Callee M. Walsh

Direct analysis of drugs in forensic applications using laser ablation electrospray ionization-tandem mass spectrometry (LAESI-MS/MS)

Laser ablation electrospray ionization tandem mass spectrometry (LAESI-MS/MS) was applied to the analysis of scheduled drugs in a variety of forensically relevant media including solutions, hair and botanic matter. LAESI-MS/MS was generally able to identify unreacted drugs directly from solutions in which common presumptive color tests had been performed. A significant correlation of 0.7 was found between the pKa of the drugs and the frequency of a positive identification in the solutions indicating that basic drugs are more favorably ionized. Basic drugs like amphetamine and methamphetamine were readily identified at 0.01 mg mL−1, well below the normal limits of detection of the color test results. For hair analysis, LAESI-MS/MS could directly identify the presence of morphine, codeine and cocaine in human hair samples at biologically relevant levels of ∼10 ng mg−1 of drug in hair. This detection was possible without any hydrolysis, extraction, derivatization, or separation of the drugs. LAESI-MS/MS could also identify the presence of tetrahydrocannabinol (THC) or cannabidiol (CBD) in cannabis leaves, in addition to mapping the spatial abundance of THC/CBD across the different leaves. The simplicity and lack of sample preparation for hair and plant analyses are noteworthy benefits, but the current detection limits are close to biologically relevant levels. These preliminary studies indicate that with some additional optimization and validation, LAESI-MS/MS could provide a direct confirmation of color spot test results at an average analysis time of 20 seconds per sample, which is considerably faster than any GC or LC run and could be a major benefit for large caseloads or backlog reduction.

Laser Ablation Electrospray Ionization-High Resolution Mass Spectrometry for Regulatory Screening of Domoic Acid in Shellfish.

Rationale Domoic acid (DA) is a potent neurotoxin that accumulates in shellfish. Routine testing involves homogenization, extraction and chromatographic analysis, with a run time of up to 30 min. Improving throughput using ambient ionization for direct analysis of DA in tissue would result in significant time savings for regulatory testing labs. Methods We assess the suitability of laser ablation electrospray ionization high‐resolution mass spectrometry (LAESI‐HRMS) for high‐throughput screening or quantitation of DA in a variety of shellfish matrices. The method was first optimized for use with HRMS detection. Challenges such as tissue sub‐sampling, isobaric interferences and method calibration were considered and practical solutions developed. Samples included 189 real shellfish samples previously analyzed by regulatory labs as well as mussel matrix certified reference materials. Results Domoic acid was selectively analyzed directly from shellfish tissue homogenates with a run time of 12 s. The limits of detection were between 0.24 and 1.6 mg DA kg−1 tissue, similar to those of LC/UV methods. The precision was between 27 and 44% relative standard deviation (RSD), making the technique more suited to screening than direct quantitation. LAESI‐MS showed good agreement with LC/UV and LC/MS and was capable of identifying samples above and below 5 mg DA kg−1 wet shellfish tissue, one quarter of the regulatory limit. Conclusions These findings demonstrate the suitability of LAESI‐MS for routine, high‐throughput screening of DA. This approach could result in significant time savings for regulatory labs carrying out shellfish safety testing on thousands of samples annually.

Abstract 4793: A novel laser ablation electrospray ionization mass spectrometry (LAESI-MS) platform for biomarker discovery in cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introductory Sentence: LAESI is a novel technique for the direct and rapid interrogation of cancer cells by mass spectrometry (MS) that can be used to identify potential biomarkers in live cell populations. Experimental: Presented in this work, the human leukemic cell line, REH, was cultured under 3 different conditions to represent clinically relevant scenarios. These included media alone or co-culture with human bone marrow or osteoblast cells to represent conditions in which leukemic disease originates and environments in which the therapeutic response of tumors is known to differ. In previous work, REH cells co-cultured with stromal cells have shown increased resistance to chemotherapeutic treatments prompting our interest in evaluating differences in tumor cells exposed to bone marrow microenvironment conditions compared to those deprived of these survival cues. Subsequently, these unique signatures may identify therapeutic targets of interest. To identify potential therapeutic targets, the cells were pelleted using centrifugation, washed twice, and pipetted upon slides for direct LAESI-MS analysis. The LAESI-MS analysis was performed using a Protea DP-1000 LAESI system interfaced to a Waters Synapt G2 mass spectrometer. The mid-IR laser in the DP-1000 had a maximum output energy of 1 mJ, a 200 µm spot size, and an operational frequency of 10 Hz. The MS was continually calibrated using a lockmass of m/z 445.1206 resulting in mass differences typically less than 5 ppm. After LAESI-MS analysis, the data was analyzed using MarkerLynx software and subjected to a principle component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) for identification of potential biomarkers from each treatment. Five replicates of each cell population were analyzed for statistical significance and potential biomarkers were searched against online databases based on accurate mass and confirmed using MS/MS where possible. Summary of Data: The LAESI-MS analysis of the REH cell pellets produced several notable differences in the cell populations that could aid in the identification of biomarkers responsible for the increased resistance to chemotherapeutic treatments. The LAESI-MS analysis was very rapid, requiring less than five minutes of analysis time per cell population to get an average “fingerprint” mass spectrum in addition to MS/MS spectra to aid in metabolite verification. Conclusions: LAESI-MS is a rapid analysis technique that can be used for cancer research as applied here to cell populations. A LAESI-MS analysis requires minimal sample pretreatment with simple washing and pelleting of the cells being the only requirement for the analysis. This method allows the direct analysis of live cells, which can ultimately lead to the determination of biomarkers of disease through the comparison of different cell populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4793. doi:1538-7445.AM2012-4793

Microprobe MS Imaging of Live Tissues, Cells, and Bacterial Colonies Using LAESI

Laser ablation electrospray ionization (LAESI) is an ambient ionization technique for mass spectrometry that is capable of performing direct spatial imaging on biological specimens with minimal-to-no sample preparation, ensuring experimental conditions that can maintain viability. Mass spectrometry imaging (MSI) by LAESI has accomplished utility in mapping the spatial distribution of small molecules including metabolites and lipids in a wide variety of biological samples under ambient conditions, ranging from sectioned animal and live plant tissues to living microbial colonies and small cohorts of cells. In this chapter, we provide a brief introduction to LAESI and offer practical guidance on performing MSI using this technique. The focus here is to discuss the main steps of custom-building a LAESI setup, to perform multidimensional imaging of tissues and cells, and to demonstrate the utility of LAESI MSI under native or native-like conditions. As recent commercialization has extended this new analytical resource to a broader user base, we anticipate two- and three-dimensional MSI by LAESI to benefit basic and applied research.

Abstract 3874: Mass spectrometry imaging determines biomarkers of early adaptive precision drug resistance in lung cancer

Drug resistance emergence is a common problem that limits long term outcome benefits in the era of precision cancer therapy. Recently, we identified an early precision drug escape mechanism with adaptive tumor cellular reprogramming emerging within days after drug initiation. Here we present a mass spectrometry imaging (MSI) approach to interrogate the biomolecular changes occurring within residual tumor cells under precision treatment with an ALK-specific kinase inhibitor TAE684 in EML4-ALK fusion (ALK+) lung adenocarcinoma xenograft. ALK+ H3122 lung adenocarcinoma murine xenograft model was established for in vivo treatment with TAE684, at a daily dose of 25mg/kg by orogastric gavage (n = 6). Diluent control was included as comparison (n = 6). Tumor measurement revealed expected remarkable tumor response with TAE684. Control tumors and drug-treated residual tumor tissues were harvested for MSI studies, at day 7 and day 14 during tumor response. MSI was carried out on formalin fixed, paraffin embedded tissues to compare peptide profiles between control tumors and 7- and 14-day ALK-TKI treated tumors using a histology guided mass spectrometry approach. Briefly, two sections were collected from each sample, one for mass spectrometry and one for histology. Mass spectrometry sections were deparaffinized, antigen retrieved, and subjected to on-tissue tryptic digestion. Tumoral areas of interest (100 μm diameter, ∼20 per sample) were annotated on digital microscopy images of the stained sections. The annotated images were merged with digital images of the unstained sections using Photoshop and this combined image was used to guide data acquisition from the areas of interest. Additionally, frozen control and day 14 TAE684 treated tumors were subjected to full section MSI to determine the ALK inhibitor drug distribution as well as the changing landscape of lipids and metabolites. Statistical analysis of the peptide data resulted in determination of 580 significant peaks using Wilcoxon rank sum test with a Bonferroni correction. A genetic algorithm classification model consisting of 24 peptide peaks was generated using a leave-20%-out cross validation over 10 iterations that resulted in an overall classification accuracy across the 3 groups of over 98%. Direct MS/MS fragmentation revealed that TAE684 was detected within the frozen dosed tumors, but was absent from the control tumors. Several lipids (notably, m/z 732.78, 744.67, and 770.72 increased, m/z 769.65 and 820.71 decreased) were found to undergo alterations in expression as a result of TAE684 treatment. MSI allowed for the direct in situ determination of biomolecules that are changing in expression landscape in ALK+ lung cancer as a result of TAE684 treatment. These results provide a rationale to advance our MSI studies to deepen our insights in mechanisms of adaptive precision drug resistance to improve treatment outcomes. Citation Format: Erin H. Seeley, Pamela S. Cantrell, Callee M. Walsh, Sijin Wen, Satoshi Komo, Xiaoliang Wu, Wei Zhang, Patrick C. Ma. Mass spectrometry imaging determines biomarkers of early adaptive precision drug resistance in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3874.

CHAPTER 19:Laser-Ablation Electrospray Ionization Mass Spectrometry (LAESI®-MS): Ambient Ionization Technology for 2D and 3D Molecular Imaging

Laser ablation electrospray ionization mass spectrometry is an ambient ionization technique applicable to plant and animal tissue imaging, live-cell imaging (bacterial and fungal colonies), and most recently to cell-by-cell imaging. This ambient pressure technique uses a mid-infrared (mid-IR) laser with a wavelength (2.94 µm) that is strongly absorbed by water to ablate samples. The resultant ablation plume contains a population of neutral molecules from the sample. Ionization occurs via coalescence of the sample molecules with an electrospray plume above the sample, and the sample ions pass into a mass spectrometer for detection. This direct analysis of the tissues alleviates the need for sample preparation, such as rinse steps, the application of a surface coating or matrix, or solvent extraction, all of which adds time to the analysis and may result in sample contamination or loss. The use of the natural water content of tissue enables both 2D and 3D imaging of plant and animal tissue sections, cell colonies on agar plates, and contact lenses. This chapter discusses the advancements in LAESI-MS technology for imaging applications, and describes the Protea LAESI DP-1000 Direct Ionization System, the first integrated commercial instrument system using LAESI technology for imaging.