Proteogenomic characterization of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.

Integrated Proteogenomic Characterization of Clear Cell Renal Cell Carcinoma.

To elucidate the deregulated functional modules that drive clear cell renal cell carcinoma (ccRCC), we performed comprehensive genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic characterization of treatment-naive ccRCC and paired normal adjacent tissue samples. Genomic analyses identified a distinct molecular subgroup associated with genomic instability. Integration of proteogenomic measurements uniquely identified protein dysregulation of cellular mechanisms impacted by genomic alterations, including oxidative phosphorylation-related metabolism, protein translation processes, and phospho-signaling modules. To assess the degree of immune infiltration in individual tumors, we identified microenvironment cell signatures that delineated four immune-based ccRCC subtypes characterized by distinct cellular pathways. This study reports a large-scale proteogenomic analysis of ccRCC to discern the functional impact of genomic alterations and provides evidence for rational treatment selection stemming from ccRCC pathobiology.

MS-PyCloud: A Cloud Computing-Based Pipeline for Proteomic and Glycoproteomic Data Analyses.

Rapid development and wide adoption of mass spectrometry-based glycoproteomic technologies have empowered scientists to study proteins and protein glycosylation in complex samples on a large scale. This progress has also created unprecedented challenges for individual laboratories to store, manage, and analyze proteomic and glycoproteomic data, both in the cost for proprietary software and high-performance computing and in the long processing time that discourages on-the-fly changes of data processing settings required in explorative and discovery analysis. We developed an open-source, cloud computing-based pipeline, MS-PyCloud, with graphical user interface (GUI), for proteomic and glycoproteomic data analysis. The major components of this pipeline include data file integrity validation, MS/MS database search for spectral assignments to peptide sequences, false discovery rate estimation, protein inference, quantitation of global protein levels, and specific glycan-modified glycopeptides as well as other modification-specific peptides such as phosphorylation, acetylation, and ubiquitination. To ensure the transparency and reproducibility of data analysis, MS-PyCloud includes open-source software tools with comprehensive testing and versioning for spectrum assignments. Leveraging public cloud computing infrastructure via Amazon Web Services (AWS), MS-PyCloud scales seamlessly based on analysis demand to achieve fast and efficient performance. Application of the pipeline to the analysis of large-scale LC-MS/MS data sets demonstrated the effectiveness and high performance of MS-PyCloud. The software can be downloaded at https://github.com/huizhanglab-jhu/ms-pycloud.

Unbiased Proteomic and Phosphoproteomic Analysis Identifies Response Signatures and Novel Susceptibilities After Combined MEK and mTOR Inhibition in BRAFV600E Mutant Glioma.

The mitogen-activated protein kinase pathway is one of the most frequently altered pathways in cancer. It is involved in the control of cell proliferation, invasion, and metabolism, and can cause resistance to therapy. A number of aggressive malignancies, including melanoma, colon cancer, and glioma, are driven by a constitutively activating missense mutation (V600E) in the v-Raf murine sarcoma viral oncogene homolog B (BRAF) component of the pathway. Mitogen-activated protein kinase kinase (MEK) inhibition is initially effective in targeting these cancers, but reflexive activation of mammalian target of rapamycin (mTOR) signaling contributes to frequent therapy resistance. We have previously demonstrated that combination treatment with the MEK inhibitor trametinib and the dual mammalian target of rapamycin complex 1/2 inhibitor TAK228 improves survival and decreases vascularization in a BRAFV600E mutant glioma model. To elucidate the mechanism of action of this combination therapy and understand the ensuing tumor response, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAFV600E mutant glioma xenografts after short-course treatment with trametinib and TAK228. We identified 13,313 proteins and 30,928 localized phosphosites, of which 12,526 proteins and 17,444 phosphosites were quantified across all samples (data available via ProteomeXchange; identifier PXD022329). We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the mitogen-activated protein kinase and mTOR pathways. Combination therapy also increased apoptotic signaling, suppressed angiogenesis signaling, and broadly suppressed the activity of the cyclin-dependent kinases. In response to combination therapy, both epidermal growth factor receptor and class 1 histone deacetylase proteins were activated. This study reports a detailed (phospho)proteomic analysis of the response of BRAFV600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies new targets for the development of rational combination therapies for BRAF-driven tumors.

Neoplastic cell enrichment of tumor tissues using coring and laser microdissection for proteomic and genomic analyses of pancreatic ductal adenocarcinoma.

BACKGROUND: The identification of differentially expressed tumor-associated proteins and genomic alterations driving neoplasia is critical in the development of clinical assays to detect cancers and forms the foundation for understanding cancer biology. One of the challenges in the analysis of pancreatic ductal adenocarcinoma (PDAC) is the low neoplastic cellularity and heterogeneous composition of bulk tumors. To enrich neoplastic cells from bulk tumor tissue, coring, and laser microdissection (LMD) sampling techniques have been employed. In this study, we assessed the protein and KRAS mutation changes associated with samples obtained by these enrichment techniques and evaluated the fraction of neoplastic cells in PDAC for proteomic and genomic analyses. METHODS: Three fresh frozen PDAC tumors and their tumor-matched normal adjacent tissues (NATs) were obtained from three sampling techniques using bulk, coring, and LMD; and analyzed by TMT-based quantitative proteomics. The protein profiles and characterizations of differentially expressed proteins in three sampling groups were determined. These three PDACs and samples of five additional PDACs obtained by the same three sampling techniques were also subjected to genomic analysis to characterize KRAS mutations. RESULTS: The neoplastic cellularity of eight PDACs ranged from less than 10% to over 80% based on morphological review. Distinctive proteomic patterns and abundances of certain tumor-associated proteins were revealed when comparing the tumors and NATs by different sampling techniques. Coring and bulk tissues had comparable proteome profiles, while LMD samples had the most distinct proteome composition compared to bulk tissues. Further genomic analysis of bulk, cored, or LMD samples demonstrated that KRAS mutations were significantly enriched in LMD samples while coring was less effective in enriching for KRAS mutations when bulk tissues contained a relatively low neoplastic cellularity. CONCLUSIONS: In addition to bulk tissues, samples from LMD and coring techniques can be used for proteogenomic studies. The greatest enrichment of neoplastic cellularity is obtained with the LMD technique.

OmicsOne: associate omics data with phenotypes in one-click.

BACKGROUND: The rapid advancements of high throughput "omics" technologies have brought a massive amount of data to process during and after experiments. Multi-omic analysis facilitates a deeper interrogation of a dataset and the discovery of interesting genes, proteins, lipids, glycans, metabolites, or pathways related to the corresponding phenotypes in a study. Many individual software tools have been developed for data analysis and visualization. However, it still lacks an efficient way to investigate the phenotypes with multiple omics data. Here, we present OmicsOne as an interactive web-based framework for rapid phenotype association analysis of multi-omic data by integrating quality control, statistical analysis, and interactive data visualization on 'one-click'. MATERIALS AND METHODS: OmicsOne was applied on the previously published proteomic and glycoproteomic data sets of high-grade serous ovarian carcinoma (HGSOC) and the published proteome data set of lung squamous cell carcinoma (LSCC) to confirm its performance. The data was analyzed through six main functional modules implemented in OmicsOne: (1) phenotype profiling, (2) data preprocessing and quality control, (3) knowledge annotation, (4) phenotype associated features discovery, (5) correlation and regression model analysis for phenotype association analysis on individual features, and (6) enrichment analysis for phenotype association analysis on interested feature sets. RESULTS: We developed an integrated software solution, OmicsOne, for the phenotype association analysis on multi-omics data sets. The application of OmicsOne on the public data set of ovarian cancer data showed that the software could confirm the previous observations consistently and discover new evidence for HNRNPU and a glycopeptide of HYOU1 as potential biomarkers for HGSOC data sets. The performance of OmicsOne was further demonstrated in the Tumor and NAT comparison study on the proteome data set of LSCC. CONCLUSIONS: OmicsOne can effectively simplify data analysis and reveal the significant associations between phenotypes and potential biomarkers, including genes, proteins, and glycopeptides, in minutes to assist users to understand aberrant biological processes.

Impact of Increased FUT8 Expression on the Extracellular Vesicle Proteome in Prostate Cancer Cells.

Extracellular vesicles (EVs) are involved in intercellular communication, transporting proteins and nucleic acids to proximal and distal regions. There is evidence of glycosylation influencing protein routing into EVs; however, the impact of aberrant cellular glycotransferase expression on EV protein profiles has yet to be evaluated. In this study, we paired extracellular vesicle characterization and quantitative proteomics to determine the systemic impact of altered α(1,6)fucosyltranferase (FUT8) expression on prostate cancer-derived EVs. Our results showed that increased cellular expression of FUT8 could reduce the number of vesicles secreted by prostate cancer cells as well as increase the abundance of proteins associated with cell motility and prostate cancer metastasis. In addition, overexpression of FUT8 resulted in altered glycans on select EV-derived glycoproteins. This study presents the first evidence of altered cellular glycosylation impacting EV protein profiles and provides further rationale for exploring the functional role of glycosylation in EV biogenesis and biology.

Glycans, Glycosite, and Intact Glycopeptide Analysis of N-Linked Glycoproteins Using Liquid Handling Systems.

Aberrant glycosylation has been shown to associate with disease progression, and with glycoproteins representing the major protein component of biological fluids this makes them attractive targets for disease monitoring. Leveraging glycoproteomic analysis via mass spectrometry (MS) could provide the insight into the altered glycosylation patterns that relate to disease progression. However, investigation of large sample cohorts requires rapid, efficient, and highly reproducible sample preparation. To address the limitation, we developed a high-throughput method for characterizing glycans, glycosites, and intact glycopeptides (IGPs) derived from N-linked glycoproteins. We combined disparate peptide enrichment strategies (i.e., hydrophilic and hydrophobic) and a liquid handling platform allowing for a high throughput and rapid enrichment of IGP in a 96-well plate format. The C18/MAX-Tip workflow reduced sample processing time and facilitated the selective enrichment of IGPs from complex samples. Furthermore, our approach enabled the analysis of deglycosylated peptides and glycans from enriched IGPs following PNGase F digest. Following development and optimization of the C18/MAX-Tip methodology using the standard glycoprotein, fetuin, we investigated normal urine samples to obtain N-linked glycoprotein information. Together, our method enables a high-throughput enrichment of glycan, glycosites, and IGPs from biological samples.

Deep Proteomics Using Two Dimensional Data Independent Acquisition Mass Spectrometry.

Methodologies that facilitate high-throughput proteomic analysis are a key step toward moving proteome investigations into clinical translation. Data independent acquisition (DIA) has potential as a high-throughput analytical method due to the reduced time needed for sample analysis, as well as its highly quantitative accuracy. However, a limiting feature of DIA methods is the sensitivity of detection of low abundant proteins and depth of coverage, which other mass spectrometry approaches address by two-dimensional fractionation (2D) to reduce sample complexity during data acquisition. In this study, we developed a 2D-DIA method intended for rapid- and deeper-proteome analysis compared to conventional 1D-DIA analysis. First, we characterized 96 individual fractions obtained from the protein standard, NCI-7, using a data-dependent approach (DDA), identifying a total of 151,366 unique peptides from 11,273 protein groups. We observed that the majority of the proteins can be identified from just a few selected fractions. By performing an optimization analysis, we identified six fractions with high peptide number and uniqueness that can account for 80% of the proteins identified in the entire experiment. These selected fractions were combined into a single sample which was then subjected to DIA (referred to as 2D-DIA) quantitative analysis. Furthermore, improved DIA quantification was achieved using a hybrid spectral library, obtained by combining peptides identified from DDA data with peptides identified directly from the DIA runs with the help of DIA-Umpire. The optimized 2D-DIA method allowed for improved identification and quantification of low abundant proteins compared to conventional unfractionated DIA analysis (1D-DIA). We then applied the 2D-DIA method to profile the proteomes of two breast cancer patient-derived xenograft (PDX) models, quantifying 6,217 and 6,167 unique proteins in basal- and luminal- tumors, respectively. Overall, this study demonstrates the potential of high-throughput quantitative proteomics using a novel 2D-DIA method.

Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis.

Mass spectrometry-based urinary proteomics is one of the most attractive strategies to discover proteins for diagnosis, prognosis, monitoring, or prediction of therapeutic responses of urological diseases involving the kidney, prostate, and bladder; however, interfering compounds found in urine necessitate sample preparation strategies that are currently not suitable for urinary proteomics in the clinical setting. Herein, we describe the C4-tip method, comprising a simple, automated strategy utilizing a reverse-phase resin tip-based format and "on-tip" digestion to examine the urine proteome. We first determined the optimal conditions for protein isolation and protease digestion on the C4-tip using the standard protein bovine fetuin. Next, we applied the C4-tip method to urinary proteomics, identifying a total of 813 protein groups using LC-MS/MS, with identified proteins from the C4-tip method displaying a similar distribution of gene ontology (GO) cellular component assignments compared to identified proteins from an ultrafiltration preparation method. Finally, we assessed the reproducibility of the C4-tip method, revealing a high Spearman correlation R-value for shared proteins identified across all tips. Together, we have shown the C4-tip method to be a simple, robust method for high-throughput analysis of the urinary proteome by mass spectrometry in the clinical setting.

Evaluation of NCI-7 Cell Line Panel as a Reference Material for Clinical Proteomics.

Reference materials are vital to benchmarking the reproducibility of clinical tests and essential for monitoring laboratory performance for clinical proteomics. The reference material utilized for mass spectrometric analysis of the human proteome would ideally contain enough proteins to be suitably representative of the human proteome, as well as exhibit a stable protein composition in different batches of sample regeneration. Previously, The Clinical Proteomic Tumor Analysis Consortium (CPTAC) utilized a PDX-derived comparative reference (CompRef) materials for the longitudinal assessment of proteomic performance; however, inherent drawbacks of PDX-derived material, including extended time needed to grow tumors and high level of expertise needed, have resulted in efforts to identify a new source of CompRef material. In this study, we examined the utility of using a panel of seven cancer cell lines, NCI-7 Cell Line Panel, as a reference material for mass spectrometric analysis of human proteome. Our results showed that not only is the NCI-7 material suitable for benchmarking laboratory sample preparation methods, but also NCI-7 sample generation is highly reproducible at both the global and phosphoprotein levels. In addition, the predicted genomic and experimental coverage of the NCI-7 proteome suggests the NCI-7 material may also have applications as a universal standard proteomic reference.

Quality Assessments of Long-Term Quantitative Proteomic Analysis of Breast Cancer Xenograft Tissues.

Clinical proteomics requires large-scale analysis of human specimens to achieve statistical significance. We evaluated the long-term reproducibility of an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomics strategy using one channel for reference across all samples in different iTRAQ sets. A total of 148 liquid chromatography tandem mass spectrometric (LC-MS/MS) analyses were completed, generating six 2D LC-MS/MS data sets for human-in-mouse breast cancer xenograft tissues representative of basal and luminal subtypes. Such large-scale studies require the implementation of robust metrics to assess the contributions of technical and biological variability in the qualitative and quantitative data. Accordingly, we derived a quantification confidence score based on the quality of each peptide-spectrum match to remove quantification outliers from each analysis. After combining confidence score filtering and statistical analysis, reproducible protein identification and quantitative results were achieved from LC-MS/MS data sets collected over a 7-month period. This study provides the first quality assessment on long-term stability and technical considerations for study design of a large-scale clinical proteomics project.

Comprehensive proteogenomic characterization of rare kidney tumors.

Non-clear cell renal cell carcinomas (non-ccRCCs) encompass diverse malignant and benign tumors. Refinement of differential diagnosis biomarkers, markers for early prognosis of aggressive disease, and therapeutic targets to complement immunotherapy are current clinical needs. Multi-omics analyses of 48 non-ccRCCs compared with 103 ccRCCs reveal proteogenomic, phosphorylation, glycosylation, and metabolic aberrations in RCC subtypes. RCCs with high genome instability display overexpression of IGF2BP3 and PYCR1. Integration of single-cell and bulk transcriptome data predicts diverse cell-of-origin and clarifies RCC subtype-specific proteogenomic signatures. Expression of biomarkers MAPRE3, ADGRF5, and GPNMB differentiates renal oncocytoma from chromophobe RCC, and PIGR and SOSTDC1 distinguish papillary RCC from MTSCC. This study expands our knowledge of proteogenomic signatures, biomarkers, and potential therapeutic targets in non-ccRCC.

Integrated glycoproteomic characterization of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC), a common form of RCC, is responsible for the high mortality rate of kidney cancer. Dysregulations of glycoproteins have been shown to associate with ccRCC. However, the molecular mechanism has not been well characterized. Here, a comprehensive glycoproteomic analysis is conducted using 103 tumors and 80 paired normal adjacent tissues. Altered glycosylation enzymes and corresponding protein glycosylation are observed, while two of the major ccRCC mutations, BAP1 and PBRM1, show distinct glycosylation profiles. Additionally, inter-tumor heterogeneity and cross-correlation between glycosylation and phosphorylation are observed. The relation of glycoproteomic features to genomic, transcriptomic, proteomic, and phosphoproteomic changes shows the role of glycosylation in ccRCC development with potential for therapeutic interventions. This study reports a large-scale tandem mass tag (TMT)-based quantitative glycoproteomic analysis of ccRCC that can serve as a valuable resource for the community.

Characterization of core fucosylation via sequential enzymatic treatments of intact glycopeptides and mass spectrometry analysis.

Core fucosylation of N-linked glycoproteins has been linked to the functions of glycoproteins in physiological and pathological processes. However, quantitative characterization of core fucosylation remains challenging due to the complexity and heterogeneity of N-linked glycosylation. Here we report a mass spectrometry-based method that employs sequential treatment of intact glycopeptides with enzymes (STAGE) to analyze site-specific core fucosylation of glycoproteins. The STAGE method utilizes Endo F3 followed by PNGase F treatment to generate mass signatures for glycosites that are formerly modified by core fucosylated N-linked glycans. We benchmark the STAGE method and use it to characterize site specific core fucosylation of glycoproteins from human hepatocellular carcinoma and pancreatic ductal adenocarcinoma, resulting in the identification of 1130 and 782 core fucosylated glycosites, respectively. These results indicate that our STAGE method enables quantitative characterization of core fucosylation events from complex protein mixtures, which may benefit our understanding of core fucosylation functions in various diseases.

Spatially restricted drivers and transitional cell populations cooperate with the microenvironment in untreated and chemo-resistant pancreatic cancer.

Pancreatic ductal adenocarcinoma is a lethal disease with limited treatment options and poor survival. We studied 83 spatial samples from 31 patients (11 treatment-naïve and 20 treated) using single-cell/nucleus RNA sequencing, bulk-proteogenomics, spatial transcriptomics and cellular imaging. Subpopulations of tumor cells exhibited signatures of proliferation, KRAS signaling, cell stress and epithelial-to-mesenchymal transition. Mapping mutations and copy number events distinguished tumor populations from normal and transitional cells, including acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasia. Pathology-assisted deconvolution of spatial transcriptomic data identified tumor and transitional subpopulations with distinct histological features. We showed coordinated expression of TIGIT in exhausted and regulatory T cells and Nectin in tumor cells. Chemo-resistant samples contain a threefold enrichment of inflammatory cancer-associated fibroblasts that upregulate metallothioneins. Our study reveals a deeper understanding of the intricate substructure of pancreatic ductal adenocarcinoma tumors that could help improve therapy for patients with this disease.

Defining candidate mRNA and protein EV biomarkers to discriminate ccRCC and pRCC from non-malignant renal cells in vitro.

Renal cell carcinoma (RCC) accounts for over 400,000 new cases and 175,000 deaths annually. Diagnostic RCC biomarkers may prevent overtreatment in patients with early disease. Extracellular vesicles (EVs) are a promising source of RCC biomarkers because EVs carry proteins and messenger RNA (mRNA) among other biomolecules. We aimed to identify biomarkers and assess biological functions of EV cargo from clear cell RCC (ccRCC), papillary RCC (pRCC), and benign kidney cell lines. EVs were enriched from conditioned cell media by size exclusion chromatography. The EV proteome was assessed using Tandem Mass Tag mass spectrometry (TMT-MS) and NanoString nCounter technology was used to profile 770 cancer-related mRNA present in EVs. The heterogeneity of protein and mRNA abundance and identification highlighted the heterogeneity of EV cargo, even between cell lines of a similar pathological group (e.g., ccRCC or pRCC). Overall, 1726 proteins were quantified across all EV samples, including 181 proteins that were detected in all samples. In the targeted profiling of mRNA by NanoString, 461 mRNAs were detected in EVs from at least one cell line, including 159 that were present in EVs from all cell lines. In addition to a shared EV cargo signature, pRCC, ccRCC, and/or benign renal cell lines also showed unique signatures. Using this multi-omics approach, we identified 34 protein candidate pRCC EV biomarkers and 20 protein and 8 mRNA candidate ccRCC EV biomarkers for clinical validation.

Proteogenomic insights into the biology and treatment of HPV-negative head and neck squamous cell carcinoma.

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.

Orthogonal Proteomic Platforms and Their Implications for the Stable Classification of High-Grade Serous Ovarian Cancer Subtypes.

The National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) established a harmonized method for large-scale clinical proteomic studies. SWATH-MS, an instance of data-independent acquisition (DIA) proteomic methods, is an alternate proteomic approach. In this study, we used SWATH-MS to analyze remnant peptides from the original retrospective TCGA samples generated for the CPTAC ovarian cancer proteogenomic study. The SWATH-MS results recapitulated the confident identification of differentially expressed proteins in enriched pathways associated with the robust Mesenchymal high-grade serous ovarian cancer subtype and the homologous recombination deficient tumors. Hence, SWATH/DIA-MS presents a promising complementary or orthogonal alternative to the CPTAC proteomic workflow, with the advantages of simpler and faster workflows and lower sample consumption, albeit with shallower proteome coverage. In summary, both analytical methods are suitable to characterize clinical samples, providing proteomic workflow alternatives for cancer researchers depending on the context-specific goals of the studies.