Pan-cancer proteogenomics characterization of tumor immunity.

Despite the successes of immunotherapy in cancer treatment over recent decades, less than <10%-20% cancer cases have demonstrated durable responses from immune checkpoint blockade. To enhance the efficacy of immunotherapies, combination therapies suppressing multiple immune evasion mechanisms are increasingly contemplated. To better understand immune cell surveillance and diverse immune evasion responses in tumor tissues, we comprehensively characterized the immune landscape of more than 1,000 tumors across ten different cancers using CPTAC pan-cancer proteogenomic data. We identified seven distinct immune subtypes based on integrative learning of cell type compositions and pathway activities. We then thoroughly categorized unique genomic, epigenetic, transcriptomic, and proteomic changes associated with each subtype. Further leveraging the deep phosphoproteomic data, we studied kinase activities in different immune subtypes, which revealed potential subtype-specific therapeutic targets. Insights from this work will facilitate the development of future immunotherapy strategies and enhance precision targeting with existing agents.

Deep Learning Prediction Boosts Phosphoproteomics-Based Discoveries Through Improved Phosphopeptide Identification.

Shotgun phosphoproteomics enables high-throughput analysis of phosphopeptides in biological samples. One of the primary challenges associated with this technology is the relatively low rate of phosphopeptide identification during data analysis. This limitation hampers the full realization of the potential offered by shotgun phosphoproteomics. Here we present DeepRescore2, a computational workflow that leverages deep learning-based retention time and fragment ion intensity predictions to improve phosphopeptide identification and phosphosite localization. Using a state-of-the-art computational workflow as a benchmark, DeepRescore2 increases the number of correctly identified peptide-spectrum matches by 17% in a synthetic dataset and identifies 19% to 46% more phosphopeptides in biological datasets. In a liver cancer dataset, 30% of the significantly altered phosphosites between tumor and normal tissues and 60% of the prognosis-associated phosphosites identified from DeepRescore2-processed data could not be identified based on the state-of-the-art workflow. Notably, DeepRescore2-processed data uniquely identifies EGFR hyperactivation as a new target in poor-prognosis liver cancer, which is validated experimentally. Integration of deep learning prediction in DeepRescore2 improves phosphopeptide identification and facilitates biological discoveries.

SEPepQuant enhances the detection of possible isoform regulations in shotgun proteomics.

Shotgun proteomics is essential for protein identification and quantification in biomedical research, but protein isoform characterization is challenging due to the extensive number of peptides shared across proteins, hindering our understanding of protein isoform regulation and their roles in normal and disease biology. We systematically assess the challenge and opportunities of shotgun proteomics-based protein isoform characterization using in silico and experimental data, and then present SEPepQuant, a graph theory-based approach to maximize isoform characterization. Using published data from one induced pluripotent stem cell study and two human hepatocellular carcinoma studies, we demonstrate the ability of SEPepQuant in addressing the key limitations of existing methods, providing more comprehensive isoform-level characterization, identifying hundreds of isoform-level regulation events, and facilitating streamlined cross-study comparisons. Our analysis provides solid evidence to support a widespread role of protein isoform regulation in normal and disease processes, and SEPepQuant has broad applications to biological and translational research.

A proteogenomics data-driven knowledge base of human cancer.

By combining mass-spectrometry-based proteomics and phosphoproteomics with genomics, epi-genomics, and transcriptomics, proteogenomics provides comprehensive molecular characterization of cancer. Using this approach, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) has characterized over 1,000 primary tumors spanning 10 cancer types, many with matched normal tissues. Here, we present LinkedOmicsKB, a proteogenomics data-driven knowledge base that makes consistently processed and systematically precomputed CPTAC pan-cancer proteogenomics data available to the public through ∼40,000 gene-, protein-, mutation-, and phenotype-centric web pages. Visualization techniques facilitate efficient exploration and reasoning of complex, interconnected data. Using three case studies, we illustrate the practical utility of LinkedOmicsKB in providing new insights into genes, phosphorylation sites, somatic mutations, and cancer phenotypes. With precomputed results of 19,701 coding genes, 125,969 phosphosites, and 256 genotypes and phenotypes, LinkedOmicsKB provides a comprehensive resource to accelerate proteogenomics data-driven discoveries to improve our understanding and treatment of human cancer. A record of this paper's transparent peer review process is included in the supplemental information.

Deep learning prediction boosts phosphoproteomics-based discoveries through improved phosphopeptide identification.

Shotgun phosphoproteomics enables high-throughput analysis of phosphopeptides in biological samples, but low phosphopeptide identification rate in data analysis limits the potential of this technology. Here we present DeepRescore2, a computational workflow that leverages deep learning-based retention time and fragment ion intensity predictions to improve phosphopeptide identification and phosphosite localization. Using a state-of-the-art computational workflow as a benchmark, DeepRescore2 increases the number of correctly identified peptide-spectrum matches by 17% in a synthetic dataset and identifies 19%-46% more phosphopeptides in biological datasets. In a liver cancer dataset, 30% of the significantly altered phosphosites between tumor and normal tissues and 60% of the prognosis-associated phosphosites identified from DeepRescore2-processed data could not be identified based on the state-of-the-art workflow. Notably, DeepRescore2-processed data uniquely identifies EGFR hyperactivation as a new target in poor-prognosis liver cancer, which is validated experimentally. Integration of deep learning prediction in DeepRescore2 improves phosphopeptide identification and facilitates biological discoveries.

Proteogenomic characterization identifies clinically relevant subgroups of intrahepatic cholangiocarcinoma.

We performed proteogenomic characterization of intrahepatic cholangiocarcinoma (iCCA) using paired tumor and adjacent liver tissues from 262 patients. Integrated proteogenomic analyses prioritized genetic aberrations and revealed hallmarks of iCCA pathogenesis. Aflatoxin signature was associated with tumor initiation, proliferation, and immune suppression. Mutation-associated signaling profiles revealed that TP53 and KRAS co-mutations may contribute to iCCA metastasis via the integrin-FAK-SRC pathway. FGFR2 fusions activated the Rho GTPase pathway and could be a potential source of neoantigens. Proteomic profiling identified four patient subgroups (S1-S4) with subgroup-specific biomarkers. These proteomic subgroups had distinct features in prognosis, genetic alterations, microenvironment dysregulation, tumor microbiota composition, and potential therapeutics. SLC16A3 and HKDC1 were further identified as potential prognostic biomarkers associated with metabolic reprogramming of iCCA cells. This study provides a valuable resource for researchers and clinicians to further identify molecular pathogenesis and therapeutic opportunities in iCCA.

caAtlas: An immunopeptidome atlas of human cancer.

Comprehensive characterization of tumor antigens is essential for the design of cancer immunotherapies, and mass spectrometry (MS)-based immunopeptidomics enables high-throughput identification of major histocompatibility complex (MHC)-bound peptide antigens in vivo. Here we construct an immunopeptidome atlas of human cancer through an extensive collection of 43 published immunopeptidomic datasets and standardized analysis of 81.6 million MS/MS spectra using an open search engine. Our analysis greatly expands the current knowledge of MHC-bound antigens, including an unprecedented characterization of post-translationally modified antigens and their cancer-association. We also perform systematic analysis of cancer-testis antigens, cancer-associated antigens, and neoantigens. We make all these data together with annotated MS/MS spectra supporting identification of each antigen in an easily browsable web portal named cancer antigen atlas (caAtlas). caAtlas provides a central resource for the selection and prioritization of MHC-bound peptides for in vitro HLA binding assay and immunogenicity testing, which will pave the way to eventual development of cancer immunotherapies.

Transfer posterior error probability estimation for peptide identification.

BACKGROUND: In shotgun proteomics, database searching of tandem mass spectra results in a great number of peptide-spectrum matches (PSMs), many of which are false positives. Quality control of PSMs is a multiple hypothesis testing problem, and the false discovery rate (FDR) or the posterior error probability (PEP) is the commonly used statistical confidence measure. PEP, also called local FDR, can evaluate the confidence of individual PSMs and thus is more desirable than FDR, which evaluates the global confidence of a collection of PSMs. Estimation of PEP can be achieved by decomposing the null and alternative distributions of PSM scores as long as the given data is sufficient. However, in many proteomic studies, only a group (subset) of PSMs, e.g. those with specific post-translational modifications, are of interest. The group can be very small, making the direct PEP estimation by the group data inaccurate, especially for the high-score area where the score threshold is taken. Using the whole set of PSMs to estimate the group PEP is inappropriate either, because the null and/or alternative distributions of the group can be very different from those of combined scores. RESULTS: The transfer PEP algorithm is proposed to more accurately estimate the PEPs of peptide identifications in small groups. Transfer PEP derives the group null distribution through its empirical relationship with the combined null distribution, and estimates the group alternative distribution, as well as the null proportion, using an iterative semi-parametric method. Validated on both simulated data and real proteomic data, transfer PEP showed remarkably higher accuracy than the direct combined and separate PEP estimation methods. CONCLUSIONS: We presented a novel approach to group PEP estimation for small groups and implemented it for the peptide identification problem in proteomics. The methodology of the approach is in principle applicable to the small-group PEP estimation problems in other fields.

Comprehensive Detection of Single Amino Acid Variants and Evaluation of Their Deleterious Potential in a PANC-1 Cell Line.

Identifying single amino acid variants (SAAVs) in cancer is critical for precision oncology. Several advanced algorithms are now available to identify SAAVs, but attempts to combine different algorithms and optimize them on large data sets to achieve a more comprehensive coverage of SAAVs have not been implemented. Herein, we report an expanded detection of SAAVs in the PANC-1 cell line using three different strategies, which results in the identification of 540 SAAVs in the mass spectrometry data. Among the set of 540 SAAVs, 79 are evaluated as deleterious SAAVs based on analysis using the novel AssVar software in which one of the driver mutations found in each protein of KRAS, TP53, and SLC37A4 is further validated using independent selected reaction monitoring (SRM) analysis. Our study represents the most comprehensive discovery of SAAVs to date and the first large-scale detection of deleterious SAAVs in the PANC-1 cell line. This work may serve as the basis for future research in pancreatic cancer and personal immunotherapy and treatment.

Single Amino Acid Variant Discovery in Small Numbers of Cells.

We have performed deep proteomic profiling down to as few as 9 Panc-1 cells using sample fractionation, TMT multiplexing, and a carrier/reference strategy. Off line fractionation of the TMT-labeled sample pooled with TMT-labeled carrier Panc-1 whole cell proteome was achieved using alkaline reversed phase spin columns. The fractionation in conjunction with the carrier/reference (C/R) proteome allowed us to detect 47 414 unique peptides derived from 6261 proteins, which provided a sufficient coverage to search for single amino acid variants (SAAVs) related to cancer. This high sample coverage is essential in order to detect a significant number of SAAVs. In order to verify genuine SAAVs versus false SAAVs, we used the SAVControl pipeline and found a total of 79 SAAVs from the 9-cell Panc-1 sample and 174 SAAVs from the 5000-cell Panc-1 C/R proteome. The SAAVs as sorted into high confidence and low confidence SAAVs were checked manually. All the high confidence SAAVs were found to be genuine SAAVs, while half of the low confidence SAAVs were found to be false SAAVs mainly related to PTMs. We identified several cancer-related SAAVs including KRAS, which is an important oncoprotein in pancreatic cancer. In addition, we were able to detect sites involved in loss or gain of glycosylation due to the enhanced coverage available in these experiments where we can detect both sites of loss and gain of glycosylation.

Quality control of single amino acid variations detected by tandem mass spectrometry.

Study of single amino acid variations (SAVs) of proteins, resulting from single nucleotide polymorphisms, is of great importance for understanding the relationships between genotype and phenotype. In mass spectrometry based shotgun proteomics, identification of peptides with SAVs often suffers from high error rates on the variant sites detected. These site errors are due to multiple reasons and can be confirmed by manual inspection or genomic sequencing. Here, we present a software tool, named SAVControl, for site-level quality control of variant peptide identifications. It mainly includes strict false discovery rate control of variant peptide identifications and variant site verification by unrestrictive mass shift relocalization. SAVControl was validated on three colorectal adenocarcinoma cell line datasets with genomic sequencing evidences and tested on a colorectal cancer dataset from The Cancer Genome Atlas. The results show that SAVControl can effectively remove false detections of SAVs. SIGNIFICANCE: Protein sequence variations caused by single nucleotide polymorphisms (SNPs) are single amino acid variations (SAVs). The investigation of SAVs may provide a chance for understanding the relationships between genotype and phenotype. Mass spectrometry (MS) based proteomics provides a large-scale way to detect SAVs. However, using the current analysis strategy to detect SAVs may lead to high rate of false positives. The SAVControl we present here is a computational workflow and software tool for site-level quality control of SAVs detected by MS. It accesses the confidence of detected variant sites by relocating the mass shift responsible for an SAV to search for alternative interpretations. In addition, it uses a strict false discovery rate control method for variant peptide identifications. The advantages of SAVControl were demonstrated on three colorectal adenocarcinoma cell line datasets and a colorectal cancer dataset. We believe that SAVControl will be a powerful tool for computational proteomics and proteogenomics.