Rashmi Kumar

Laser capture microdissection (LCM) - based spatial mass spectrometry proteomics is a rapidly emerging technique with strong potential for use in formalin-fixed, paraffin-embedded (FFPE) tissues. Several sample-preparation methods have been developed to decrosslink FFPE proteins for spatial proteomics; however, residual crosslinks often remain, and depth can remain impaired relative to fresh frozen tissue samples. To increase proteome coverage in spatially resolved LCM-FFPE samples, we investigated a panel of chemical compounds with the potential to catalyze the decrosslinking of nucleophilic functional groups on proteins. Systematic screening and optimization of temperature, incubation time, and reagent concentration led to the identification of 3,4-diaminobenzoic acid as an effective agent for improving proteome coverage in FFPE pancreatic tissue. This compound could boost precursor identifications by more than 10% at both reduced (70 °C) and high (90 °C) temperatures. Application of this chemical-decrosslinking strategy to a pancreatic ductal adenocarcinoma tissue section enabled the identification of numerous cell-type–enriched proteins with clinical and therapeutic relevance. Taken together, our findings show that chemical decrosslinking can increase proteome coverage in FFPE tissues, thereby advancing our understanding of tissue microenvironments in physiological and pathological contexts.

Single-cell proteomic (scProteomic) measurements of peripheral blood mononuclear cells (PBMCs) are of considerable value in human health, given their involvement in the maintenance of healthy and diseased states. However, the high heterogeneity and relatively small size of immune cell types demand maximal throughput and sensitivity in proteomic measurements that have yet to be fully realized. Here, we describe an approach that addresses sensitivity and throughput through the implementation of Real-Time spectral Library Searching (RTLS), TMTpro 32-plex labelling, an updated nested-nanodroplet processing in One pot for Trace Samples (N2), and a dual-column liquid chromatography system. By prioritizing tandem mass spectrometry (MS2) features with high similarity to library spectra, RTLS enables greater identification depth and feature reproducibility than a standard shotgun MS2 approach in low-input and single-cell samples. The platform permitted 660 single PBMCs to be measured per day, with an average of 750 protein identifications per cell and 1,648 proteins in total, achieving the necessary throughput and depth to characterize immune cell populations. Application of this scProteomic method and a new cell typing informatics approach to 2,130 PBMCs enabled the identification of both major and low-frequency cell types (∼1-2%), as well as associated proteomic markers.

Antibodies conjugated to metal chelating polymers are routinely used in high-dimensional multiplexed single cell mass spectrometric imaging techniques, such as immunohistochemistry-based multiplexed ion beam imaging by time-of-flight (MIBI-TOF) mass spectrometry, imaging mass cytometry (IMC), and flow cytometry-based CyTOF. However, successful multiplexed capability of these techniques is heavily dependent on the stability of the metal-chelates used. Chelate stability is governed by the ionic radius of the metal used, which in some cases can fall below or exceed the optimal range for commercially available DTPA-based polymers. In this study, we have developed and optimized macrocyclic chelators for metals with relatively small (i.e., Ga) or large (i.e., Tl) atomic radii. In agreement with previously published studies, we observed NOTA to be a suitable chelator for Ga, whereas DOTA was found to be an ideal chelator for Tl and larger lanthanides, such as La, Ce, and Pr. DOTA and DTPA chelator dendrimers were synthesized and conjugated to primary antibodies that were subsequently used for tissue staining. Antibodies conjugated with the DOTA-dendrimer were more stable and exhibited more specific staining than those modified with the corresponding DTPA-dendrimer. With these new chelates, we incorporated seven new reporter channels into a highly multiplexed MIBI-TOF imaging study containing 44 protein epitope markers on various tissues. To the best of our knowledge, this is the largest multiplexed panel used to date for MIBI-TOF applications.

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. 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. We developed an integrated workflow to directly identify proteoforms and reveal their spatial distributions. 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) while 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.

RATIONALE Universally, the most common method of embedding dissected or resected fresh frozen (FF) tissues is within optimal cutting temperature (OCT) media. Unfortunately, OCT is not ideal for mass spectrometry (MS) assays. The media perfuses through tissues, resulting in persistent polymer contamination causing issues for sensitive analyses. Complete removal of all OCT media background would enable techniques such as MS imaging (MSI) to be bridged to clinically relevant samples stored in biobanks which possess extensive medical histories. Even though methods have been outlined for spatial lipidomics and proteomics, many still avoid these tissues despite vast potential for biological discoveries applying MS or MSI. METHODS Reference FF human pancreas was prepared within OCT media and cryosectioned for matrix-assisted laser desorption/ionization (MALDI)-MSI. Sample preparation protocols using aqueous ammonium formate for spatial lipidomics, and a variety of solutions for fixation, delipidation, and salt removal enabled spatial proteomics as well. MSI was performed on a UHMR HF Orbitrap MS detected broad classes of lipids and intact proteins, enabling an assessment of polymer perfusion and signal delocalization over a broad mass range to evaluate any deleterious artifacts. RESULTS We observe lower polymer contamination from OCT tissues with ammonium formate washes as expected, with several caveats. This was most effective in negative ion mode, where in tandem with the boosted lipid signal positive results were achieved for phospholipids (600-900 Da) and higher mass lipids (>1250 Da). Positive ion mode analyses can be completed for phospholipids, but the condensation of alkali adducts to protonated forms not only increased lipid signals, but also increased signals of larger, persistent PEG oligomers (>1250 Da) which were not previously reported. Even with extensive tissue washing completing a spatial proteomics protocol, the removal of all remnant artifacts of OCT media was not feasible. CONCLUSIONS The finding of these persistent remnant polymers from the OCT embedding process has several consequences for MSI, directly effecting primary performance of the MALDI source and lipid and protein analyses. This does not mean OCT tissues should not be or cannot be used for spatial omics by MSI, but several quality control and quality assurance measures should be implemented, and we discuss paths forward and several considerations.

Biological organisms are multifaceted, intricate systems where slight perturbations can result in extensive changes in gene expression, protein abundance and/or activity, and metabolic flux. These changes occur at different timescales, spatially across cells of heterogenous origins, and within single cells. Hence multimodal measurements at the smallest biological scales are necessary to capture dynamic changes in heterogenous biological systems. Of the analytical techniques used to measure biomolecules, mass spectrometry has proven to be a powerful option due to its sensitivity, robustness, and flexibility with regards to breadth of biomolecules that can be analyzed. Recently many studies have coupled mass spectrometry to other analytical techniques with the goal of measuring multiple modalities from the same single-cell. It is with these concepts in mind that we focus this Review on mass spectrometry-enabled multiomic measurements at single-cell or near-single-cell resolution.

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.

High-dimensional single-cell mass spectrometric imaging techniques such as multiplexed ion beam imaging by time-of-flight mass spectrometry (MIBI-TOF), imaging mass cytometry (IMC), and flow cytometry-based CyTOF utilize antibodies conjugated to linear metal-chelating polymers. Here, we report on the synthesis and characterization of a dendrimer-based polymer and its utilization in tissue imaging using MIBI-TOF. We compared the staining performance in FFPE tissue of antibodies for lineage-specific immune proteins (CD20, CD3, CD45, FoxP3) that were conjugated with dendrimer or linear polymer. Staining of serial tissue sections with dendron-conjugated and linear-polymer-conjugated antibodies revealed comparable avidities of dendrons and linear polymers with log₂ (ratio of mean positive pixel intensity of staining for linear polymers to dendrons) within the range ±0.25. Interestingly, dendron-conjugated antibodies were observed to have some advantages over linear polymer-conjugated antibodies. For example, tissue staining of a nuclear protein, FoxP3 with dendron-conjugated antibodies showed notably less background staining than that of linear-polymer-conjugated antibodies. Additionally, dendron-conjugated antibodies did not exhibit off-target cytosolic binding in neural tissue typically observed when using linear polymer conjugates. Taken together, this work provides a versatile framework for using third-generation dendron-conjugated antibodies with improved staining over conventional linear polymers.