193 nm Ultraviolet Photodissociation for the Characterization of Singly Charged Proteoforms Generated by MALDI

Zemaitis, Kevin; Zhou, Mowei; Kew, Will; Paša‐Tolić, Ljiljana

doi:10.1021/jasms.2c00302

Cited by 3 publications

(2 citation statements)

References 29 publications

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“…Briefly, intact proteins were analyzed after a tissue fixation and lipid depletion with a protocol, and tryptic peptides were analyzed after in-situ deposition of trypsin directly on the tissue and overnight incubation, this is described in great depth elsewhere. 30,32 Glycan data was collected from formalin-fixed paraffin-embedded (FFPE) soybean nodules (Glycine max cv. Williams 82).…”

Section: Methodsmentioning

confidence: 99%

“…33 Both glycans and intact proteins were analyzed on a custom UHMR Q Exactive HF Orbitrap (Thermo Scientific, Bremen, DE) fitted with an elevated pressure MALDI source (Spectroglyph, Kennewick, WA) operated under a custom privilege license. 32 Tryptic peptides were analyzed on a 12T solariX Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Bremen, DE). All raw MALDI-MS data were summed with Mozaic (v.2022.5.1.b1; Spectroswiss, Lausanne, CH).…”

Section: Methodsmentioning

confidence: 99%

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IsoMatchMS: Open-Source Software for Automated Annotation and Visualization of High Resolution MALDI-MS Spectra

Degnan

Zemaitis

Lewis

et al. 2023

Preprint

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

IsoMatchMS: Open-Source Software for Automated Annotation and Visualization of High Resolution MALDI-MS Spectra

Degnan

Zemaitis

Lewis

et al. 2023

Preprint

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Current Challenges and Future Directions in Peptidomics

Schrader,

Fricker

2024

Methods in Molecular Biology

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Spatial top-down proteomics for the functional characterization of human kidney

Zemaitis,

Fulcher,

Kumar

et al. 2024

Preprint

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Background: The Human Proteome Project has credibly detected nearly 93% of the roughly 20,000 proteins which are predicted by the human genome. However, the proteome is enigmatic, where alterations in amino acid sequences from polymorphisms and alternative splicing, errors in translation, and post-translational modifications result in a proteome depth estimated at several million unique proteoforms. Recently mass spectrometry has been demonstrated in several landmark efforts mapping the human proteoform landscape in bulk analyses. Herein, we developed an integrated workflow for characterizing proteoforms from human tissue in a spatially resolved manner by coupling laser capture microdissection, nanoliter-scale sample preparation, and mass spectrometry imaging. Results: Using healthy human kidney sections as the case study, we focused our analyses on the major functional tissue units including glomeruli, tubules, and medullary rays. After laser capture microdissection, these isolated functional tissue units were processed with microPOTS (microdroplet processing in one-pot for trace samples) for sensitive top-down proteomics measurement. This provided a quantitative database of 616 proteoforms that was further leveraged as a library for mass spectrometry imaging with near-cellular spatial resolution over the entire section. Notably, several mitochondrial proteoforms were found to be differentially abundant between glomeruli and convoluted tubules, and further spatial contextualization was provided by mass spectrometry imaging confirming unique differences identified by microPOTS, and further expanding the field-of-view for unique distributions such as enhanced abundance of a truncated form (1-74) of ubiquitin within cortical regions. Conclusions: We developed an integrated workflow to directly identify proteoforms and reveal their spatial distributions. Where of the 20 differentially abundant proteoforms identified as discriminate between tubules and glomeruli by microPOTS, the vast majority of tubular proteoforms were of mitochondrial origin (8 of 10) where discriminate proteoforms in glomeruli were primarily hemoglobin subunits (9 of 10). These trends were also identified within ion images demonstrating spatially resolved characterization of proteoforms that has the potential to reshape discovery-based proteomics because the proteoforms are the ultimate effector of cellular functions. Applications of this technology have the potential to unravel etiology and pathophysiology of disease states, informing on biologically active proteoforms, which remodel the proteomic landscape in chronic and acute disorders.

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193 nm Ultraviolet Photodissociation for the Characterization of Singly Charged Proteoforms Generated by MALDI

Cited by 3 publications

References 29 publications

IsoMatchMS: Open-Source Software for Automated Annotation and Visualization of High Resolution MALDI-MS Spectra

IsoMatchMS: Open-Source Software for Automated Annotation and Visualization of High Resolution MALDI-MS Spectra

Current Challenges and Future Directions in Peptidomics

Spatial top-down proteomics for the functional characterization of human kidney

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