Enhanced Spatially Resolved Proteomics Using On-Tissue Hydrogel-Mediated Protein Digestion

Abstract

The identification of proteins from tissue specimens is a challenging area of biological research. Many current techniques for identification forfeit some level of spatial information during the sample preparation process. Recently, hydrogel technologies have been developed that perform spatially localized protein extraction and digestion prior to downstream proteomic analysis. Regiospecific protein identifications acquired using this approach have thus far been limited to 1-2 mm diameter areas. The need to target smaller populations of cells with this technology necessitates the production of smaller diameter hydrogels. Herein, we demonstrate hydrogel fabrication processes that allow hydrogel applications down to a diameter of ∼260 μm, approximately 1/15 of the area of previous approaches. Parameters such as the percent polyacrylamide used in hydrogel construction as well as the concentration of trypsin with which the hydrogel is loaded are investigated to maximize the number of protein identifications from subsequent liquid chromatography tandem MS (LC-MS/MS) analysis of hydrogel extracts. An 18% polyacrylamide concentration is shown to provide for a more rigid polymer network than the conventional 7.5% polyacrylamide concentration and supports the fabrication of individual hydrogels using the small punch biopsies. Over 600 protein identifications are still achieved at the smallest hydrogel diameters of 260 μm. The utility of these small hydrogels is demonstrated through the analysis of sub regions of a rat cerebellum tissue section. While over 900 protein identifications are made from each hydrogel, approximately 20% of the proteins identified are unique to each of the two regions, highlighting the importance of targeting tissue subtypes to accurately characterize tissue biology. These newly improved methods to the hydrogel process will allow researchers to target smaller biological features for robust spatially localized proteomic analyses.

Beta cell heterogeneity: an evolving concept

“…Notably, recent technological advances are merging omics with histological analysis; these approaches are known as ‘spatial genomics’ [39] or ‘spatial proteomics’ [40, 41]. Single-cell RNA sequencing or mass spectrometry in situ (preserving the spatial location of the studied cells) promises to expose new layers of beta cell biology and heterogeneity.…”

Protein identification in imaging mass spectrometry through spatially targeted liquid micro‐extractions

“…The number of identifications is comparable with those obtained from other spatially targeted approaches such as hydrogels. A hydrogel fabricated with a diameter of 1.66 mm has been shown to be capable of identifying 1052 proteins, while the LESA experiment at 1.0 μL (~1.71 mm diameter measured previously) identified 1119 proteins . The top‐down experiments can be completed in as little as 1–2 h, where the majority of the time is devoted to LC/MS/MS analysis, as the tissue washes and extractions only take ~10 min to complete.…”

“…Utilization of a glass capillary for extractions allows for improved droplet resolutions (<600 μm on water‐sensitive paper) and reproducibility (relative standard deviations of <5%) for most experiments. Comparisons of spatially targeted proteomic techniques such as hydrogel experiments, laser capture laser microdissection (LCM), and tissue homogenization have been discussed elsewhere . In these studies, 1.5 mm LCM, 1.5 mm tissue punch/digestion, and 1.67 mm hydrogel experiments yielded ~800–1000 protein identifications whereas the 1.0 μL LESA experiment (1.71 mm on tissue) described herein yielded ~1120 protein identifications.…”

“…Finally, spatially directed approaches, such as enzyme‐loaded hydrogels and liquid extraction surface analyses (LESA), allow for the interrogation of discrete regions of the tissue surface, retaining the spatial integrity of an IMS analysis. Hydrogels, small gel polymers that are loaded with trypsin and placed on a tissue for digestion and extraction of peptides, have been shown to be an efficient method of generating protein identifications but they are difficult to manipulate due to their small size . LESA workflows involve the dispensing of small volumes of solvent onto the tissue surface, allowing for the diffusion of analytes into the liquid which can be followed by traditional proteomic workflows providing for a sensitive, high‐throughput approach to performing spatially targeted proteomics experiments …”

β‐Cells are not uniform after all—Novel insights into molecular heterogeneity of insulin‐secreting cells

“…As introduced briefly above with the example of imaging mass cytometry, medium and high throughput methodologies are begging to be applied with spatial resolution. Exciting recent advances include “spatial genomics” and “spatial proteomics” . In addition to in situ mass spectrometry, the multiplexing of single molecule RNA fluorescence in situ hybridization promises to allow for the simultaneous detection and quantification of dozens or even hundreds of mRNA species in tissue sections .…”