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.

Glycoproteomics-based signatures for tumor subtyping and clinical outcome prediction of high-grade serous ovarian cancer.

Inter-tumor heterogeneity is a result of genomic, transcriptional, translational, and post-translational molecular features. To investigate the roles of protein glycosylation in the heterogeneity of high-grade serous ovarian carcinoma (HGSC), we perform mass spectrometry-based glycoproteomic characterization of 119 TCGA HGSC tissues. Cluster analysis of intact glycoproteomic profiles delineates 3 major tumor clusters and 5 groups of intact glycopeptides. It also shows a strong relationship between N-glycan structures and tumor molecular subtypes, one example of which being the association of fucosylation with mesenchymal subtype. Further survival analysis reveals that intact glycopeptide signatures of mesenchymal subtype are associated with a poor clinical outcome of HGSC. In addition, we study the expression of mRNAs, proteins, glycosites, and intact glycopeptides, as well as the expression levels of glycosylation enzymes involved in glycoprotein biosynthesis pathways in each tumor. The results show that glycoprotein levels are mainly controlled by the expression of their individual proteins, and, furthermore, that the glycoprotein-modifying glycans correspond to the protein levels of glycosylation enzymes. The variation in glycan types further shows coordination to the tumor heterogeneity. Deeper understanding of the glycosylation process and glycosylation production in different subtypes of HGSC may provide important clues for precision medicine and tumor-targeted therapy.

Integrated Proteomic and Glycoproteomic Characterization of Human High-Grade Serous Ovarian Carcinoma.

Many gene products exhibit great structural heterogeneity because of an array of modifications. These modifications are not directly encoded in the genomic template but often affect the functionality of proteins. Protein glycosylation plays a vital role in proper protein functions. However, the analysis of glycoproteins has been challenging compared with other protein modifications, such as phosphorylation. Here, we perform an integrated proteomic and glycoproteomic analysis of 83 prospectively collected high-grade serous ovarian carcinoma (HGSC) and 23 non-tumor tissues. Integration of the expression data from global proteomics and glycoproteomics reveals tumor-specific glycosylation, uncovers different glycosylation associated with three tumor clusters, and identifies glycosylation enzymes that were correlated with the altered glycosylation. In addition to providing a valuable resource, these results provide insights into the potential roles of glycosylation in the pathogenesis of HGSC, with the possibility of distinguishing pathological outcomes of ovarian tumors from non-tumors, as well as classifying tumor clusters.

Proteomic Analysis of the Air-Way Fluid in Lung Cancer. Detection of Periostin in Bronchoalveolar Lavage (BAL).

Background: Bronchoalveolar lavage (BAL) is a specific type of air-way fluid. It is a commonly used clinical specimen for the diagnosis of benign diseases and cancers of the lung. Although previous studies have identified several disease-associated proteins in the BAL, the potential utility of BAL in lung cancer is still not well-studied. Based upon the fact that the majority of secreted proteins are glycoproteins, we have profiled N-glycoproteins in BAL collected from lung cancers, and investigated the expression of glycoproteins such as the matrix N-glycoprotein, periostin, in lung cancers. Methods: BAL specimens (n = 16) were collected from lung cancer patients, and analyzed using mass spectrometry-based quantitative N-glycoproteomic technique. Additional BAL specimens (n = 39) were independently collected to further evaluate the expression of periostin by using an enzyme-linked immunosorbent assay (ELISA). Results: A total of 462 glycoproteins were identified in BAL samples using N-glycoproteomic technique, including 290 in lung adenocarcinoma (ADC, n = 5), 376 in squamous cell carcinoma (SQCC, n = 4), 309 in small cell lung carcinoma (SCLC, n = 4), and 316 in benign lung disease (n = 3). The expressions of several glycoproteins were elevated, including 8 in ADC, 12 in SQCC, and 17 in SCLC, compared to benign BALs. The expression of periostin was detected in all subtypes of lung cancers. To further investigate the expression of periostin, an ELISA assay was performed using additional independently collected BALs (n = 39) The normalized levels of periostin in benign disease, ADC, SQCC, and SCLC were 255 ± 104 (mean ± SE) and 4,002 ± 2,181, 3,496 ± 1,765, and 1,772 ± 1,119 ng/mg of total BAL proteins. Conclusion: Our findings demonstrate that proteomic analysis of BAL can be used for the study of cancer-associated extracellular proteins in air-way fluid from lung cancer patients.

Mass Spectrometric Mapping of Glycoproteins Modified by Tn-Antigen Using Solid-Phase Capture and Enzymatic Release.

Tn-antigen (Tn), a single N-acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser/Thr residues, is found on most cancer yet rarely detected in adult normal tissues as reported in previous studies, featuring it as one of the most distinctive signatures of cancer. Although it is important in cancer, Tn modified glycoproteins are not entirely clear owing to the lack of a suitable method. Knowing the Tn-glycosylated proteins and glycosylation sites are essential to the prevention, diagnosis, and therapy of cancer associated with the expression of Tn. Here, we introduce a method named EXoO-Tn for large-scale mapping of Tn-glycosylated proteins and glycosylation sites. EXoO-Tn utilizes solid-phase immobilization of proteolytic peptides of proteins, which modifies Tn by glycosyltransferase C1GalT1 with isotopically labeled UDP-Gal(13C6), to tag and convert Tn to Gal(13C6)-Tn, which gives rise to a unique glycan mass. The exquisite Gal(13C6) modified Tn are then recognized by a human-gut-bacterial enzyme, OpeRATOR, and released at the N-termini of the Gal(13C6)-Tn-occupied Ser/Thr residues from immobilized peptides to yield site-containing glycopeptides. The effectiveness of EXoO-Tn was benchmarked by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. The EXoO-Tn was further applied to the analysis of pancreatic cancer sera, where Tn-glycoproteins were identified. Given the significance of Tn in cancer, EXoO-Tn is anticipated to have broad translational and clinical utilities.

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.

Expression of p16 and p53 in non-small-cell lung cancer: clinicopathological correlation and potential prognostic impact.

Aim: p16 and p53 are frequently altered intracellular pathways in cancers. We investigated the aberrant expression of p16 and its relationship with p53 and HPV status in primary non-small-cell lung carcinoma. Patients & methods: Lung tumor tissue microarray (n = 163), immunohistochemical study of p16 and p53, and HPV in-situ hybridization were analyzed. Results: p16 and p53 were detected in 50.7 and 57.3% of adenocarcinoma (ADCs; n = 75), and 35.2 and 63.6% of squamous cell carcinoma (n = 88). HPV was detected in 16 and 10.2% of ADC and squamous cell carcinoma. In ADCs, p16 positive tumors demonstrated a favorable median overall survival time of 60.9 months, compared with p16 negative tumors of 46.9 months (p < 0.05). Furthermore, we did not find significant relationships between p16 expression and HPV status, nor with p53 expression. Conclusion: p16 play an unique role in lung cancer survival. The mechanism of p16 needs to be further studied.

Mapping the O-glycoproteome using site-specific extraction of O-linked glycopeptides (EXoO).

Protein glycosylation is one of the most abundant post-translational modifications. However, detailed analysis of O-linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of suitable methodologies. Here, a chemoenzymatic method is introduced for the site-specific extraction of O-linked glycopeptides (EXoO), which enabled the mapping of over 3,000 O-linked glycosylation sites and definition of their glycans on over 1,000 proteins in human kidney tissues, T cells, and serum. This large-scale localization of O-linked glycosylation sites demonstrated that EXoO is an effective method for defining the site-specific O-linked glycoproteome in different types of sample. Detailed structural analysis of the sites identified revealed conserved motifs and topological orientations facing extracellular space, the cell surface, the lumen of the Golgi, and the endoplasmic reticulum (ER). EXoO was also able to reveal significant differences in the O-linked glycoproteome of tumor and normal kidney tissues pointing to its broader use in clinical diagnostics and therapeutics.

Reanalysis of Global Proteomic and Phosphoproteomic Data Identified a Large Number of Glycopeptides.

Protein glycosylation plays fundamental roles in many cellular processes, and previous reports have shown dysregulation to be associated with several human diseases, including diabetes, cancer, and neurodegenerative disorders. Despite the vital role of glycosylation for proper protein function, the analysis of glycoproteins has been lagged behind to other protein modifications. In this study, we describe the reanalysis of global proteomic data from breast cancer xenograft tissues using recently developed software package GPQuest 2.0, revealing a large number of previously unidentified N-linked glycopeptides. More importantly, we found that using immobilized metal affinity chromatography (IMAC) technology for the enrichment of phosphopeptides had coenriched a substantial number of sialoglycopeptides, allowing for a large-scale analysis of sialoglycopeptides in conjunction with the analysis of phosphopeptides. Collectively, combined tandem mass spectrometry (MS/MS) analyses of global proteomic and phosphoproteomic data sets resulted in the identification of 6 724 N-linked glycopeptides from 617 glycoproteins derived from two breast cancer xenograft tissues. Next, we utilized GPQuest 2.0 for the reanalysis of global and phosphoproteomic data generated from 108 human breast cancer tissues that were previously analyzed by Clinical Proteomic Analysis Consortium (CPTAC). Reanalysis of the CPTAC data set resulted in the identification of 2 683 glycopeptides from the global proteomic data set and 4 554 glycopeptides from phosphoproteomic data set, respectively. Together, 11 292 N-linked glycopeptides corresponding to 1 731 N-linked glycosites from 883 human glycoproteins were identified from the two data sets. This analysis revealed an extensive number of glycopeptides hidden in the global and enriched in IMAC-based phosphopeptide-enriched proteomic data, information which would have remained unknown from the original study otherwise. The reanalysis described herein can be readily applied to identify glycopeptides from already existing data sets, providing insight into many important facets of protein glycosylation in different biological, physiological, and pathological processes.

Expression of P40 and P63 in lung cancers using fine needle aspiration cases. Understanding clinical pitfalls and limitations.

BACKGROUND: Fine-needle aspiration (FNA) biopsy of lung lesions is a highly accurate method for diagnosing and staging of lung cancers, particularly in patients with advanced cancer. Although, the majority of FNA cases of non-small cell lung carcinoma (NSCLC) can be subclassified by hematoxylin and eosin (H&E) sections, immunohistochemical (IHC) markers are usually necessary for difficult cases. Our previous study has shown that both P40 and P63 demonstrate differential sensitivity and specificity in the subclassification of squamous cell carcinoma (SqCC) using tumor tissue microarrays (TMA). In the present study, we further evaluated the utility of P40 and P63 and the potential pitfalls and limitations associated with the usefulness of these stains in FNA cases. METHODS: By a computer search of pathology archives, 144 FNA biopsies with diagnoses of lung cancers and P40/P63 stains were identified, including 50 adenocarcinomas (ADCs), 56 SqCCs, 8 small cell lung carcinomas (SCLCs), and 12 cases of poorly differentiated carcinoma (PD CA). Ten benign FNA lung lesions and 8 other malignant neoplasms were also included as controls. Nuclear staining patterns of P40 and P63 were scored semi-quantitatively as 0 (negative), 1 (<10%, weak and focal), or 2 (>10%, strong and diffuse). RESULTS: In lung SqCCs, P40 and P63 were positive in 77.3% and 89.5% cases, respectively. In ADCs, P40 was weakly and focally positive in 6.1% cases, and P63 was variably positive in 62.8% cases. In SCLCs, P40 and P63 were focally positive in 12.5% and 50% cases. In PD CAs, no P40 or P63 immunoreactivity was detected. In the group of other neoplasms (n=8) both P40 and P63 were positive in the case of metastatic non-seminomatous germ cell tumor (NSGCT) (n=1), and P63 was positive in the case of metastatic Merkel cell carcinoma (n=1). The sensitivity and specificity of P40 and P63 were 76.9%/93.3%, and 90.2%/50.7% in the lung SqCC. CONCLUSIONS: P63 has a better sensitivity, and P40 has a better specificity for SqCC. A positive staining pattern with both markers was also found in certain non-SqCC cases. Recognizing limitations of these markers are particularly important in FNA cases.

Serum fucosylated prostate-specific antigen (PSA) improves the differentiation of aggressive from non-aggressive prostate cancers.

BACKGROUND: Clinically, it is still challenging to differentiate aggressive from non-aggressive prostate cancers (Pca) by non-invasive approaches. Our recent studies showed that overexpression of alpha (1-6) fucosyltransferase played an important role in Pca cells. In this study, we have investigated levels of glycoproteins and their fucosylated glycoforms in sera of Pca patients, as well as the potential utility of fucosylated glycoproteins in the identification of aggressive Pca. MATERIAL AND METHODS: Serum samples from histomorphology-proven Pca cases were included. Prostate-specific antigen (PSA), tissue inhibitor of metallopeptidase 1 (TIMP1) and tissue plasminogen activator (tPA), and their fucosylated glycoforms were captured by Aleuria Aurantia Lectin (AAL), followed by the multiplex magnetic bead-based immunoassay. The level of fucosylated glycoproteins was correlated with patients' Gleason score of the tumor. RESULT: Among three fucosylated glycoproteins, the fucosylated PSA was significantly increased and correlated with the tumor Gleason score (p<0.05). The ratio of fucosylated PSA showed a marked increase in aggressive tumors in comparison to non-aggressive tumors. ROC analysis also showed an improved predictive power of fucosylated PSA in the identification of aggressive Pca. CONCLUSIONS: Our data demonstrated that fucosylated PSA has a better predictive power to differentiate aggressive tumors from non-aggressive tumors, than that of native PSA and two other glycoproteins. The fucosylated PSA has the potential to be used as a surrogate biomarker.

Expression of transcript factors SALL4 and OCT4 in a subset of non-small cell lung carcinomas (NSCLC).

BACKGROUND: SALL4 and OCT4 are transcription factors and play essential roles in stem cell development and oncogenesis. However, the expression of these transcription factors has not been well studied in lung cancers. In this study, we evaluated the expression of SALL4 and OCT4 in non-small cell lung carcinomas (NSCLC) by immunochemistry. NSCLC tissue microarrays (TMAs) were constructed with a total of 77 primary lung adenocarcinomas (ADCs) and 90 primary lung squamous cell carcinomas (SqCCs). A mouse monoclonal anti-human SALL4 (1:400 dilution) and a polyclonal anti-human OCT4 (1:200 dilution) antibodies were used. Nuclear staining of SALL4 and OCT4 was scored semi-quantitatively using a three tiered scale. The expressions of SALL4 and OCT4 were correlated with the tumor differentiation, pathological stage, and patients' clinical information. RESULTS: In primary ADCs, the stronger expression of SALL4 and OCT4 was 7.8% and 9.1%, respectively. The stronger expression of SALL4 was inversely correlated with tumor differentiations. In primary SqCCs, the stronger expressions of SALL4 and OCT4 were 16.7% and 0%, respectively. The expression of SALL4 is correlated with the expression of OCT4, but inversely correlated with the tumor stage in SqCCs. CONCLUSIONS: We found that both SALL4 and OCT4 were differentially expressed in a subset of primary ADC and SqCC. Our finding suggest that different stem cell markers may be expressed and/or play differential role in different subtypes of NSCLC. The potential role of SALL4 and OCT4 needs to be further investigated in NSCLC.

The utility of a novel triple marker (combination of TTF1, napsin A, and p40) in the subclassification of non-small cell lung cancer.

In lung cancer, targeted therapies depend on accurate histological subclassification of the tumor. The majority of lung cancers can be subclassified based on hematoxylin and eosin staining; however, classification may be difficult in small biopsies. In this study, we investigated the utility of a newly developed triple marker (combination of TTF1/Napsin A/p40) and compared the sensitivity and specificity of this novel marker with individual markers in the subclassification of non-small cell lung carcinomas. Lung cancer tissue microarrays were constructed using surgical resection material from the Johns Hopkins Hospital. They included 77 adenocarcinomas (ADCs), 77 squamous cell carcinomas (SqCCs), and 46 cases of metastatic lung ADCs. Immunostaining patterns of all markers were scored semi-quantitatively and compared. In ADCs, the sensitivity and specificity of the triple marker were 93.5% and 77.5%, respectively. The sensitivity and specificity of TTF1 and Napsin A were 85.7% and 75.0%, and 89.6% and 90.0%. In SqCCs, the sensitivity and specificity of the triple marker were 88.3% and 92.5%, while the p40, p63 and CK5/6 showed 80.5% and 90.0%; 93.5% and 80.0%; and 89.6% and 80.0%. In addition, the sensitivity and specificity of the triple marker in metastatic ADCs showed 71.7% and 73.5%, respectively. Our triple marker (combination of TTF1/Napsin A/p40) showed a similar sensitivity and specificity for the subclassification of NSCLC when compared to individual markers. Our study not only demonstrates a useful combination of immunomarkers but also optimally conserves tissue for molecular marker testing.

Glycoproteomic analysis identifies human glycoproteins secreted from HIV latently infected T cells and reveals their presence in HIV+ plasma.

Glycoproteins secreted into plasma from T cells infected with human immunodeficiency virus (HIV) latent infection may provide insight into understanding the host response to HIV infection in vivo. Glycoproteomics, which evaluates the level of the glycoproteome, remains a novel approach to study this host response to HIV. In order to identify human glycoproteins secreted from T cells with latent HIV infection, the medium from cultured HIV replication-competent T cells was compared with the medium from cultured parental A3.01 cells via solid phase extraction of glycopeptides (SPEG) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Using these methods, 59 human glycoproteins were identified as having significantly different abundance levels between the media from these two cell lines. The relevance of these 59 proteins to HIV infection in vivo was assessed in plasma from HIV+ and HIV- subjects. Comparison between T cell and plasma revealed that six glycoproteins (galectin-3-binding protein, L-selectin, neogenin, adenosine deaminase CECR1, ICOS ligand and phospholipid transfer protein) were significantly elevated in the HIV+ T cells and plasma studies. These findings suggest that the response of T cells harboring latent HIV infection contributed, in part, to the glycoprotein changes in HIV+ plasma. These proteins, once validated, could provide insight into host-HIV interaction.

Aberrant Mucin5B expression in lung adenocarcinomas detected by iTRAQ labeling quantitative proteomics and immunohistochemistry.

BACKGROUND: Lung cancer is the number one cause of cancer-related deaths in the United States and worldwide. The complex protein changes and/or signature of protein expression in lung cancer, particularly in non-small cell lung cancer (NSCLC) has not been well defined. Although several studies have investigated the protein profile in lung cancers, the knowledge is far from complete. Among early studies, mucin5B (MUC5B) has been suggested to play an important role in the tumor progression. MUC5B is the major gel-forming mucin in the airway. In this study, we investigated the overall protein profile and MUC5B expression in lung adenocarcinomas, the most common type of NSCLCs. METHODS: Lung adenocarcinoma tissue in formalin-fixed paraffin-embedded (FFPE) blocks was collected and microdissected. Peptides from 8 tumors and 8 tumor-matched normal lung tissue were extracted and labeled with 8-channel iTRAQ reagents. The labeled peptides were identified and quantified by LC-MS/MS using an LTQ Orbitrap Velos mass spectrometer. MUC5B expression identified by iTRAQ labeling was further validated using immunohistochemistry (IHC) on tumor tissue microarray (TMA). RESULTS: A total of 1288 peptides from 210 proteins were identified and quantified in tumor tissues. Twenty-two proteins showed a greater than 1.5-fold differences between tumor and tumor-matched normal lung tissues. Fifteen proteins, including MUC5B, showed significant changes in tumor tissues. The aberrant expression of MUC5B was further identified in 71.1% of lung adenocarcinomas in the TMA. DISCUSSIONS: A subset of tumor-associated proteins was differentially expressed in lung adenocarcinomas. The differential expression of MUC5B in lung adenocarcinomas suggests its role as a potential biomarker in the detection of adenocarcinomas.

Conserved amino acids within the adenovirus 2 E3/19K protein differentially affect downregulation of MHC class I and MICA/B proteins.

Successful establishment and persistence of adenovirus (Ad) infections are facilitated by immunosubversive functions encoded in the early transcription unit 3 (E3). The E3/19K protein has a dual role, preventing cell surface transport of MHC class I/HLA class I (MHC-I/HLA-I) Ags and the MHC-I-like molecules (MHC-I chain-related chain A and B [MICA/B]), thereby inhibiting both recognition by CD8 T cells and NK cells. Although some crucial functional elements in E3/19K have been identified, a systematic analysis of the functional importance of individual amino acids is missing. We now have substituted alanine for each of 21 aas in the luminal domain of Ad2 E3/19K conserved among Ads and investigated the effects on HLA-I downregulation by coimmunoprecipitation, pulse-chase analysis, and/or flow cytometry. Potential structural alterations were monitored using conformation-dependent E3/19K-specific mAbs. The results revealed that only a small number of mutations abrogated HLA-I complex formation (e.g., substitutions W52, M87, and W96). Mutants M87 and W96 were particularly interesting as they exhibited only minimal structural changes suggesting that these amino acids make direct contacts with HLA-I. The considerable number of substitutions with little functional defects implied that E3/19K may have additional cellular target molecules. Indeed, when assessing MICA/B cell-surface expression we found that mutation of T14 and M82 selectively compromised MICA/B downregulation with essentially no effect on HLA-I modulation. In general, downregulation of HLA-I was more severely affected than that of MICA/B; for example, substitutions W52, M87, and W96 essentially abrogated HLA-I modulation while largely retaining the ability to sequester MICA/B. Thus, distinct conserved amino acids seem preferentially important for a particular functional activity of E3/19K.