Integration of Ion Mobility MSE after Fully Automated, Online, High-Resolution Liquid Extraction Surface Analysis Micro-Liquid Chromatography.

Direct analysis by mass spectrometry (imaging) has become increasingly deployed in preclinical and clinical research due to its rapid and accurate readouts. However, when it comes to biomarker discovery or histopathological diagnostics, more sensitive and in-depth profiling from localized areas is required. We developed a comprehensive, fully automated online platform for high-resolution liquid extraction surface analysis (HR-LESA) followed by micro-liquid chromatography (LC) separation and a data-independent acquisition strategy for untargeted and low abundant analyte identification directly from tissue sections. Applied to tissue sections of rat pituitary, the platform demonstrated improved spatial resolution, allowing sample areas as small as 400 µm to be studied, a major advantage over conventional LESA. The platform integrates an online buffer exchange and washing step for removal of salts and other endogenous contamination that originates from local tissue extraction. Our carry over-free platform showed high reproducibility, with an interextraction variability below 30%. Another strength of the platform is the additional selectivity provided by a postsampling gas-phase ion mobility separation. This allowed distinguishing coeluted isobaric compounds without requiring additional separation time. Furthermore, we identified untargeted and low-abundance analytes, including neuropeptides deriving from the pro-opiomelanocortin precursor protein and localized a specific area of the pituitary gland (i.e., adenohypophysis) known to secrete neuropeptides and other small metabolites related to development, growth, and metabolism. This platform can thus be applied for the in-depth study of small samples of complex tissues with histologic features of ∼400 µm or more, including potential neuropeptide markers involved in many diseases such as neurodegenerative diseases, obesity, bulimia, and anorexia nervosa.

Sequencing and Identification of Endogenous Neuropeptides with Matrix-Enhanced Secondary Ion Mass Spectrometry Tandem Mass Spectrometry.

Matrix-enhanced secondary ion mass spectrometry (ME-SIMS) has overcome one of the biggest disadvantages of SIMS analysis by providing the ability to detect intact biomolecules at high spatial resolution. By increasing ionization efficiency and minimizing primary ion beam-induced fragmentation of analytes, ME-SIMS has proven useful for detection of numerous biorelevant species, now including peptides. We report here the first demonstration of tandem ME-SIMS for de novo sequencing of endogenous neuropeptides from tissue in situ (i.e., rat pituitary gland). The peptide ions were isolated for tandem MS analysis using a 1 Da mass isolation window, followed by collision-induced dissociation (CID) at 1.5 keV in a collision cell filled with argon gas, for confident identification of the detected peptide. Using this method, neuropeptides up to m/z 2000 were detected and sequenced from the posterior lobe of the rat pituitary gland. These results demonstrate the potential for ME-SIMS tandem MS development in bottom-up proteomics imaging at high-spatial resolution.

Spatial Autocorrelation in Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) is a powerful molecular imaging technique. In microprobe MSI, images are created through a grid-wise interrogation of individual spots by mass spectrometry across a surface. Classical statistical tests for within-sample comparisons fail as close-by measurement spots violate the assumption of independence of these tests, which can lead to an increased false-discovery rate. For spatial data, this effect is referred to as spatial autocorrelation. In this study, we investigated spatial autocorrelation in three different matrix-assisted laser desorption/ionization MSI data sets. These data sets cover different molecular classes (metabolites/drugs, lipids, and proteins) and different spatial resolutions ranging from 20 to 100 µm. Significant spatial autocorrelation was detected in all three data sets and found to increase with decreasing pixel size. To enable statistical testing for differences in mass signal intensities between regions of interest within MSI data sets, we propose the use of Conditional Autoregressive (CAR) models. We show that, by accounting for spatial autocorrelation, discovery rates (i.e., the ratio between the features identified and the total number of features) could be reduced between 21% and 69%. The reliability of this approach was validated by control mass signals based on prior knowledge. In light of the advent of larger MSI data sets based on either an increased spatial resolution or 3D data sets, accounting for effects due to spatial autocorrelation becomes even more indispensable. Here, we propose a generic and easily applicable workflow to enable within-sample statistical comparisons.

A New Method and Mass Spectrometer Design for TOF-SIMS Parallel Imaging MS/MS.

We report a method for the unambiguous identification of molecules in biological and materials specimens at high practical lateral resolution using a new TOF-SIMS parallel imaging MS/MS spectrometer. The tandem mass spectrometry imaging reported here is based on the precise monoisotopic selection of precursor ions from a TOF-SIMS secondary ion stream followed by the parallel and synchronous collection of the product ion data. Thus, our new method enables simultaneous surface screening of a complex matrix chemistry with TOF-SIMS (MS(1)) imaging and targeted identification of matrix components with MS/MS (MS(2)) imaging. This approach takes optimal advantage of all ions produced from a multicomponent sample, compared to classical tandem mass spectrometric methods that discard all ions with the exception of specific ions of interest. We have applied this approach for molecular surface analysis and molecular identification on the nanometer scale. High abundance sensitivity is achieved at low primary ion dose density; therefore, one-of-a-kind samples may be relentlessly probed before ion-beam-induced molecular damage is observed.

Use of advantageous, volatile matrices enabled by next-generation high-speed matrix-assisted laser desorption/ionization time-of-flight imaging employing a scanning laser beam.

RATIONALE: In mass spectrometry imaging (MSI) it is often desirable to analyse the same sample in both polarities to extract the most information. However, many matrices that produce high-quality spectra in matrix-assisted laser desorption/ionization (MALDI) are volatile, greatly limiting their use in long imaging experiments. We demonstrate that using a new high speed MALDI-MSI instrument, volatile matrices, including those that produce intense lipid signals in both positive and negative ion mode, can now be effectively used in MSI. METHODS: A prototype Bruker rapifleX MALDI Tissuetyper™ time-of-flight (TOF) instrument was used for high-speed imaging. This allows acquisition rates up to 50 pixels/s made possible by use of a 10 kHz laser and two rotating mirrors that allow the laser beam to be moved over, and synchronised with, the rapidly moving sample. MSI experiments were performed on mouse brain sections using non-vacuum stable dithranol and 2,6-dihydroxyacetophenone (DHA) matrices with pixel sizes ranging from 10 × 10 µm(2) to 50 × 50 µm(2). RESULTS: Both DHA and dithranol produced rich, complementary lipid spectra in both positive and negative ion modes. Due to the rapid acquisition speed of the instrument, both matrices could be effectively used for MSI despite their volatility. For example, an entire mouse brain could be imaged consecutively in both positive and negative ion mode with 50 × 50 µm(2) pixels in ~35 min. We demonstrate that these speeds make possible both faster and higher resolution imaging of biological tissues on practical timescales. CONCLUSIONS: These high acquisition speeds now make possible whole new classes of matrices that are unstable under high vacuum for MALDI-MSI studies. This provides researchers with far greater range and flexibility in choosing the best matrix for the given sample and analytes that they wish to detect. In addition, such instruments allow MSI to be performed at higher resolution across larger areas on practical time scales.

Gold sputtered fiducial markers for combined secondary ion mass spectrometry and MALDI imaging of tissue samples.

Mass spectrometry imaging (MSI) is a label free technique capable of providing simultaneous identification and localization of biomolecules. A multimodal approach is required that allows for the study of the complexity of biological tissue samples to overcome the limitations of a single MSI technique. Secondary ion mass spectrometry (SIMS) allows for high spatial resolution imaging while matrix-assisted laser desorption (MALDI) offers a significantly wider mass range. The combination of coregistered SIMS and MALDI images results in detailed and unique biomolecular information. In this Technical Note, we describe how gold sputtered/implanted fiducial markers (FM) are created and can be used to ensure a proper overlay and coregistration of the two-dimensional images provided by the two MSI modalities.