Reciprocal Regulation of the TOR Kinase and ABA Receptor Balances Plant Growth and Stress Response.

As sessile organisms, plants must adapt to variations in the environment. Environmental stress triggers various responses, including growth inhibition, mediated by the plant hormone abscisic acid (ABA). The mechanisms that integrate stress responses with growth are poorly understood. Here, we discovered that the Target of Rapamycin (TOR) kinase phosphorylates PYL ABA receptors at a conserved serine residue to prevent activation of the stress response in unstressed plants. This phosphorylation disrupts PYL association with ABA and with PP2C phosphatase effectors, leading to inactivation of SnRK2 kinases. Under stress, ABA-activated SnRK2s phosphorylate Raptor, a component of the TOR complex, triggering TOR complex dissociation and inhibition. Thus, TOR signaling represses ABA signaling and stress responses in unstressed conditions, whereas ABA signaling represses TOR signaling and growth during times of stress. Plants utilize this conserved phospho-regulatory feedback mechanism to optimize the balance of growth and stress responses.

DIA-Based Phosphoproteomics Identifies Early Phosphorylation Events in Response to EGTA and Mannitol in Arabidopsis.

Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous research has demonstrated that osmotic stress rapidly induces calcium influx and signaling, along with the activation of a specific subset of protein kinases, notably the Raf-like protein (RAF)-sucrose nonfermenting-1-related protein kinase 2 (SnRK2) kinase cascades within minutes. However, the intricate interplay between calcium signaling and the activation of RAF-SnRK2 kinase cascades remains elusive. Here, in this study, we discovered that Raf-like protein (RAF) kinases undergo hyperphosphorylation in response to osmotic shocks. Intriguingly, treatment with the calcium chelator EGTA robustly activates RAF-SnRK2 cascades, mirroring the effects of osmotic treatment. Utilizing high-throughput data-independent acquisition-based phosphoproteomics, we unveiled the global impact of EGTA on protein phosphorylation. Beyond the activation of RAFs and SnRK2s, EGTA treatment also activates mitogen-activated protein kinase cascades, Calcium-dependent protein kinases, and receptor-like protein kinases, etc. Through overlapping assays, we identified potential roles of mitogen-activated protein kinase kinase kinase kinases and receptor-like protein kinases in the osmotic stress-induced activation of RAF-SnRK2 cascades. Our findings illuminate the regulation of phosphorylation and cellular events by Ca2+ signaling, offering insights into the (exocellular) Ca2+ deprivation during early hyperosmolality sensing and signaling.

Data-Independent Acquisition Phosphoproteomics of Urinary Extracellular Vesicles Enables Renal Cell Carcinoma Grade Differentiation.

Translating the research capability and knowledge in cancer signaling into clinical settings has been slow and ineffective. Recently, extracellular vesicles (EVs) have emerged as a promising source for developing disease phosphoprotein markers to monitor disease status. This study focuses on the development of a robust data-independent acquisition (DIA) using mass spectrometry to profile urinary EV phosphoproteomics for renal cell cancer (RCC) grades differentiation. We examined gas-phase fractionated library, direct DIA (library-free), forbidden zones, and several different windowing schemes. After the development of a DIA mass spectrometry method for EV phosphoproteomics, we applied the strategy to identify and quantify urinary EV phosphoproteomes from 57 individuals representing low-grade clear cell RCC, high-grade clear cell RCC, chronic kidney disease, and healthy control individuals. Urinary EVs were efficiently isolated by functional magnetic beads, and EV phosphopeptides were subsequently enriched by PolyMAC. We quantified 2584 unique phosphosites and observed that multiple prominent cancer-related pathways, such as ErbB signaling, renal cell carcinoma, and regulation of actin cytoskeleton, were only upregulated in high-grade clear cell RCC. These results show that EV phosphoproteome analysis utilizing our optimized procedure of EV isolation, phosphopeptide enrichment, and DIA method provides a powerful tool for future clinical applications.

Meeting Report on the 2nd Chinese American Society for Mass Spectrometry Conference: Advancing Biological and Pharmaceutical Mass Spectrometry.

The 2nd CASMS conference was held virtually through Gather. Town platform from October 17 to 21, 2022, with a total of 363 registrants including an outstanding and diverse group of scientists at the forefront of their research fields from both academia and industry worldwide, especially in the United States and China. The conference offered a 5-day agenda with an exciting scientific program consisting of two plenary lectures, 14 parallel symposia, and 4 special sessions in which a total of 97 invited speakers presented technological innovations and their applications in proteomics & biological mass spectrometry and metabo-lipidomics & pharmaceutical mass spectrometry. In addition, 18 invited speakers/panelists presented at 3 research-focused and 2 career development workshops. Moreover, 144 posters, 54 lightning talks, 5 sponsored workshops, and 14 exhibitions were presented, from which 20 posters and 8 lightning talks received presentation awards. Furthermore, the conference featured 1 MCP lectureship and 5 young investigator awardees for the first time to highlight outstanding mid-career and early-career rising stars in mass spectrometry from our society. The conference provided a unique scientific platform for young scientists (i.e., graduate students, postdocs and junior faculty/investigators) to present their research, meet with prominent scientists, and learn about career development and job opportunities (http://casms.org).

Targeted Phosphoproteomics of Human Saliva Extracellular Vesicles via Multiple Reaction Monitoring Cubed (MRM3).

Oral squamous cell carcinoma (OSCC) has become a global health problem due to its increasing incidence and high mortality rate. Early intervention through monitoring of the diagnostic biomarker levels during OSCC treatment is critical. Extracellular vesicles (EVs) are emerging surrogates in intercellular communication through transporting biomolecule cargo and have recently been identified as a potential source of biomarkers such as phosphoproteins for many diseases. Here, we developed a multiple reaction monitoring cubed (MRM3) method coupled with a novel sample preparation strategy, extracellular vesicles to phosphoproteins (EVTOP), to quantify phosphoproteins using a minimal amount of saliva (50 µL) samples from OSCC patients with high specificity and sensitivity. Our results established differential patterns in the phosphopeptide content of healthy, presurgery, and postsurgery OSCC patient groups. Notably, we discovered significantly increased salivary phosphorylated alpha-amylase (AMY) in the postsurgery group compared to the presurgery group. We hereby present the first targeted MS method with extremely high sensitivity for measuring endogenous phosphoproteins in human saliva EVs.

Assessment of urine sample collection and processing variables for extracellular vesicle-based proteomics.

Extracellular vesicles (EVs) in urine are a promising source for developing non-invasive biomarkers. However, urine concentration and content are highly variable and dynamic, and actual urine collection and handling often is nonideal. Furthermore, patients such as those with prostate diseases have challenges in sample collection due to difficulties in holding urine at designated time points. Here, we simulated the actual situation of clinical sample collection to examine the stability of EVs in urine under different circumstances, including urine collection time and temporary storage temperature, as well as daily urine sampling under different diet conditions. EVs were isolated using functionalized EVtrap magnetic beads and characterized by nanoparticle tracking analysis (NTA), western blotting, electron microscopy, and mass spectrometry (MS). EVs in urine remained relatively stable during temporary storage for 6 hours at room temperature and for 12 hours at 4 °C, while significant fluctuations were observed in EV amounts from urine samples collected at different time points from the same individuals, especially under certain diets. Sample normalization with creatinine reduced the coefficient of variation (CV) values among EV samples from 17% to approximately 6% and facilitated downstream MS analyses. Finally, based on the results, we applied them to evaluate potential biomarker panels in prostate cancer by data-independent acquisition (DIA) MS, presenting the recommendation that can facilitate biomarker discovery with nonideal handling conditions.

Identification of Novel Kinases of Tau Using Fluorescence Complementation Mass Spectrometry (FCMS).

Hyperphosphorylation of the microtubule-associated protein Tau is a major hallmark of Alzheimer's disease and other tauopathies. Understanding the protein kinases that phosphorylate Tau is critical for the development of new drugs that target Tau phosphorylation. At present, the repertoire of the Tau kinases remains incomplete, and methods to uncover novel upstream protein kinases are still limited. Here, we apply our newly developed proteomic strategy, fluorescence complementation mass spectrometry, to identify novel kinase candidates of Tau. By constructing Tau- and kinase-fluorescent fragment library, we detected 59 Tau-associated kinases, including 23 known kinases of Tau and 36 novel candidate kinases. In the validation phase using in vitro phosphorylation, among 15 candidate kinases we attempted to purify and test, four candidate kinases, OXSR1 (oxidative-stress responsive gene 1), DAPK2 (death-associated protein kinase 2), CSK (C-terminal SRC kinase), and ZAP70 (zeta chain of T-cell receptor-associated protein kinase 70), displayed the ability to phosphorylate Tau in time-course experiments. Furthermore, coexpression of these four kinases along with Tau increased the phosphorylation of Tau in human neuroglioma H4 cells. We demonstrate that fluorescence complementation mass spectrometry is a powerful proteomic strategy to systematically identify potential kinases that can phosphorylate Tau in cells. Our discovery of new candidate kinases of Tau can present new opportunities for developing Alzheimer's disease therapeutic strategies.

Proteomic and phosphoproteomic landscape of salivary extracellular vesicles to assess OSCC therapeutical outcomes.

Circulating extracellular vesicles (EVs) have emerged as an appealing source for surrogates to evaluate the disease status. Herein, we present a novel proteomic strategy to identify proteins and phosphoproteins from salivary EVs to distinguish oral squamous cell carcinoma (OSCC) patients from healthy individuals and explore the feasibility to evaluate therapeutical outcomes. Bi-functionalized magnetic beads (BiMBs) with Ti (IV) ions and a lipid analog, 1,2-Distearoyl-3-sn-glycerophosphoethanolamine (DSPE) are developed to efficiently isolate EVs from small volume of saliva. In the discovery stage, label-free proteomics and phosphoproteomics quantification showed 315 upregulated proteins and 132 upregulated phosphoproteins in OSCC patients among more than 2500 EV proteins and 1000 EV phosphoproteins, respectively. We further applied targeted proteomics by coupling parallel reaction monitoring with parallel accumulation-serial fragmentation (prm-PASEF) to measure panels of proteins and phosphoproteins from salivary EVs collected before and after surgical resection. A panel of three total proteins and three phosphoproteins, most of which have previously been associated with OSCC and other cancer types, show sensitive response to the therapy in individual patients. Our study presents a novel strategy to the discovery of effective biomarkers for non-invasive assessment of OSCC surgical outcomes with small amount of saliva.

One-Pot Analytical Pipeline for Efficient and Sensitive Proteomic Analysis of Extracellular Vesicles.

Extracellular vesicle (EV) proteomics emerges as an effective tool for discovering potential biomarkers for disease diagnosis, monitoring, and therapeutics. However, the current workflow of mass spectrometry-based EV proteome analysis is not fully compatible in a clinical setting due to inefficient EV isolation methods and a tedious sample preparation process. To streamline and improve the efficiency of EV proteome analysis, here we introduce a one-pot analytical pipeline integrating a robust EV isolation approach, EV total recovery and purification (EVtrap), with in situ protein sample preparation, to detect urinary EV proteome. By incorporating solvent-driven protein capture and fast on-bead digestion, the one-pot pipeline enabled the whole EV proteome analysis to be completed within one day. In comparison with the existing workflow, the one-pot pipeline was able to obtain better peptide yield and identify the equivalent number of unique EV proteins from 1 mL of urine. Finally, we applied the one-pot pipeline to profile proteomes in urinary EVs of bladder cancer patients. A total of 2774 unique proteins were identified in 53 urine samples using a 15 min gradient library-free data-independent acquisition method. Taken altogether, our novel one-pot analytical pipeline demonstrated its potential for routine and robust EV proteomics in biomedical applications.

Proteomic Discovery and Array-Based Validation of Biomarkers from Urinary Exosome by Supramolecular Probe.

Exosomes are nanoscale, membrane-enclosed vesicles with contents similar to their parent cells, which are rich in potential biomarkers. Urine, as a noninvasive sampling body fluid, has the advantages of being simple to collect, stable in protein, diverse and not regulated by homeostatic mechanisms of the body, making it a favorable target for studying tumor biomarkers. In this report, the urinary exosomal proteome was analyzed and high-throughput downstream validation was performed using a supramolecular probe-based capture and in situ detection. The technology demonstrated the efficient enrichment of exosomes with a high concentration (5.5 × 1010 particles/mL) and a high purity (2.607 × 1010 particles/mg) of exosomes from urine samples. Proteomic analysis of urine samples from patients with hepatocellular carcinoma and healthy individuals combined with proteomic screening techniques revealed that 68 proteins were up-regulated in patients with hepatocellular carcinoma. As a proof-of-principle study, three of these differentially expressed proteins, including OLFM4, HDGF and GDF15, were validated using the supramolecular probe-based array (48 samples per batch). These findings demonstrate the great potential of this approach toward a liquid biopsy for the discovery and validation of biomarkers from urinary exosomes, and it can be extended to various biological samples with lower content of exosomes.