Milad Nazari

Influence of C-Trap Ion Accumulation Time on the Detectability of Analytes in IR-MALDESI MSI.

Laser desorption followed by post electrospray ionization requires synchronized timing of the key events (sample desorption/ionization, mass spectrometry analysis, and sample translation) necessary to conduct mass spectrometry imaging (MSI) with adequate analyte sensitivity. In infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI analyses, two laser pulses are used for analysis at each volumetric element, or voxel, of a biological sample and ion accumulation in the C-trap exceeding 100 ms is necessary to capture all sample-associated ions using an infrared laser with a 20 Hz repetition rate. When coupled to an Orbitrap-based mass spectrometer like the Q Exactive Plus, this time window for ion accumulation exceeds dynamically controlled trapping of samples with comparable ion flux by Automatic Gain Control (AGC), which cannot be used during MSI analysis. In this work, a next-generation IR-MALDESI source has been designed and constructed that incorporates a mid-infrared OPO laser capable of operating at 100 Hz and allows requisite C-trap inject time during MSI to be reduced to 30 ms. Analyte detectability of the next-generation IR-MALDESI integrated source has been evaluated as a function of laser repetition rate (100-20 Hz) with corresponding C-trap ion accumulation times (30-110 ms) in both untargeted and targeted analysis of biological samples. Reducing the C-trap ion accumulation time resulted in increased ion abundance by up to 3 orders of magnitude for analytes ranging from xenobiotics to endogenous lipids, and facilitated the reduction of voxel-to-voxel variability by more than 3-fold.

MSiReader v1.0: Evolving Open-Source Mass Spectrometry Imaging Software for Targeted and Untargeted Analyses

AbstractA major update to the mass spectrometry imaging (MSI) software MSiReader is presented, offering a multitude of newly added features critical to MSI analyses. MSiReader is a free, open-source, and vendor-neutral software written in the MATLAB platform and is capable of analyzing most common MSI data formats. A standalone version of the software, which does not require a MATLAB license, is also distributed. The newly incorporated data analysis features expand the utility of MSiReader beyond simple visualization of molecular distributions. The MSiQuantification tool allows researchers to calculate absolute concentrations from quantification MSI experiments exclusively through MSiReader software, significantly reducing data analysis time. An image overlay feature allows the incorporation of complementary imaging modalities to be displayed with the MSI data. A polarity filter has also been incorporated into the data loading step, allowing the facile analysis of polarity switching experiments without the need for data parsing prior to loading the data file into MSiReader. A quality assurance feature to generate a mass measurement accuracy (MMA) heatmap for an analyte of interest has also been added to allow for the investigation of MMA across the imaging experiment. Most importantly, as new features have been added performance has not degraded, in fact it has been dramatically improved. These new tools and the improvements to the performance in MSiReader v1.0 enable the MSI community to evaluate their data in greater depth and in less time. Graphical Abstractᅟ

Enhanced Lipidome Coverage in Shotgun Analyses by using Gas-Phase Fractionation.

AbstractA high resolving power shotgun lipidomics strategy using gas-phase fractionation and data-dependent acquisition (DDA) was applied toward comprehensive characterization of lipids in a hen ovarian tissue in an untargeted fashion. Using this approach, a total of 822 unique lipids across a diverse range of lipid categories and classes were identified based on their MS/MS fragmentation patterns. Classes of glycerophospholipids and glycerolipids, such as glycerophosphocholines (PC), glycerophosphoethanolamines (PE), and triglycerides (TG), are often the most abundant peaks observed in shotgun lipidomics analyses. These ions suppress the signal from low abundance ions and hinder the chances of characterizing low abundant lipids when DDA is used. These issues were circumvented by utilizing gas-phase fractionation, where DDA was performed on narrow m/z ranges instead of a broad m/z range. Employing gas-phase fractionation resulted in an increase in sensitivity by more than an order of magnitude in both positive- and negative-ion modes. Furthermore, the enhanced sensitivity increased the number of lipids identified by a factor of ≈4, and facilitated identification of low abundant lipids from classes such as cardiolipins that are often difficult to observe in untargeted shotgun analyses and require sample-specific preparation steps prior to analysis. This method serves as a resource for comprehensive profiling of lipids from many different categories and classes in an untargeted manner, as well as for targeted and quantitative analyses of individual lipids. Furthermore, this comprehensive analysis of the lipidome can serve as a species- and tissue-specific database for confident identification of other MS-based datasets, such as mass spectrometry imaging. Graphical Abstractᅟ

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI).

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Characterization of the Spectral Accuracy of an Orbitrap Mass Analyzer Using Isotope Ratio Mass Spectrometry

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source coupled to the Q Exactive Plus has been extensively used in untargeted mass spectrometry imaging (MSI) analyses of biological tissue sections. Although the Orbitrap is a high-resolution and accurate-mass (HRAM) mass analyzer, these attributes alone cannot be used for the reliable identification of unknown analytes observed in complex biological matrices. Spectral accuracy (SA) is the ability of the mass spectrometer to accurately measure the isotopic distributions which, when used with high mass measurement accuracy (MMA), can facilitate the elucidation of a single elemental composition. To investigate the effects of different ion populations on an Orbitraps SA and MMA, a solution of caffeine, the tetrapeptide MRFA, and ultramark was analyzed using a Q Exactive Plus across eight distinct automatic gain control (AGC) targets. The same compounds from the same lot numbers were also individually analyzed using isotope ratio mass spectrometry (IRMS) to accurately determine the isotopic abundance of 13C, 15N, and 34S. We demonstrated that at optimum absolute ion abundances the Orbitrap can be used to accurately count carbons, nitrogens, and sulfurs in samples with varying masses. Additionally, absolute monoisotopic ion abundances required for high SA were empirically determined by using the expected (IRMS) and experimental (Orbitrap) isotopic distributions to calculate the Pearson chi-square test. These thresholds for absolute ion abundances can be used in untargeted MSI studies to shorten an identification list by rapidly screening for isotopic distributions whose absolute ion abundances are high enough to accurately estimate the number of atoms.

Mass spectrometric detection of chlorophyll a and the tetrapyrrole secondary metabolite tolyporphin A in the filamentous cyanobacterium HT-58-2. Approaches to high-throughput screening of intact cyanobacteria

Tolyporphins are unusual tetrapyrrole macrocycles produced by the filamentous cyanobacterium–microbial community HT-58-2, the only known source to date. Numerous cyanobacterial samples have been collected worldwide but most have not been screened for secondary metabolites. Identification of tolyporphins typically has entailed lipophilic extraction followed by chromatographic fractionation and spectroscopic and/or mass spectrometric analysis. For quantitation, lengthy lipophilic extraction, sample processing and HPLC separation are needed. Examination by MALDI-TOF-MS (with the matrix 1,5-diaminonaphthalene) of lipophilic crude extracts of small-scale HT-58-2 samples (2 mL) without chromatographic fractionation enabled semi-quantitation of tolyporphin A over a 41-day growth period. Screening for tolyporphin A in intact or slightly sheared and vortexed HT-58-2 samples (no lipophilic extraction), and confirmation of identity by tandem MS, were carried out by IR-MALDESI-FTMS. Tolyporphin A was identified by th...

Direct analysis of terpenes from biological buffer systems using SESI and IR-MALDESI

Terpenes are the largest class of natural products with a wide range of applications including use as pharmaceuticals, fragrances, flavorings, and agricultural products. Terpenes are biosynthesized by the condensation of a variable number of isoprene units resulting in linear polyisoprene diphosphate units, which can then be cyclized by terpene synthases into a range of complex structures. While these cyclic structures have immense diversity and potential in different applications, their direct analysis in biological buffer systems requires intensive sample preparation steps such as salt cleanup, extraction with organic solvents, and chromatographic separations. Electrospray post-ionization can be used to circumvent many sample cleanup and desalting steps. SESI and IR-MALDESI are two examples of ionization methods that employ electrospray post-ionization at atmospheric pressure and temperature. By coupling the two techniques and doping the electrospray solvent with silver ions, olefinic terpenes of different classes and varying degrees of volatility were directly analyzed from a biological buffer system with no sample workup steps.

Direct Screening of Enzyme Activity using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

RATIONALE High-throughput screening (HTS) is a critical step in the drug discovery process. However, most mass spectrometry (MS)-based HTS methods require sample cleanup steps prior to analysis. In this work we present the utility of infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) for monitoring an enzymatic reaction directly from a biological buffer system with no sample cleanup and at high throughput. METHODS IR-MALDESI was used to directly analyze reaction mixtures from a well plate at different time points after reaction initiation. The percent conversion of precursors to products was used to screen the enzyme activity. The reaction was performed with two different concentrations of precursors and enzyme in order to assess the dynamic range of the assay. Eventually, a pseudo-HTS study was designed to investigate the utility of IR-MALDESI screening enzyme activity in a high-throughput manner. RESULTS IR-MALDESI was able to readily monitor the activity of IDH1 over time at two different concentrations of precursors and enzyme. The calculated Z-factors of 0.65 and 0.41 confirmed the suitability of the developed method for screening enzyme activity in HTS manner. Finally, in a single-blind pseudo-HTS analysis IR-MALDESI was able to correctly predict the identity of all samples, where 8/10 samples were identified with high confidence and the other two samples with lower confidence. CONCLUSIONS The enzymatic activity of IDH1 was screened by directly analyzing the reaction content from the buffer in well plates with no sample cleanup steps. This proof-of-concept study demonstrates the robustness of IR-MALDESI for direct analysis of enzymatic reactions from biological buffers with no sample cleanup and its immense potential for HTS applications.

IR-MALDESI Mass Spectrometry Imaging at 50 Micron Spatial Resolution

AbstractHigh spatial resolution in mass spectrometry imaging (MSI) is crucial to understanding the biology dictated by molecular distributions in complex tissue systems. Here, we present MSI using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) at 50 μm resolution. An adjustable iris, beam expander, and an aspherical focusing lens were used to reduce tissue ablation diameters for MSI at high resolution. The laser beam caustic was modeled using laser ablation paper to calculate relevant laser beam characteristics. The minimum laser spot diameter on the tissue was determined using tissue staining and microscopy. Finally, the newly constructed optical system was used to image hen ovarian tissue with and without oversampling, detailing tissue features at 50 μm resolution. Graphical Abstractᅟ

MALDESI: Fundamentals, Direct Analysis, and MS Imaging

Mass spectrometry (MS) has become one of the most important tools in analytical and bioanalytical fields. One of the areas of research in MS is the development of ambient ionization methods that allow for the analysis of samples with little to no sample preparation. Matrix-assisted laser desorption electrospray ionization (MALDESI) is a hybrid ionization method combining attributes from matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). The vast experimental space governing the MALDESI process has been investigated thoroughly, while its applications in direct analysis of biomolecules were explored.