where it is gaseous) remains unclear. Here, we show that fluorescence (Förster) resonance energy transfer (FRET) measurements combined with mass spectrometry provides intramolecular distance constraints in gaseous, ionized proteins. Using an experimental setup which combines trapping mass spectrometry and laser-induced fluorescence spectroscopy, the structure of a fluorescently labeled mutant variant of the protein GB1 was probed as a function of charge state. Steady-state fluorescence emission spectra and time-resolved donor fluorescence measurements of mass-selected GB1 show a marked decrease in the FRET efficiency with increasing number of charges on the gaseous protein, which suggests a Coulombically driven unfolding and expansion of its structure. This lies in stark contrast to the pH stability of GB1 in solution. Comparison with solution-phase single-molecule FRET measurements show lower FRET efficiency for all charge states of the gaseous protein examined, indicating that the ensemble of conformations present in the gas phase is, on average, more expanded than the native form. These results represent the first FRET measurements on a mass-selected protein and illustrate the utility of FRET for obtaining a new kind of structural information for large, desolvated biomolecules.

Ambient Mass Spectrometry Imaging with Picosecond Infrared Laser Ablation Electrospray Ionization (PIR-LAESI).

A picosecond infrared laser (PIRL) is capable of cutting through biological tissues in the absence of significant thermal damage. As such, PIRL is a standalone surgical scalpel with the added bonus of minimal postoperative scar tissue formation. In this work, a tandem of PIRL ablation with electrospray ionization (PIR-LAESI) mass spectrometry is demonstrated and characterized for tissue molecular imaging, with a limit of detection in the range of 100 nM for reserpine or better than 5 nM for verapamil in aqueous solution. We characterized PIRL crater size using agar films containing Rhodamine. PIR-LAESI offers a 20-30 µm vertical resolution (∼3 µm removal per pulse) and a lateral resolution of ∼100 µm. We were able to detect 25 fmol of Rhodamine in agar ablation experiments. PIR-LAESI was used to map the distribution of endogenous methoxykaempferol glucoronide in zebra plant (Aphelandra squarrosa) leaves producing a localization map that is corroborated by the literature. PIR-LAESI was further used to image the distribution inside mouse kidneys of gadoteridol, an exogenous magnetic resonance contrast agent intravenously injected. Parallel mass spectrometry imaging (MSI) using desorption electrospray ionization (DESI) and matrix assisted laser desorption ionization (MALDI) were performed to corroborate PIR-LAESI images of the exogenous agent. We further show that PIR-LAESI is capable of desorption ionization of proteins as well as phospholipids. This comparative study illustrates that PIR-LAESI is an ion source for ambient mass spectrometry applications. As such, a future PIRL scalpel combined with secondary ionization such as ESI and mass spectrometry has the potential to provide molecular feedback to guide PIRL surgery.

A residue in helical conformation in the native state adopts a ß-strand conformation in the folding transition state despite its high and canonical Φ-value.

Delineating structures of the transition states in protein folding reactions has provided great insight into the mechanisms by which proteins fold. The most common method for obtaining this information is Φ-value analysis, which is carried out by measuring the changes in the folding and unfolding rates caused by single amino acid substitutions at various positions within a given protein. Canonical Φ-values range between 0 and 1, and residues displaying high values within this range are interpreted to be important in stabilizing the transition state structure, and to elicit this stabilization through native-like interactions. Although very successful in defining the general features of transition state structures, Φ-value analysis can be confounded when non-native interactions stabilize this state. In addition, direct information on backbone conformation within the transition state is not provided. In the work described here, we have investigated structure formation at a conserved ß-bulge (with helical conformation) in the Fyn SH3 domain by characterizing the effects of substituting all natural amino acids at one position within this structural motif. By comparing the effects on folding rates of these substitutions with database-derived local structure propensity values, we have determined that this position adopts a non-native backbone conformation in the folding transition state. This result is surprising because this position displays a high and canonical Φ-value of 0.7. This work emphasizes the potential role of non-native conformations in folding pathways and demonstrates that even positions displaying high and canonical Φ-values may, nevertheless, adopt a non-native conformation in the transition state.

Kinetic consequences of native state optimization of surface-exposed electrostatic interactions in the Fyn SH3 domain.

Optimization of surface exposed charge-charge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface charge-charge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of charge-charge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture.

Crystallography on a chip.

A new chip-based crystal-mounting approach for rapid room-temperature data collection from numerous crystals is described. This work was motivated by the recent development of X-ray free-electron lasers. These novel sources deliver very intense femtosecond X-ray pulses that promise to yield high-resolution diffraction data of nanocrystals before their destruction by radiation damage. Thus, the concept of `diffraction before destruction' requires rapid replenishment of the sample for each exposure. The chip promotes the self-assembly of an array of protein crystals on a surface. Rough features on the surface cause the crystals to adopt random orientations, allowing efficient sampling of reciprocal space.

Self-localizing stabilized mega-pixel picoliter arrays with size-exclusion sorting capabilities.

We report on a liquid self-localizing process capable of producing Mega-pixel arrays of picoliter volumes on a 1 cm(2) area, within seconds, for high throughput sampling. The chip is based on principles of spatially varying wetting and stabilization. The key is to develop differential surface contact regions, which lead to both localization of the solution and increasing the surface adsorption energy to further pin the liquid to the surface, as highlighted by other studies. By exploiting surface roughness for enhanced wettability, we demonstrate wetting of wells with the aspect ratio of 100. The high precision of reactive ion etching (RIE) of silicon substrates allows for an extremely reproducible method of preparing the array of identical well structures and increased contact area to increase surface adsorption in the wells. "Dynamic wetting" is then readily achieved through inducing contact line instability by simply moving a drop of liquid on the top surface of the array. Liquid samples self-localize into the array pattern with the associated liquid flow leading to self-localization of suspended particles or analyte. The resulting picoliter volumes are both spatially ordered and stable for long periods of time, even for such small volumes, to permit selective measurements of the contents. This development will be particularly important in the assembly of the massive amounts of crystalline particles needed for atomically resolved structural dynamics using the latest advances in high number density electron and X-ray sources.

Theoretical and experimental demonstration of the importance of specific nonnative interactions in protein folding.

Many experimental and theoretical studies have suggested a significant role for nonnative interactions in protein folding pathways, but the energetic contributions of these interactions are not well understood. We have addressed the energetics and the position specificity of nonnative hydrophobic interactions by developing a continuum coarse-grained chain model with a native-centric potential augmented by sequence-dependent hydrophobic interactions. By modeling the effect of different hydrophobicity values at various positions in the Fyn SH3 domain, we predicted energetically significant nonnative interactions that led to acceleration or deceleration of the folding rate depending on whether they were more populated in the transition state or unfolded state. These nonnative contacts were centered on position 53 in the Fyn SH3 domain, which lies in an exposed position in a 3(10)-helix. The energetic importance of the predicted nonnative interactions was confirmed experimentally by folding kinetics studies combined with double mutant thermodynamic cycles. By attaining agreement of theoretical and experimental investigations, this study provides a compelling demonstration that specific nonnative interactions can significantly influence folding energetics. Moreover, we show that a coarse-grained model with a simple consideration of hydrophobicity is sufficient for the accurate prediction of kinetically important nonnative interactions.