Biology subjects

Velickovic, M.

Publications and source records attributed to Velickovic, M..

4 recordsLinked to original sources

bioRxiv2023

Robust collection and processing for label-free single voxel proteomics

With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk tissues. However, such bulk measurement lacks spatial resolution and obscures important tissue heterogeneity, which make it impossible for proteome mapping of tissue microenvironment. Here we report an integrated wet collection of single tissue voxel and Surfactant-assisted One-Pot voxel processing method termed wcSOP for robust label-free single voxel proteomics. wcSOP capitalizes on buffer droplet-assisted wet collection of single tissue voxel dissected by LCM into the PCR tube cap and MS-compatible surfactant-assisted one-pot voxel processing in the collection cap. This convenient method allows reproducible label-free quantification of [~]900 and [~]4,600 proteins for single voxel from fresh frozen human spleen tissue at 20 m x 20 m x 10 m (close to single cells) and 200 m x 200 m x 10 m ([~]100 cells), respectively. 100s-1000s of protein signatures with differential expression levels were identified to be spatially resolved between spleen red and white pulp regions depending on the voxel size. Region-specific signaling pathways were enriched from single voxel proteomics data. Antibody-based CODEX imaging was used to validate label-free MS quantitation for single voxel analysis. The wcSOP-MS method paves the way for routine robust single voxel proteomics and spatial proteomics.

molecular biology↗

bioRxiv2023

Coupling microdroplet-based sample preparation, multiplexed isobaric labeling, and nanoflow peptide fractionation for deep proteome profiling of tissue microenvironment

There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity at a cell-type-specific level to better understand and predict the function of complex biological systems, such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverages due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (Microdroplet Processing in One pot for Trace Samples), the multiplexed isobaric labelling, and a nanoflow peptide fractionation approach. The integrated workflow allowed to maximize proteome coverage of laser-isolated tissue samples containing nanogram proteins. We demonstrated the deep spatial proteomics can quantify more than 5,000 unique proteins from a small-sized human pancreatic tissue pixel ([~]60,000 {micro}m2) and reveal unique islet microenvironments.

biochemistry↗

bioRxiv2022

Proteome mapping of the human pancreatic islet microenvironment reveals endocrine-exocrine signaling sphere of influence

The need for a clinically accessible method with the ability to match protein activity within heterogeneous tissues is currently unmet by existing technologies. Our proteomics sample preparation platform, named microPOTS (Microdroplet Processing in One pot for Trace Samples), can be used to measure relative protein abundance in micron-scale samples alongside the spatial location of each measurement, thereby tying biologically interesting proteins and pathways to distinct regions. However, given the smaller sample number and amount of tissue measu red, standard mass spectrometric analysis pipelines have proven inadequate. Here we describe how existing computational approaches can be adapted to focus on the specific biological questions asked in spatial proteomics experiments. We apply this approach to present an unbiased characterization of the human islet microenvironment comprising the entire complex array of tissues involved while maintaining spatial information and the degree of the islets sphere of influence. We identify specific functional activity unique to the pancreatic islet cells and demonstrate how far their signature can be measured. Our results show that we can distinguish pancreatic islet cells from the neighboring exocrine tissue environment, recapitulate known biological functions of islet cells, and identify a spatial gradient in the expression of RNA processing proteins within the islet microenvironment.

systems biology↗

bioRxiv2022

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We developed a novel tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3,000 (>9,500) phosphopeptides from 1 (10) {micro}g of cell lysate using the label-free method without a spectral library. It also enabled precise quantification of [~]600 phosphopeptides from 100 cells sorted by FACS (single-cell level input for the enriched phosphopeptides) and [~]700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 {micro}m (equivalent to [~]100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.

biochemistry↗