Systems Biology

In plants, the apoplast contains a diverse set of proteins that underpin mechanisms for maintaining cell homeostasis, cell wall remodeling, cell signaling, and pathogen defense. Apoplast protein composition is highly regulated, primarily through the control of secretory traffic in response to endogenous and environmental factors. Dynamic changes in apoplast proteome facilitate plant survival in a changing climate. Even so, the apoplast proteome profiles in plants remain poorly characterized due to technological limitations. Recent progress in quantitative proteomics has significantly advanced the resolution of proteomic profiling in mammalian systems and has the potential for application in plant systems. In this protocol, we provide a detailed and efficient protocol for tandem mass tag (TMT)-based quantitative analysis of Arabidopsis thaliana secretory proteome to resolve dynamic changes in leaf apoplast proteome profiles. The protocol employs apoplast flush collection followed by protein cleaning using filter-aided sample preparation (FASP), protein digestion, TMT-labeling of peptides, and mass spectrometry (MS) analysis. Subsequent data analysis for peptide detection and quantification uses Proteome Discoverer software (PD) 3.0. Additionally, we have incorporated in silico–generated spectral libraries using PD 3.0, which enables rapid and efficient analysis of proteomic data. Our optimized protocol offers a robust framework for quantitative secretory proteomic analysis in plants, with potential applications in functional proteomics and the study of trafficking systems that impact plant growth, survival, and health.

Recent advances in single-cell technologies have provided limited insight into the role of protein phosphorylation in T-cell fate and function. Dysregulated protein phosphorylation is associated with adverse clinical outcomes, emphasizing the need for reliable methods to unravel the complexities of T-cell signal transduction and disease-related alterations. While flow cytometry is widely used, it is constrained by spectral overlap, limiting the number of protein targets for simultaneous analysis. To overcome this, we present a robust protocol for whole blood T-cell stimulation and subsequent analysis using mass cytometry by time-of-flight (CyTOF). CyTOF minimizes spillover into adjacent channels by employing highly pure, stable, heavy metal–conjugated antibodies for protein detection. This protocol offers a high-dimensional approach for phenotypic and phospho-protein characterization of key signaling pathways, including JAK/STAT, MAPK, PI3K/mTOR, PKC, and NF-κB. A key feature is the T-cell stimulation reagent, which mimics endogenous activation by engaging the T-cell receptor (TCR)/CD3 complex and providing co-stimulation via an anti-CD28 antibody. Further, we enhance reproducibility and enable batch processing through the implementation of the Prot1/Thaw-Lyse system for immediate cryopreservation of stimulated blood samples. By employing CyTOF, this method permits the simultaneous analysis of 31 protein targets with single-cell resolution, minimizing spillover and providing superior specificity, sensitivity, and resolution over flow cytometric methods. This approach facilitates the robust assessment of TCR activation and its effect on bystander populations, which has been challenging with spectral flow cytometry due to the limited availability of methanol-resistant fluorophores. This protocol is a precise and reproducible method for elucidating the downstream effects of T-cell stimulation and immune status, with significant potential for clinical applications, including the assessment of T-cell-targeted therapies.

Protein isolation combined with two-dimensional electrophoresis (2-DE) is a powerful technique for analyzing complex protein mixtures, enabling the simultaneous separation of thousands of proteins. This method involves two distinct steps: isoelectric focusing (IEF), which separates proteins based on their isoelectric points (pI), and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins by their relative molecular weights. However, the success of 2-DE is highly dependent on the quality of the starting material. Isolating proteins from plant mature roots is challenging due to interfering compounds and a thick, lignin-rich cell wall. Bacterial proteins and metabolites further complicate extraction in legumes, which form symbiotic relationships with bacteria. Endogenous proteases can degrade proteins, and microbial contaminants may co-purify with plant proteins. Therefore, comparing extraction methods is essential to minimize contaminants, maximize yield, and preserve protein integrity. In this study, we compare two protein isolation techniques for lupine roots and optimize a protein precipitation protocol to enhance the yield for downstream proteomic analyses. The effectiveness of each method was evaluated based on the quality and resolution of 2-DE gel images. The optimized protocol provides a reliable platform for comparative proteomics and functional studies of lupine root responses to stress, e.g., drought or salinity, and symbiotic interactions with bacteria.

OtUBD is a high-affinity ubiquitin-binding domain (UBD) derived from a large protein produced by the microorganism Orientia tsutsugamushi. The following protocol describes a step-by-step process for the enrichment of ubiquitinated proteins from baker's yeast and mammalian cell lysates using OtUBD. The OtUBD affinity resin can strongly enrich both mono- and poly-ubiquitinated proteins from crude lysates. The protocol further describes the use of different buffer formulations to specifically enrich for proteins covalently modified by ubiquitin with or without proteins that associate with them. Combining different OtUBD-mediated enrichment protocols with liquid chromatography–tandem mass spectrometry (LC–MS/MS) helps distinguish the pool of covalently ubiquitinated proteins (the ubiquitinome) from ubiquitin- or ubiquitinated protein-interacting proteins (the ubiquitin interactome). The OtUBD tool described in the protocol has been used successfully with downstream applications such as immunoblotting and differential proteomics. It provides researchers with a versatile and economical tool for the study of ubiquitin biology.

Brain endothelial cells, which constitute the cerebrovasculature, form the first interface between the blood and brain and play essential roles in maintaining central nervous system (CNS) homeostasis. These cells exhibit strong apicobasal polarity, with distinct luminal and abluminal membrane compositions that crucially mediate compartmentalized functions of the vasculature. Existing transcriptomic and proteomic profiling techniques often lack the spatial resolution to discriminate between these membrane compartments, limiting insights into their distinct molecular compositions and functions. To overcome these limitations, we developed an in vivo proteomic strategy to selectively label and enrich luminal cerebrovascular proteins. In this approach, we perfuse a membrane-impermeable biotinylation reagent into the vasculature to covalently tag cell surface proteins exposed on the luminal side. This is followed by microvessel isolation and streptavidin-based enrichment of biotinylated proteins for downstream mass spectrometry analysis. Using this method, we robustly identified over 1,000 luminally localized proteins via standard liquid chromatography–tandem mass spectrometry (LC–MS/MS) techniques, achieving substantially improved enrichment of canonical luminal markers compared with conventional vascular proteomic approaches. Our method enables the generation of a high-confidence, compartment-resolved atlas of the luminal cerebrovascular proteome and offers a scalable platform for investigating endothelial surface biology in both healthy and disease contexts.

This manuscript details protocols for the ZnCl₂ precipitation-assisted sample preparation (ZASP) for proteomic analysis. By inducing protein precipitation with ZASP precipitation buffer (ZPB, final concentration of ZnCl ₂ at 100 mM and 50% methanol), ZASP depletes harsh detergents and impurities, such as sodium dodecyl sulfate (SDS), Triton X-100, and urea, at high concentrations in solution from protein solutions prior to trypsin digestion. It is a practical, robust, and cost-effective approach for proteomic sample preparation. It has been observed that 90.2% of the proteins can be recovered from lysates by incubating with an equal volume of ZPB at room temperature for 10 min. In 1 h of data-dependent acquisition (DDA) analysis on an Exploris 480, 4,037 proteins and 25,626 peptides were quantified from 1 μg of mouse small intestine proteins, reaching a peak of 4,500 proteins and up to 30,000 peptides with 5 μg of input. Additionally, ZASP outperformed other common sample preparation methods such as sodium deoxycholate (SDC)-based in-solution digestion, acetone precipitation, filter-aided sample preparation (FASP), and single-pot, solid-phase-enhanced sample preparation (SP3). It demonstrated superior performance in protein (4,456 proteins) and peptide identification (29,871 peptides), lower missing cleavage rates (16.3%), and high reproducibility (Pearson correlation coefficients of 0.96 between replicates) with similar protein distributions and cellular localization patterns. Significantly, the cost of ZASP per sample with 100 μg of protein as input is lower than 30 RMB, including the expense of trypsin.

Within a cell, proteins have distinct and highly variable half-lives. As a result, the molecular ages of proteins can range from seconds to years. How the age of a protein influences its environmental interactions is a largely unexplored area of biology. To facilitate such studies, we recently developed a technique termed “proteome birthdating” that differentially labels proteins based on their time of synthesis. Proteome birthdating enables analyses of age distributions of the proteome by tandem mass spectrometry (LC–MS/MS) and provides a methodology for investigating the protein age selectivity of diverse cellular pathways. Proteome birthdating can also provide measurements of protein turnover kinetics from single, sequentially labeled samples. Here, we provide a practical guide for conducting proteome birthdating in in vitro model systems. The outlined workflow covers cell culture, isotopic labeling, protein extraction, enzymatic digestion, peptide cleanup, mass spectrometry, data processing, and theoretical considerations for interpretation of the resulting data.

Plants rely on metabolite regulation of proteins to control their metabolism and adapt to environmental changes, but studying these complex interaction networks remains challenging. The proteome integral solubility alteration (PISA) assay, a high-throughput chemoproteomic technique, was originally developed for mammalian systems to investigate drug targets. PISA detects changes in protein stability upon interaction with small molecules, quantified through LC–MS. Here, we present an adapted PISA protocol for Arabidopsis thaliana chloroplasts to identify potential protein interactions with ascorbate. Chloroplasts are extracted using a linear Percoll gradient, treated with multiple ascorbate concentrations, and subjected to heat-induced protein denaturation. Soluble proteins are extracted via ultracentrifugation, and proteome-wide stability changes are quantified using multiplexed LC–MS. We provide instructions for deconvolution of LC–MS spectra and statistical analysis using freely available software. This protocol enables unbiased screening of protein regulation by small molecules in plants without requiring prior knowledge of interaction partners, chemical probe design, or genetic modifications.

Known as the cell’s antenna and signaling hub, the primary cilium is a hair-like organelle with a few micrometers in length and 200–300 nm in diameter. Due to the small size of the primary cilium, it is technically challenging to profile ciliary proteins from mammalian cells. Traditional methods, such as physical isolation of cilia, are susceptible to contamination from other cellular components. Other proximity-based labeling methods via APEX or BioID have been used to map ciliary proteins. However, these approaches have their inherent limitations, including the use of toxic reagents like H₂O₂ and prolonged labeling kinetics. Here, we show a new proximity-based labeling technique for primary cilia with TurboID. TurboID presents a distinct advantage over BioID and APEX2 due to its expedited labeling kinetics, taking minutes instead of hours, and its use of a non-toxic biotin substrate, which eliminates the need for H₂O₂. When targeted to the cilium, TurboID selectively labels ciliary proteins with biotin. The biotinylated proteins are then enriched with streptavidin beads and labeled with tandem mass tags (TMT), followed by mass spectrometry (MS) detection. This protocol eliminates the requirement of toxic labeling reagents and significantly reduces the labeling time, thus providing advantages in mapping signaling proteins with high temporal resolution in live cells.

Glioblastoma (GBM) is the most aggressive brain tumor, and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. A label-free, mass spectrometry–based quantitative proteomics has been developed to identify and characterize proteins that are differentially expressed in GBM to gain a better understanding of the interactions and functions that lead to the pathological state focusing on the extracellular matrix (ECM). The main challenge in GBM research has been to identify novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. To better investigate the GBM secretome upon in vitro treatment with histone deacetylase inhibitor (iHDAC), we employed a high-throughput label-free methodology of protein identification and quantification based on mass spectrometry followed by in silico studies. Our analysis revealed significant changes in the ECM protein profile, particularly those associated with the angiogenic matrisome. Proteins such as decorin, ADAM10, ADAM12, and ADAM15 were differentially regulated upon in silico analysis. In contrast, key angiogenesis markers such as VEGF and ECM proteins like fibronectin and integrins did not display significant changes. These results suggest that iHDAC inhibitors may modulate or suppress tumor behavior growth by targeting ECM proteins’ secretion rather than directly inhibiting angiogenesis.