Brent R. Reschke

Top‐down mass spectrometry imaging of intact proteins by laser ablation ESI FT‐ICR MS

Laser ablation ESI (LAESI) is a recent development in MS imaging. It has been shown that lipids and small metabolites can be imaged in various samples such as plant material, tissue sections or bacterial colonies without any sample pretreatment. Further, LAESI has been shown to produce multiply charged protein ions from liquids or solid surfaces. This presents a means to address one of the biggest challenges in MS imaging; the identification of proteins directly from biological tissue surfaces. Such identification is hindered by the lack of multiply charged proteins in common MALDI ion sources and the difficulty of performing tandem MS on such large, singly charged ions. We present here top‐down identification of intact proteins from tissue with a LAESI ion source combined with a hybrid ion‐trap FT‐ICR mass spectrometer. The performance of the system was first tested with a standard protein with electron capture dissociation and infrared multiphoton dissociation fragmentation to prove the viability of LAESI FT‐ICR for top‐down proteomics. Finally, the imaging of a tissue section was performed, where a number of intact proteins were measured and the hemoglobin α chain was identified directly from tissue using CID and infrared multiphoton dissociation fragmentation.

Remote laser ablation electrospray ionization mass spectrometry for non‐proximate analysis of biological tissues

RATIONALE We introduce remote laser ablation electrospray ionization (LAESI), a novel, non-proximate ambient sampling technique. Remote LAESI allows additional analytical instrumentation to be incorporated during sample analysis. This work demonstrates the utility of remote LAESI and, when combined with optical microscopy, allows for the microscopy-guided sampling of biological tissues. METHODS Rapid prototyping using a 3D printer was applied to produce various ablation chamber geometries. A focused 5 ns, 2.94 µm laser pulse kept at 10 Hz ablated the sample within the chamber, remote to the mass spectrometer inlet. Ablated particulates were carried through a transfer tube by N2 gas, delivered to the electrospray plume and ionized. A long-distance microscope was used to capture images of tissues before, during and after ablation. RESULTS Optimized remote LAESI was found to have a 27% transport efficiency compared with conventional LAESI, sufficient for many applications. A comparable molecular coverage was obtained with remote LAESI for the analysis of plant tissue. Proof-of-principle experiments using a pansy flower and a maple leaf indicated the functionality of this approach for selecting domains of interest for analysis by optical microscopy and obtaining chemical information from those selected regions by remote LAESI-MS. CONCLUSIONS Remote LAESI is an ambient non-proximate sampling technique, proven to detect metabolites in biological tissues. When combined with optical microscopy, remote LAESI allows for the simultaneous acquisition of morphological and chemical information. This technique has important implications for histology, where chemical information for specific locations within a tissue is critical.

Subcellular Metabolite and Lipid Analysis of Xenopus laevis Eggs by LAESI Mass Spectrometry

Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.

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.

Analyte transport past a nanofluidic intermediate electrode junction in a microfluidic device

A glass microfluidic device is presented in which a microchannel is split into two regions with different electric fields by a nanochannel intermediate electrode junction formed by dielectric breakdown. The objective is to sink current through the nanochannel junction without sample loss or broadening of the band as it passes the junction. This type of performance is desired in many microfluidic applications, including the coupling of microchannel/CE with ESI‐MS, electrochemical detection, and electric field gradient focusing. The voltage offsets in this study are suitable for microchannel/CE‐ESI‐MS. Imaging of the transport of model anions and cations through the junction indicates that the junction exhibits nanofluidic behavior and the mean depth of the nanochannel is estimated to be ∼105 nm. The ion permselectivity of the nanochannel induces concentration polarization and enriched and depleted concentration polarization zones form on opposite sides of the nanochannel, altering the current and electric field distributions along the main microchannel. Anion transport efficiency past the junction was high, 96.0%, and varied little over the pH range of 4.0–8.0. In contrast, cation transport is much lower, and decreases from 72 to 11% from pH 4.0 to 8.0. Band broadening increases with increasing pH less than 70% over the pH range of 4.0–8.0. It is anticipated that this characterization will aid in the understanding and optimization of such junctions made from permselective membranes and porous glass.

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

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.