Isobaric labeling using tandem mass tags (TMTs) is increasingly applied for deep-scale proteomic studies in a multitude of organisms and addressing diverse research questions. The cost of labeling ...reagents represents a substantial proportion of the total expenses for conducting such experiments. Here, Zecha et al. present an economically optimized TMT labeling approach that reduces the quantity of required labeling reagent by a factor of eight and reproducibly achieves complete labeling.
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Highlights
•TMT labeling protocol with excellent intra- and interlaboratory reproducibility.•Complete in-solution labeling of peptides using 1/8 of recommended TMT quantities.•Demonstration of utility for deep-scale (phospho)proteome analysis.
Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Experiments focusing on proteoform analysis in drug time course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an experiment. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and overlabeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800 μg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concentrations. When reaction volumes were reduced to maintain TMT and peptide concentrations of at least 10 mm and 2 g/l, respectively, TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99% and excellent intra- and interlaboratory reproducibility. The utility of the optimized protocol was further demonstrated in a deep-scale proteome and phosphoproteome analysis of patient-derived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chemistry and provide guidance on adopting efficient labeling protocols for different peptide quantities.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The autophagy-lysosomal pathway plays an essential role in cellular homeostasis as well as a protective function against a variety of diseases including neurodegeneration. Conversely, inhibition of ...autophagy, for example due to lysosomal dysfunction, can lead to pathological accumulation of dysfunctional autophagosomes and consequent neuronal cell death. We previously reported that autophagy is inhibited and contributes to neuronal cell death following spinal cord injury (SCI). In this study, we examined lysosomal function and explored the mechanism of lysosomal defects following SCI. Our data demonstrated that expression levels and processing of the lysosomal enzyme cathepsin D (CTSD) are decreased by 2 h after SCI. Enzymatic activity levels of CTSD and another lysosomal enzyme, N-acetyl-alpha-glucosaminidase, are both decreased 24 h post injury, indicating general lysosomal dysfunction. Subcellular fractionation and immunohistochemistry analysis demonstrated that this dysfunction is due to lysosomal membrane permeabilization and leakage of lysosomal contents into the cytosol. To directly assess extent and mechanisms of damage to lysosomal membranes, we performed mass spectrometry-based lipidomic analysis of lysosomes purified from SCI and control spinal cord. At 2 h post injury our data demonstrated increase in several classes of lysosophospholipids, the products of phospholipases (PLAs), as well as accumulation of PLA activators, ceramides. Phospholipase cPLA2, the main PLA species expressed in the CNS, has been previously implicated in mediation of secondary injury after SCI, but the mechanisms of its involvement remain unclear. Our data demonstrate that cPLA2 is activated within 2 h after SCI preferentially in the lysosomal fraction, where it colocalizes with lysosomal-associated membrane protein 2 in neurons. Inhibition of cPLA2 in vivo decreased lysosomal damage, restored autophagy flux, and reduced neuronal cell damage. Taken together our data implicate lysosomal defects in pathophysiology of SCI and for the first time indicate that cPLA2 activation leads to lysosomal damage causing neuronal autophagosome accumulation associated with neuronal cell death.
Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic options. We characterized the proteogenomic landscape of LSCC, providing a deeper exposition of LSCC ...biology with potential therapeutic implications. We identify NSD3 as an alternative driver in FGFR1-amplified tumors and low-p63 tumors overexpressing the therapeutic target survivin. SOX2 is considered undruggable, but our analyses provide rationale for exploring chromatin modifiers such as LSD1 and EZH2 to target SOX2-overexpressing tumors. Our data support complex regulation of metabolic pathways by crosstalk between post-translational modifications including ubiquitylation. Numerous immune-related proteogenomic observations suggest directions for further investigation. Proteogenomic dissection of CDKN2A mutations argue for more nuanced assessment of RB1 protein expression and phosphorylation before declaring CDK4/6 inhibition unsuccessful. Finally, triangulation between LSCC, LUAD, and HNSCC identified both unique and common therapeutic vulnerabilities. These observations and proteogenomics data resources may guide research into the biology and treatment of LSCC.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Serial multi-omic analysis of proteome, phosphoproteome, and acetylome provides insights into changes in protein expression, cell signaling, cross-talk and epigenetic pathways involved in disease ...pathology and treatment. However, ubiquitylome and HLA peptidome data collection used to understand protein degradation and antigen presentation have not together been serialized, and instead require separate samples for parallel processing using distinct protocols. Here we present MONTE, a highly sensitive multi-omic native tissue enrichment workflow, that enables serial, deep-scale analysis of HLA-I and HLA-II immunopeptidome, ubiquitylome, proteome, phosphoproteome, and acetylome from the same tissue sample. We demonstrate that the depth of coverage and quantitative precision of each 'ome is not compromised by serialization, and the addition of HLA immunopeptidomics enables the identification of peptides derived from cancer/testis antigens and patient specific neoantigens. We evaluate the technical feasibility of the MONTE workflow using a small cohort of patient lung adenocarcinoma tumors.
Saliby et al. show that a machine learning approach can accurately classify clear cell renal cell carcinoma (RCC) into distinct molecular subtypes using transcriptomic data. When applied to tumors ...biospecimens from the JAVELIN Renal 101 (JR101) trial, a benefit is observed with immune checkpoint inhibitor (ICI)-based therapy across all molecular subtypes.
Renal cell carcinoma (RCC) of variant histology comprises approximately 20% of kidney cancer diagnoses, yet the optimal therapy for these patients and the factors that impact immunotherapy response ...remain largely unknown. To better understand the determinants of immunotherapy response in this population, we characterized blood- and tissue-based immune markers for patients with variant histology RCC, or any RCC histology with sarcomatoid differentiation, enrolled in a phase II clinical trial of atezolizumab and bevacizumab. Baseline circulating (plasma) inflammatory cytokines were highly correlated with one another, forming an "inflammatory module" that was increased in International Metastatic RCC Database Consortium poor-risk patients and was associated with worse progression-free survival (PFS; P = 0.028). At baseline, an elevated circulating vascular endothelial growth factor A (VEGF-A) level was associated with a lack of response (P = 0.03) and worse PFS (P = 0.021). However, a larger increase in on-treatment levels of circulating VEGF-A was associated with clinical benefit (P = 0.01) and improved overall survival (P = 0.0058). Among peripheral immune cell populations, an on-treatment decrease in circulating PD-L1+ T cells was associated with improved outcomes, with a reduction in CD4+PD-L1+ HR, 0.62; 95% confidence interval (CI), 0.49-0.91; P = 0.016 and CD8+PD-L1+ T cells (HR, 0.59; 95% CI, 0.39-0.87; P = 0.009) correlated with improved PFS. Within the tumor itself, a higher percentage of terminally exhausted (PD-1+ and either TIM-3+ or LAG-3+) CD8+ T cells was associated with worse PFS (P = 0.028). Overall, these findings support the value of tumor and blood-based immune assessments in determining therapeutic benefit for patients with RCC receiving atezolizumab plus bevacizumab and provide a foundation for future biomarker studies for patients with variant histology RCC receiving immunotherapy-based combinations.
To explore the biology of lung adenocarcinoma (LUAD) and identify new therapeutic opportunities, we performed comprehensive proteogenomic characterization of 110 tumors and 101 matched normal ...adjacent tissues (NATs) incorporating genomics, epigenomics, deep-scale proteomics, phosphoproteomics, and acetylproteomics. Multi-omics clustering revealed four subgroups defined by key driver mutations, country, and gender. Proteomic and phosphoproteomic data illuminated biology downstream of copy number aberrations, somatic mutations, and fusions and identified therapeutic vulnerabilities associated with driver events involving KRAS, EGFR, and ALK. Immune subtyping revealed a complex landscape, reinforced the association of STK11 with immune-cold behavior, and underscored a potential immunosuppressive role of neutrophil degranulation. Smoking-associated LUADs showed correlation with other environmental exposure signatures and a field effect in NATs. Matched NATs allowed identification of differentially expressed proteins with potential diagnostic and therapeutic utility. This proteogenomics dataset represents a unique public resource for researchers and clinicians seeking to better understand and treat lung adenocarcinomas.
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•Comprehensive LUAD proteogenomics exposes multi-omic clusters and immune subtypes•Phosphoproteomics identifies candidate ALK-fusion diagnostic markers and targets•Candidate drug targets: PTPN11 (EGFR), SOS1 (KRAS), neutrophil degranulation (STK11)•Phospho and acetyl modifications denote tumor-specific markers and druggable proteins
Comprehensive proteogenomic characterization of lung adenocarcinomas and paired normal adjacent tissues from patients of diverse smoking status and country of origin yields insights into cancer taxonomy, oncogenesis, and immune response; offers novel candidate biomarkers and therapeutic targets; and provides a community resource for further discovery.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We present a proteogenomic study of 108 human papilloma virus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs). Proteomic analysis systematically catalogs HNSCC-associated proteins and ...phosphosites, prioritizes copy number drivers, and highlights an oncogenic role for RNA processing genes. Proteomic investigation of mutual exclusivity between FAT1 truncating mutations and 11q13.3 amplifications reveals dysregulated actin dynamics as a common functional consequence. Phosphoproteomics characterizes two modes of EGFR activation, suggesting a new strategy to stratify HNSCCs based on EGFR ligand abundance for effective treatment with inhibitory EGFR monoclonal antibodies. Widespread deletion of immune modulatory genes accounts for low immune infiltration in immune-cold tumors, whereas concordant upregulation of multiple immune checkpoint proteins may underlie resistance to anti-programmed cell death protein 1 monotherapy in immune-hot tumors. Multi-omic analysis identifies three molecular subtypes with high potential for treatment with CDK inhibitors, anti-EGFR antibody therapy, and immunotherapy, respectively. Altogether, proteogenomics provides a systematic framework to inform HNSCC biology and treatment.
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•A systematic inventory of HNSCC-associated proteins, phosphosites, and pathways•Three multi-omic subtypes linked to targeted treatment approaches and immunotherapy•Widespread deletion of immune modulatory genes accounts for loss of immunogenicity•Two modes of EGFR activation inform response to anti-EGFR monoclonal antibodies
Huang et al. report a proteogenomic study on 108 HPV-negative head and neck squamous cell carcinomas (HNSCCs). In addition to creating a comprehensive resource for pathogenic insights, multi-omic analysis identifies therapeutic hypotheses that may inform more precise approaches to treatment.
The integration of mass spectrometry-based proteomics with next-generation DNA and RNA sequencing profiles tumors more comprehensively. Here this “proteogenomics” approach was applied to 122 ...treatment-naive primary breast cancers accrued to preserve post-translational modifications, including protein phosphorylation and acetylation. Proteogenomics challenged standard breast cancer diagnoses, provided detailed analysis of the ERBB2 amplicon, defined tumor subsets that could benefit from immune checkpoint therapy, and allowed more accurate assessment of Rb status for prediction of CDK4/6 inhibitor responsiveness. Phosphoproteomics profiles uncovered novel associations between tumor suppressor loss and targetable kinases. Acetylproteome analysis highlighted acetylation on key nuclear proteins involved in the DNA damage response and revealed cross-talk between cytoplasmic and mitochondrial acetylation and metabolism. Our results underscore the potential of proteogenomics for clinical investigation of breast cancer through more accurate annotation of targetable pathways and biological features of this remarkably heterogeneous malignancy.
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•Comprehensive proteogenomics resource from prospectively collected breast tumors•Proteogenomics defines ERBB2 and Rb status with clinical implications•Acetylproteome profiling yields insights into subtype-specific cancer metabolism•Immune profiling nominates subsets of luminal tumors for immune therapy
Breast cancer is a highly heterogeneous disease with variable outcomes and subtype-driven treatment approaches, making precision medicine a considerable challenge. Proteogenomic analyses of 122 primary breast cancers provide insights into clinically relevant biology, including cell cycle dysregulation, tumor immunogenicity, aberrant metabolism, and heterogeneity in therapeutic target expression.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Background
Renal cell carcinoma of variant histology (vRCC) encompasses approximately 20% of RCC diagnoses. Due to a poor understanding of the different biologies of vRCCs, there is ...currently no standard of care for this type of tumor and guidelines are extrapolated from clear-cell RCC trials. A phase II trial of atezolizumab plus bevacizumab in patients with vRCC or sarcomatoid differentiation had an acceptable safety profile and evidence of clinical activity, particularly in tumors with positive PD-L1 expression or sarcomatoid differentiation. To better understand the determinants of immunotherapy response in this population, we characterized blood- and tissue-based immune markers for patients with vRCC, or any RCC histology with sarcomatoid differentiation, enrolled in this phase II trial of atezolizumab and bevacizumab.
Methods
We used patient data and biospecimens (blood and tumor) from a prospective phase-II clinical trial of the anti-PD-L1 antibody atezolizumab and the anti-VEGF-A antibody bevacizumab in patients with metastatic RCC with variant histology and/or sarcomatoid differentiation (NCT02724878). Blood samples were collected at baseline, prior to therapy (C1D1: cycle one day one), and on-treatment, following two cycles of therapy (C3D1: cycle three day one). Plasma was assayed for the levels of soluble factors (e.g., cytokines and growth factors), and peripheral blood mononuclear cells (PBMCs) were immunophenotyped using flow cytometry. In addition, formalin-fixed, paraffin-embedded (FFPE) archival or pre-treatment study tumor biopsies were analyzed by immunohistochemistry and immunofluorescence for enrolled subjects with available biospecimen. Baseline demographics, clinical characteristics, and treatment outcomes, including progression-free survival (PFS), overall survival (OS), and clinical benefit, were collected, and analyzed from the trial database. This study was approved by each participating institution's institutional review board, and all patients provided written informed consent. We used R (version 4.1.1) to carry out our analyses. We assessed the relationships between biomarker variables were analyzed using Pearson correlations and hierarchical clustering using Euclidean distance. All biomarker variables were Z-score normalized. We calculated an inflammatory module, the systemic inflammation score (SIS), which corresponds to the average score of the five highly correlated inflammatory cytokines: MIP-1b, IL-1, MCP-1, IL-6, and IL-13. We applied pair-wise Wilcoxon tests to evaluate the association of biomarkers with clinical benefit groups and log-rank-tests and univariate Cox regression models to evaluate the relationship of biomarkers with PFS and OS.
Results
With the exception of two angiogenic cytokines (VEGF-A and Angiopoietin 2) that were more abundant in patients with non-sarcomatoid tumors, baseline circulating cytokines (plasma) had similar distributions between sarcomatoid and non-sarcomatoid subgroups. Baseline circulating inflammatory cytokines were highly correlated with one another, forming an “inflammatory module” that was increased in IMDC-poor risk patients and was associated with worse PFS (p=0.028) and with resistance to therapy (p=0.033). At baseline, an elevated circulating VEGF-A level was associated with a lack of response (p=0.03) and worse PFS (p=0.021). However, a larger increase in on-treatment levels of circulating VEGF-A was associated with clinical benefit (p=0.01) and improved overall survival (p=0.0058). Among peripheral immune cell populations, an on-treatment decrease in circulating PD-L1+ T cells was associated with improved outcomes, with a reduction in CD4+ PD-L1+ (HR:0.620.49-0.91, p=0.016) and CD8+ PD-L1+ T cells (HR:0.590.39-0.87, p=0.009) correlated with improved PFS. Within the tumor itself, a higher percentage of terminally exhausted (PD-1+ and either TIM-3+ or LAG-3+) CD8+ T cells was associated with worse PFS (p=0.028). We observed similar trends for all of these results in different subgroups according to sarcomatoid status and histology.
Conclusions
Overall, these findings support the value of tumor and blood-based immune assessments in determining therapeutic benefit for patients with RCC receiving atezolizumab plus bevacizumab. Furthermore, they provide a foundation for future biomarker studies for patients with variant histology RCC receiving immunotherapy-based combinations.
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