Phosphoprotein dynamics of interacting T cells and tumor cells by HySic.

Functional interactions between cytotoxic T cells and tumor cells are central to anti-cancer immunity. However, our understanding of the proteins involved is limited. Here, we present HySic (hybrid quantification of stable isotope labeling by amino acids in cell culture [SILAC]-labeled interacting cells) as a method to quantify protein and phosphorylation dynamics between and within physically interacting cells. Using co-cultured T cells and tumor cells, we directly measure the proteome and phosphoproteome of engaged cells without the need for physical separation. We identify proteins whose abundance or activation status changes upon T cell:tumor cell interaction and validate our method with established signal transduction pathways including interferon γ (IFNγ) and tumor necrosis factor (TNF). Furthermore, we identify the RHO/RAC/PAK1 signaling pathway to be activated upon cell engagement and show that pharmacologic inhibition of PAK1 sensitizes tumor cells to T cell killing. Thus, HySic is a simple method to study rapid protein signaling dynamics in physically interacting cells that is easily extended to other biological systems.

Deep (phospho)proteomics profiling of pre- treatment needle biopsies identifies signatures of treatment resistance in HER2+ breast cancer.

Patients with early-stage HER2-overexpressing breast cancer struggle with treatment resistance in 20%-40% of cases. More information is needed to predict HER2 therapy response and resistance in vivo. In this study, we perform (phospho)proteomics analysis of pre-treatment HER2+ needle biopsies of early-stage invasive breast cancer to identify molecular signatures predictive of treatment response to trastuzumab, pertuzumab, and chemotherapy. Our data show that accurate quantification of the estrogen receptor (ER) and HER2 biomarkers, combined with the assessment of associated biological features, has the potential to enable better treatment outcome prediction. In addition, we identify cellular mechanisms that potentially precondition tumors to resist therapy. We find proteins with expression changes that correlate with resistance and constitute to a strong predictive signature for treatment success in our patient cohort. Our results highlight the multifactorial nature of drug resistance in vivo and demonstrate the necessity of deep tumor profiling.

Optimized Suspension Trapping Method for Phosphoproteomics Sample Preparation.

A successful mass spectrometry-based phosphoproteomics analysis relies on effective sample preparation strategies. Suspension trapping (S-Trap) is a novel, rapid, and universal method of sample preparation that is increasingly applied in bottom-up proteomics studies. However, the performance of the S-Trap protocol for phosphoproteomics studies is unclear. In the existing S-Trap protocol, the addition of phosphoric acid (PA) and methanol buffer creates a fine protein suspension to capture proteins on a filter and is a critical step for subsequent protein digestion. Herein, we demonstrate that this addition of PA is detrimental to downstream phosphopeptide enrichment, rendering the standard S-Trap protocol suboptimal for phosphoproteomics. In this study, the performance of the S-Trap digestion for proteomics and phosphoproteomics is systematically evaluated in large-scale and small-scale samples. The results of this comparative analysis show that an optimized S-Trap approach, where trifluoroacetic acid is substituted for PA, is a simple and effective method to prepare samples for phosphoproteomics. Our optimized S-Trap protocol is applied to extracellular vesicles to demonstrate superior sample preparation workflow for low-abundance, membrane-rich samples.

CSPP1 stabilizes growing microtubule ends and damaged lattices from the luminal side.

Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling the formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates the elongation and stability of ciliary axonemes and other microtubule-based structures.

High-Throughput Assessment of Kinome-wide Activation States.

Aberrant kinase activity has been linked to a variety of disorders; however, methods to probe kinase activation states in cells have been lacking. Until now, kinase activity has mainly been deduced from either protein expression or substrate phosphorylation levels. Here, we describe a strategy to directly infer kinase activation through targeted quantification of T-loop phosphorylation, which serves as a critical activation switch in a majority of protein kinases. Combining selective phosphopeptide enrichment with robust targeted mass spectrometry, we provide highly specific assays for 248 peptides, covering 221 phosphosites in the T-loop region of 178 human kinases. Using these assays, we monitored the activation of 63 kinases through 73 T-loop phosphosites across different cell types, primary cells, and patient-derived tissue material. The sensitivity of our assays is highlighted by the reproducible detection of TNF-α-induced RIPK1 activation and the detection of 46 T-loop phosphorylation sites from a breast tumor needle biopsy.

Augmenting Immunotherapy Impact by Lowering Tumor TNF Cytotoxicity Threshold.

New opportunities are needed to increase immune checkpoint blockade (ICB) benefit. Whereas the interferon (IFN)γ pathway harbors both ICB resistance factors and therapeutic opportunities, this has not been systematically investigated for IFNγ-independent signaling routes. A genome-wide CRISPR/Cas9 screen to sensitize IFNγ receptor-deficient tumor cells to CD8 T cell elimination uncovered several hits mapping to the tumor necrosis factor (TNF) pathway. Clinically, we show that TNF antitumor activity is only limited in tumors at baseline and in ICB non-responders, correlating with its low abundance. Taking advantage of the genetic screen, we demonstrate that ablation of the top hit, TRAF2, lowers the TNF cytotoxicity threshold in tumors by redirecting TNF signaling to favor RIPK1-dependent apoptosis. TRAF2 loss greatly enhanced the therapeutic potential of pharmacologic inhibition of its interaction partner cIAP, another screen hit, thereby cooperating with ICB. Our results suggest that selective reduction of the TNF cytotoxicity threshold increases the susceptibility of tumors to immunotherapy.

Proteome-wide Analysis of Protein Thermal Stability in the Model Higher Plant Arabidopsis thaliana.

Modern tandem MS-based sequencing technologies allow for the parallel measurement of concentration and covalent modifications for proteins within a complex sample. Recently, this capability has been extended to probe a proteome's three-dimensional structure and conformational state by determining the thermal denaturation profile of thousands of proteins simultaneously. Although many animals and their resident microbes exist under a relatively narrow, regulated physiological temperature range, plants take on the often widely ranging temperature of their surroundings, possibly influencing the evolution of protein thermal stability. In this report we present the first in-depth look at the thermal proteome of a plant species, the model organism Arabidopsis thaliana By profiling the melting curves of over 1700 Arabidopsis proteins using six biological replicates, we have observed significant correlation between protein thermostability and several known protein characteristics, including molecular weight and the composition ratio of charged to polar amino acids. We also report on a divergence of the thermostability of the core and regulatory domains of the plant 26S proteasome that may reflect a unique property of the way protein turnover is regulated during temperature stress. Lastly, the highly replicated database of Arabidopsis melting temperatures reported herein provides baseline data on the variability of protein behavior in the assay. Unfolding behavior and experiment-to-experiment variability were observed to be protein-specific traits, and thus this data can serve to inform the design and interpretation of future targeted assays to probe the conformational status of proteins from plants exposed to different chemical, environmental and genetic challenges.

Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants.

Abscisic acid (ABA)¹ is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs rapid phosphoproteomic changes.

Phosphoproteomic Analyses Reveal Early Signaling Events in the Osmotic Stress Response.

Elucidating how plants sense and respond to water loss is important for identifying genetic and chemical interventions that may help sustain crop yields in water-limiting environments. Currently, the molecular mechanisms involved in the initial perception and response to dehydration are not well understood. Modern mass spectrometric methods for quantifying changes in the phosphoproteome provide an opportunity to identify key phosphorylation events involved in this process. Here, we have used both untargeted and targeted isotope-assisted mass spectrometric methods of phosphopeptide quantitation to characterize proteins in Arabidopsis (Arabidopsis thaliana) whose degree of phosphorylation is rapidly altered by hyperosmotic treatment. Thus, protein phosphorylation events responsive to 5 min of 0.3 m mannitol treatment were first identified using 15N metabolic labeling and untargeted mass spectrometry with a high-resolution ion-trap instrument. The results from these discovery experiments were then validated using targeted Selected Reaction Monitoring mass spectrometry with a triple quadrupole. Targeted Selected Reaction Monitoring experiments were conducted with plants treated under nine different environmental perturbations to determine whether the phosphorylation changes were specific for osmosignaling or involved cross talk with other signaling pathways. The results indicate that regulatory proteins such as members of the mitogen-activated protein kinase family are specifically phosphorylated in response to osmotic stress. Proteins involved in 5' messenger RNA decapping and phosphatidylinositol 3,5-bisphosphate synthesis were also identified as targets of dehydration-induced phosphoregulation. The results of these experiments demonstrate the utility of targeted phosphoproteomic analysis in understanding protein regulation networks and provide new insight into cellular processes involved in the osmotic stress response.