Search

The annotated protein database was curated to annotate MS/MS data in the manuscript titled 'Mapping Microhabitats: Metabolome Informed Proteome Imaging of Lignocellulose Decomposition by a Naturally Evolved Microbial Consortium'.&nbsp

Due to its speed, accuracy, and adaptability to various sample types, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become a popular method to identify molecular isotope profiles from biological samples. Often MALDI-MS data does not include tandem MS fragmentation data, and thus the identification of compounds in samples requires external databases so that the accurate mass of detected signals can be matched to known molecular compounds. Most relevant software tools are focused on small molecules (e.g., metabolites, lipids), and cannot be easily adapted to protein data due to their more complex isotopic distributions. Here, we present an R package called IsoMatchMS for the automated annotation of MALDI-MS data for multiple datatypes (e.g., intact proteins, lipids, metabolites, etc.). This tool accepts already computed molecular formulas, or for proteomics applications, can compute molecular formulas from a list of input peptides or proteins including proteins with post-translational modifications. Visualization of all matched isotopic profiles are provided in a highly accessible HTML format called a trelliscope display, which allows users to filter and sort by several parameters such as match scores and the number of peaks matched. IsoMatchMS simplifies the annotation and visualization of MALDI-MS data for downstream analyses.

Probing the entirety of any species metabolome is an analytical grand challenge, especially at a cellular scale. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a common spatial metabolomics assay, but this technique has limited molecular coverage for several reasons. To expand the application space of spatial metabolomics, we developed an on-tissue chemical derivatization (OTCD) workflow using 4-APEBA for confident identification of several dozen elusive phytocompounds. Overall, this new OTCD method enabled the annotation of roughly 280 metabolites, with only 10% overlap in metabolic coverage when compared to analog negative ion mode MALDI-MSI on serial sections. We demonstrate that 4-APEBA outperforms other derivatization agents providing: (1) broad specificity towards carbonyls, (2) low background, and (3) introduction of bromine isotopes. Notably, the latter two attributes also facilitate more confidence in our bioinformatics for data processing. The workflow detailed here trailblazes a path towards spatial hormonomics within plant samples, enhancing detection of carboxylates, aldehydes, and plausibly other carbonyls. As such, several phytohormones, which have various roles within stress responses and cellular communication can now be spatially profiled, as demonstrated in poplar root and soybean root nodule.

Probing the entirety of any species metabolome is an analytical grand challenge, especially at a cellular scale. Where spatial metabolomics, completed primarily by matrix-assisted laser desorption/ionization (MALDI), has limited molecular coverage for several reasons. To expand the scope of spatial metabolomics, we developed an on-tissue chemical derivatization (OTCD) workflow using 4-APEBA for confident identification of several dozen elusive phytocompounds, including several phytohormones, which have various roles within stress responses and cellular communication. Superiority of 4-APEBA is established in comparison to other derivatization agents with (1) broad specificity towards carbonyls, (2) low background, and (3) introduction of bromine isotopes, where the latter two facilitate confident bioinformatics. The outlined workflow trailblazes a path towards spatial hormonomics within plant samples, enhancing detection of carboxylates, aldehydes, ketones, and plausibly phenols.

Scope: An overall protocol outlining several nested sub-protocols for the 2D mapping of intact proteoforms from human tissue by MALDI imaging on an UHMR Q Exactive Orbitrap MS. Expected Outcomes: Annotated ion images of intact proteoforms ready to be ingested and disseminated.

Scope: A detailed protocol entailing the overall protocols developed for human tissue sections for the UHMR HF Orbitrap platform. This includes QC metrics for tissue sectioning, an overview of different sample washing steps, an optional tissue acidification protocol, as well as matrix application. Expected Outcomes: Tissue sections fully prepared for MALDI imaging using UHMR HF Orbitrap.

Scope: A detailed protocol entailing the sample washing protocols developed for human kidney, this includes several timed washing steps for fixing the tissue, removing small molecule interferents, and desalting the tissue surface optimized for use with a Spectroglyph EP-MALDI-2 source mounted on a custom Thermo Scientific UHMR Q Exactive HF Orbitrap. Expected Outcomes: Human kidney slides ready for pre-extraction and/or matrix application.

Scope: A detailed protocol entailing the sample washing protocols developed for human pancreas, this includes several timed washing steps for fixing the tissue, removing small molecule interferents, and desalting the tissue surface optimized for use with a Spectroglyph EP-MALDI-2 source mounted on a custom Thermo Scientific UHMR Q Exactive HF Orbitrap. Expected Outcomes: Human pancreas slides ready for pre-extraction and/or matrix application.

Scope: The protocol describes a workflow for processing mass spectrometry imaging (MSI) data of high resolution intact proteins/proteoforms by MALDI. It involves the visualization and image generation using commercial software and also peak annotation using open source code. Expected Outcomes: Tabular output of matched proteoforms, as well as a trelliscope display of overlaid isotopic distributions allowing for the annotation of high-resolution accurate mass distributions of proteoforms.

Conventional proteomic approaches measure the averaged signal from mixed cell populations or bulk tissues, leading to the dilution of signals arising from subpopulations of cells that might serve as important biomarkers. Recent developments in bottom-up proteomics have enabled spatial mapping of cellular heterogeneity in tissue microenvironments. However, bottom-up proteomics cannot unambiguously define and quantify proteoforms, which are intact (i.e. functional) forms of proteins capturing genetic variations, alternatively spliced transcripts and post-translational modifications. Herein, we described a spatially resolved top-down proteomics (TDP) platform for proteoform identification and quantitation directly from tissue sections. The spatial TDP platform consisted of a nanoPOTS (nanodroplet Processing in One pot for Trace Samples)-based sample preparation system and an LCM (laser capture microdissection)-based cell isolation system. We improved the nanoPOTS sample preparation by adding benzonase in the extraction buffer to enhance the coverage of nucleus proteins. Using ~200 cultured cells as test samples, this approach increased total proteoform identifications from 493 to 700; with newly identified proteoforms primarily corresponding to nuclear proteins. To demonstrate the spatial TDP platform in tissue samples, we analyzed LCM-isolated tissue voxels from rat brain cortex and hypothalamus regions. We quantified 509 proteoforms within the union of TopPIC and TDPortal identifications to match with features from ProMex. Several proteoforms corresponding to the same gene exhibited mixed abundance profiles between two tissue regions, suggesting potential PTM-specific spatial distributions. The spatial TDP workflow has prospects for biomarker discovery at proteoform level from small tissue sections.

Scope: A detailed protocol entailing the calibration, operation, and verification of the custom UHMR Q Exactive HF Orbitrap with a Spectroglyph EP-MALDI-2 source for high-spatial resolution imaging. This entails various procedures for instrument calibration and checks for the MALDI source and spectrometer for the intact proteoform analyses at near-cellular spatial resolution (> 15 µm) for human tissue sections without oversampling the tissue. Expected Outcomes: Creation of several dozen ion images which correspond to proteoforms visually localized within physiological regions of the profiled tissue sections.

Core histones including H2A, H2B, H3, and H4 are key modulators of cellular repair, transcription, and replication within eukaryotic cells, playing vital roles within the pathogenesis of disease and cellular responses to environmental stimuli. Traditional mass spectrometry (MS) based bottom-up and top-down proteomics allows for the comprehensive identification of proteins and of post-translational modification (PTM) harboring proteoforms. However, these methodologies have difficulties preserving near cellular spatial distributions because they typically require laser capture microdissection (LCM) and advanced sample preparation techniques. Herein, we coupled a matrix-assisted laser desorption/ionization (MALDI) source with a Thermo Scientific™ Q Exactive™ HF Orbitrap™ MS upgraded with ultra-high mass range (UHMR) boards for the first demonstration of complementary high-resolution accurate mass (HR/AM) measurements of proteoforms up to 16.5 kDa directly from tissue using this benchtop mass spectrometer. The platform achieved isotopic resolution throughout the detected mass range, providing confident assignments of proteoforms with low ppm mass error and considerable increase in duty cycle over other Fourier trans-form mass analyzers. Proteoform mapping of core histones was demonstrated on sections of human kidney at near-cellular spatial resolution, with several key distributions of histone and other proteoforms noted within both healthy biopsy and a section from a renal cell carcinoma (RCC) containing nephrectomy. The use of MALDI-MS imaging (MSI) for proteoform map-ping demonstrates several steps towards high-throughput accurate identification of proteoforms and provides a new tool for mapping biomolecule distributions throughout tissue sections in extended mass ranges.