Proteogenomic Analysis of Breast Cancer Transcriptomic and Proteomic Data, Using De Novo Transcript Assembly: Genome-Wide Identification of Novel Peptides and Clinical Implications.

We have carried out proteogenomic analysis of the breast cancer transcriptomic and proteomic data, available at The Clinical Proteomic Tumor Analysis Consortium resource, to identify novel peptides arising from alternatively spliced events as well as other noncanonical expressions. We used a pipeline that consisted of de novo transcript assembly, six frame-translated custom database, and a combination of search engines to identify novel peptides. A portfolio of 4,387 novel peptide sequences initially identified was further screened through PepQuery validation tool (Clinical Proteomic Tumor Analysis Consortium), which yielded 1,558 novel peptides. We considered the dataset of 1,558 validated through PepQuery to understand their functional and clinical significance, leaving the rest to be further verified using other validation tools and approaches. The novel peptides mapped to the known gene sequences as well as to genomic regions yet undefined for translation, 580 novel peptides mapped to known protein-coding genes, 147 to non-protein-coding genes, and 831 belonged to novel translational sequences. The novel peptides belonging to protein-coding genes represented alternatively spliced events or 5' or 3' extensions, whereas others represented translation from pseudogenes, long noncoding RNAs, or novel peptides originating from uncharacterized protein-coding sequences-mostly from the intronic regions of known genes. Seventy-six of the 580 protein-coding genes were associated with cancer hallmark genes, which included key oncogenes, transcription factors, kinases, and cell surface receptors. Survival association analysis of the 76 novel peptide sequences revealed 10 of them to be significant, and we present a panel of six novel peptides, whose high expression was found to be strongly associated with poor survival of patients with human epidermal growth factor receptor 2-enriched subtype. Our analysis represents a landscape of novel peptides of different types that may be expressed in breast cancer tissues, whereas their presence in full-length functional proteins needs further investigations.

A pathway map of signaling events triggered upon SARS-CoV infection.

Severe acute respiratory syndrome coronaviruses (SARS-CoVs) caused worldwide epidemics over the past few decades. Extensive studies on various strains of coronaviruses provided a basic understanding of the pathogenesis of the disease. Presently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is leading a global pandemic with unprecedented challenges. This is the third coronavirus outbreak of this century. A signaling pathway map of signaling events induced by SARS-CoV infection is not yet available. In this study, we present a literature-annotated signaling pathway map of reactions induced by SARS-CoV infected cells. Multiple signaling modules were found to be orchestrated including PI3K-AKT, Ras-MAPK, JAK-STAT, Type 1 IFN and NFκB. The signaling pathway map of SARS-CoV consists of 110 molecules and 101 reactions mediated by SARS-CoV proteins. The pathway reaction data are available in various community standard data exchange formats including Systems Biology Graphical Notation (SBGN). The pathway map is publicly available through the GitHub repository and data in various formats can be freely downloadable.

Identification of a Novel Splice Variant of Neural Cell Adhesion Molecule in Glioblastoma Through Proteogenomics Analysis.

Splice variants are known to be important in the pathophysiology of tumors, including the brain cancers. We applied a proteogenomics pipeline to identify splice variants in glioblastoma (GBM, grade IV glioma), a highly malignant brain tumor, using in-house generated mass spectrometric proteomic data and public domain RNASeq dataset. Our analysis led to the identification of a novel exon that maps to the long isoform of Neural cell adhesion molecule 1 (NCAM1), expressed on the surface of glial cells and neurons, important for cell adhesion and cell signaling. The presence of the novel exon is supported with the identification of five peptides spanning it. Additional peptides were also detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel separated proteins from GBM patient tissue, underscoring the presence of the novel peptides in the intact brain protein. The novel exon was detected in the RNASeq dataset in 18 of 25 GBM samples and separately validated in additional 10 GBM tumor tissues using quantitative real-time-polymerase chain reaction (qRT-PCR). Both transcriptomic and proteomic data indicate downregulation of NCAM1, including the novel variant, in GBM. Domain analysis of the novel NCAM1 sequence indicates that the insertion of the novel exon contributes extra low-complexity region in the protein that may be important for protein-protein interactions and hence for cell signaling associated with tumor development. Taken together, the novel NCAM1 variant reported in this study exemplifies the importance of future multiomics research and systems biology applications in GBM.

Sample preparation method considerations for integrated transcriptomic and proteomic analysis of tumors.

Sample processing protocols that enable compatible recovery of differentially expressed transcripts and proteins are necessary for integration of the multiomics data applied in the analysis of tumors. In this pilot study, we compared two different isolation methods for extracting RNA and protein from laryngopharyngeal tumor tissues and the corresponding adjacent normal sections. In Method 1, RNA and protein were isolated from a single tissue section sequentially and in Method 2, the extraction was carried out using two different sections and two independent and parallel protocols for RNA and protein. RNA and protein from both methods were subjected to RNA-seq and iTRAQ-based LC-MS/MS analysis, respectively. Analysis of data revealed that a higher number of differentially expressed transcripts and proteins were concordant in their regulation trends in Method 1 as compared to Method 2. Cross-method comparison of concordant entities revealed that RNA and protein extraction from the same tissue section (Method 1) recovered more concordant entities that are missed in the other extraction method (Method 2) indicating heterogeneity in distribution of these entities in different tissue sections. Method 1 could thus be the method of choice for integrated analysis of transcriptome and proteome data.

Synovial fluid proteome in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoinflammatory disorder that affects small joints. Despite intense efforts, there are currently no definitive markers for early diagnosis of RA and for monitoring the progression of this disease, though some of the markers like anti CCP antibodies and anti vimentin antibodies are promising. We sought to catalogue the proteins present in the synovial fluid of patients with RA. It was done with the aim of identifying newer biomarkers, if any, that might prove promising in future. METHODS: To enrich the low abundance proteins, we undertook two approaches-multiple affinity removal system (MARS14) to deplete some of the most abundant proteins and lectin affinity chromatography for enrichment of glycoproteins. The peptides were analyzed by LC-MS/MS on a high resolution Fourier transform mass spectrometer. RESULTS: This effort was the first total profiling of the synovial fluid proteome in RA that led to identification of 956 proteins. From the list, we identified a number of functionally significant proteins including vascular cell adhesion molecule-1, S100 proteins, AXL receptor protein tyrosine kinase, macrophage colony stimulating factor (M-CSF), programmed cell death ligand 2 (PDCD1LG2), TNF receptor 2, (TNFRSF1B) and many novel proteins including hyaluronan-binding protein 2, semaphorin 4A (SEMA4D) and osteoclast stimulating factor 1. Overall, our findings illustrate the complex and dynamic nature of RA in which multiple pathways seems to be participating actively. CONCLUSIONS: The use of high resolution mass spectrometry thus, enabled identification of proteins which might be critical to the progression of RA.

A network map of Interleukin-10 signaling pathway.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine with important immunoregulatory functions. It is primarily secreted by antigen-presenting cells such as activated T-cells, monocytes, B-cells and macrophages. In biologically functional form, it exists as a homodimer that binds to tetrameric heterodimer IL-10 receptor and induces downstream signaling. IL-10 is associated with survival, proliferation and anti-apoptotic activities of various cancers such as Burkitt lymphoma, non-Hodgkins lymphoma and non-small scell lung cancer. In addition, it plays a central role in survival and persistence of intracellular pathogens such as Leishmania donovani, Mycobacterium tuberculosis and Trypanosoma cruzi inside the host. The signaling mechanisms of IL-10 cytokine are not well explored and a well annotated pathway map has been lacking. To this end, we developed a pathway resource by manually annotating the IL-10 induced signaling molecules derived from literature. The reactions were categorized under molecular associations, activation/inhibition, catalysis, transport and gene regulation. In all, 37 molecules and 76 reactions were annotated. The IL-10 signaling pathway can be freely accessed through NetPath, a resource of signal transduction pathways previously developed by our group.

Data from human salivary proteome - A resource of potential biomarkers for oral cancer.

Salivary proteins are an important source for developing marker-based assays for oral cancers. To get an insight into the proteins present in human saliva, we applied multiple strategies involving affinity-based depletion of abundant proteins, fractionation of the resulting proteins or their tryptic peptides followed by LC-MS/MS analysis, using high resolution mass spectrometry. By integrating the protein identifications observed by us with those from similar workflows employed in earlier investigations, we compiled an updated salivary proteome. We have mapped the salivary proteome to the published data on differentially expressed proteins from oral cancer tissues and also for their secretory features using prediction tools, SignalP 4.1, TMHMM 2c and Exocarta. Proteotypic peptides for the subset of proteins implicated in oral cancer and mapped to any two of the prediction tools for secretory potential have been listed. The data here are related to the research article "Human saliva proteome - a resource of potential biomarkers for oral cancer" in the Journal of Proteomics [1].

A knowledgebase resource for interleukin-17 family mediated signaling.

Interleukin-17 (IL-17) belongs to a relatively new family of cytokines that has garnered attention as the signature cytokine of Th17 cells. This cytokine family consists of 6 ligands, which bind to 5 receptor subtypes and induce downstream signaling. Although the receptors are ubiquitously expressed, cellular responses to ligands vary across tissues. The cytokine family is associated with various autoimmune disorders including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma and psoriasis in addition to being implicated in the pathogenesis of cancer. In addition, this family plays a role in host defense against bacterial and fungal infections. The signaling mechanisms of the IL-17 family of proinflammatory cytokines are not well explored. In this study, we present a resource of literature-annotated reactions induced by IL-17. The reactions are catalogued under 5 categories, namely; molecular association, catalysis, transport, activation/inhibition and gene regulation. A total of 93 molecules and 122 reactions have been annotated. The IL-17 pathway is freely available through NetPath, a resource of signal transduction pathways previously developed by our group.

Human salivary proteome--a resource of potential biomarkers for oral cancer.

Proteins present in human saliva offer an immense potential for clinical applications. However, exploring salivary proteome is technically challenged due to the presence of amylase and albumin in high abundance. In this study, we used four workflows to analyze human saliva from healthy individuals which involved depletion of abundant proteins using affinity-based separation methods followed by protein or peptide fractionation and high resolution mass spectrometry analysis. We identified a total of 1256 human salivary proteins, 292 of them being reported for the first time. All identifications were verified for any shared proteins/peptides from the salivary microbiome that may conflict with the human protein identifications. On integration of our results with the analyses reported earlier, we arrived at an updated human salivary proteome containing 3449 proteins, 808 of them have been reported as differentially expressed proteins in oral cancer tissues. The secretory nature of 598 of the 808 proteins has also been supported on the basis of the presence of signal sequence, transmembrane domain or association with exosomes. From this subset, we provide a priority list of 139 proteins along with their proteotypic peptides, which may serve as a reference for targeted investigations as secretory markers for clinical applications in oral malignancies. This article is part of a Special Issue entitled: Proteomics in India.

Proteomics of human aqueous humor.

The aqueous humor is a colorless, transparent fluid that fills the anterior chamber of the eye. It plays an important role in maintaining the intraocular pressure and providing nourishment to the lens and cornea. The constitution of the aqueous humor is controlled by the blood-aqueous barrier. Though this ocular fluid has been extensively studied, its role in ocular physiology is still not completely understood. In this study, aqueous humor samples were collected from 250 patients undergoing cataract surgery, subjected to multiple fractionation strategies and analyzed on a Fourier transform LTQ-Orbitrap Velos mass spectrometer. In all, we identified 763 proteins, of which 386 have been identified for the first time in this study. Sorbitol dehydrogenase (SORD), filensin (BFSP1), and phakinin (BFSP2) are some of the proteins that have not been previously reported in the aqueous humor. Gene Ontology analysis revealed 35% of the identified proteins to be extracellular, with a majority of them involved in cell communication and signal transduction. This study comprehensively reports 386 novel proteins that have important potential as biomarker candidates for future research into personalized medicine and diagnostics aimed towards improving visual health.

Annotation of the zebrafish genome through an integrated transcriptomic and proteomic analysis.

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼ 69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes.

A draft map of the human proteome.

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

A Network Map of FGF-1/FGFR Signaling System.

Fibroblast growth factor-1 (FGF-1) is a well characterized growth factor among the 22 members of the FGF superfamily in humans. It binds to all the four known FGF receptors and regulates a plethora of functions including cell growth, proliferation, migration, differentiation, and survival in different cell types. FGF-1 is involved in the regulation of diverse physiological processes such as development, angiogenesis, wound healing, adipogenesis, and neurogenesis. Deregulation of FGF-1 signaling is not only implicated in tumorigenesis but also is associated with tumor invasion and metastasis. Given the biomedical significance of FGFs and the fact that individual FGFs have different roles in diverse physiological processes, the analysis of signaling pathways induced by the binding of specific FGFs to their cognate receptors demands more focused efforts. Currently, there are no resources in the public domain that facilitate the analysis of signaling pathways induced by individual FGFs in the FGF/FGFR signaling system. Towards this, we have developed a resource of signaling reactions triggered by FGF-1/FGFR system in various cell types/tissues. The pathway data and the reaction map are made available for download in different community standard data exchange formats through NetPath and NetSlim signaling pathway resources.

Pancreatic Cancer Database: an integrative resource for pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer-related death in the world. The etiology of pancreatic cancer is heterogeneous with a wide range of alterations that have already been reported at the level of the genome, transcriptome, and proteome. The past decade has witnessed a large number of experimental studies using high-throughput technology platforms to identify genes whose expression at the transcript or protein levels is altered in pancreatic cancer. Based on expression studies, a number of molecules have also been proposed as potential biomarkers for diagnosis and prognosis of this deadly cancer. Currently, there are no repositories which provide an integrative view of multiple Omics data sets from published research on pancreatic cancer. Here, we describe the development of a web-based resource, Pancreatic Cancer Database (http://www.pancreaticcancerdatabase.org), as a unified platform for pancreatic cancer research. PCD contains manually curated information pertaining to quantitative alterations in miRNA, mRNA, and proteins obtained from small-scale as well as high-throughput studies of pancreatic cancer tissues and cell lines. We believe that PCD will serve as an integrative platform for scientific community involved in pancreatic cancer research.

Functional annotation of proteome encoded by human chromosome 22.

As part of the chromosome-centric human proteome project (C-HPP) initiative, we report our progress on the annotation of chromosome 22. Chromosome 22, spanning 51 million base pairs, was the first chromosome to be sequenced. Gene dosage alterations on this chromosome have been shown to be associated with a number of congenital anomalies. In addition, several rare but aggressive tumors have been associated with this chromosome. A number of important gene families including immunoglobulin lambda locus, Crystallin beta family, and APOBEC gene family are located on this chromosome. On the basis of proteomic profiling of 30 histologically normal tissues and cells using high-resolution mass spectrometry, we show protein evidence of 367 genes on chromosome 22. Importantly, this includes 47 proteins, which are currently annotated as "missing" proteins. We also confirmed the translation start sites of 120 chromosome 22-encoded proteins. Employing a comprehensive proteogenomics analysis pipeline, we provide evidence of novel coding regions on this chromosome which include upstream ORFs and novel exons in addition to correcting existing gene structures. We describe tissue-wise expression of the proteins and the distribution of gene families on this chromosome. These data have been deposited to ProteomeXchange with the identifier PXD000561.

Proteomic analysis of human osteoarthritis synovial fluid.

BACKGROUND: Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography. RESULTS: We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples. CONCLUSIONS: We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis.

Differential proteomic analysis of synovial fluid from rheumatoid arthritis and osteoarthritis patients.

BACKGROUND: Rheumatoid arthritis and osteoarthritis are two common musculoskeletal disorders that affect the joints. Despite high prevalence rates, etiological factors involved in these disorders remain largely unknown. Dissecting the molecular aspects of these disorders will significantly contribute to improving their diagnosis and clinical management. In order to identify proteins that are differentially expressed between these two conditions, a quantitative proteomic profiling of synovial fluid obtained from rheumatoid arthritis and osteoarthritis patients was carried out by using iTRAQ labeling followed by high resolution mass spectrometry analysis. RESULTS: We have identified 575 proteins out of which 135 proteins were found to be differentially expressed by ≥3-fold in the synovial fluid of rheumatoid arthritis and osteoarthritis patients. Proteins not previously reported to be associated with rheumatoid arthritis including, coronin-1A (CORO1A), fibrinogen like-2 (FGL2), and macrophage capping protein (CAPG) were found to be upregulated in rheumatoid arthritis. Proteins such as CD5 molecule-like protein (CD5L), soluble scavenger receptor cysteine-rich domain-containing protein (SSC5D), and TTK protein kinase (TTK) were found to be upregulated in the synovial fluid of osteoarthritis patients. We confirmed the upregulation of CAPG in rheumatoid arthritis synovial fluid by multiple reaction monitoring assay as well as by Western blot. Pathway analysis of differentially expressed proteins revealed a significant enrichment of genes involved in glycolytic pathway in rheumatoid arthritis. CONCLUSIONS: We report here the largest identification of proteins from the synovial fluid of rheumatoid arthritis and osteoarthritis patients using a quantitative proteomics approach. The novel proteins identified from our study needs to be explored further for their role in the disease pathogenesis of rheumatoid arthritis and osteoarthritis.Sartaj Ahmad and Raja Sekhar Nirujogi contributed equally to this article.

Plasma Proteome Database as a resource for proteomics research: 2014 update.

Plasma Proteome Database (PPD; http://www.plasmaproteomedatabase.org/) was initially described in the year 2005 as a part of Human Proteome Organization's (HUPO's) pilot initiative on Human Plasma Proteome Project. Since then, improvements in proteomic technologies and increased throughput have led to identification of a large number of novel plasma proteins. To keep up with this increase in data, we have significantly enriched the proteomic information in PPD. This database currently contains information on 10,546 proteins detected in serum/plasma of which 3784 have been reported in two or more studies. The latest version of the database also incorporates mass spectrometry-derived data including experimentally verified proteotypic peptides used for multiple reaction monitoring assays. Other novel features include published plasma/serum concentrations for 1278 proteins along with a separate category of plasma-derived extracellular vesicle proteins. As plasma proteins have become a major thrust in the field of biomarkers, we have enabled a batch-based query designated Plasma Proteome Explorer, which will permit the users in screening a list of proteins or peptides against known plasma proteins to assess novelty of their data set. We believe that PPD will facilitate both clinical and basic research by serving as a comprehensive reference of plasma proteins in humans and accelerate biomarker discovery and translation efforts.