Minkun Wang

LC-MS/MS-based serum proteomics for identification of candidate biomarkers for hepatocellular carcinoma

Associating changes in protein levels with the onset of cancer has been widely investigated to identify clinically relevant diagnostic biomarkers. In the present study, we analyzed sera from 205 patients recruited in the United States and Egypt for biomarker discovery using label‐free proteomic analysis by LC‐MS/MS. We performed untargeted proteomic analysis of sera to identify candidate proteins with statistically significant differences between hepatocellular carcinoma (HCC) and patients with liver cirrhosis. We further evaluated the significance of 101 proteins in sera from the same 205 patients through targeted quantitation by MRM on a triple quadrupole mass spectrometer. This led to the identification of 21 candidate protein biomarkers that were significantly altered in both the United States and Egyptian cohorts. Among the 21 candidates, ten were previously reported as HCC‐associated proteins (eight exhibiting consistent trends with our observation), whereas 11 are new candidates discovered by this study. Pathway analysis based on the significant proteins reveals upregulation of the complement and coagulation cascades pathway and downregulation of the antigen processing and presentation pathway in HCC cases versus patients with liver cirrhosis. The results of this study demonstrate the power of combining untargeted and targeted quantitation methods for a comprehensive serum proteomic analysis, to evaluate changes in protein levels and discover novel diagnostic biomarkers. All MS data have been deposited in the ProteomeXchange with identifier PXD001171 (http://proteomecentral.proteomexchange.org/dataset/PXD001171).

LC-MS profiling of N-Glycans derived from human serum samples for biomarker discovery in hepatocellular carcinoma.

Defining clinically relevant biomarkers for early stage hepatocellular carcinoma (HCC) in a high-risk population of cirrhotic patients has potentially far-reaching implications for disease management and patient health. Changes in glycan levels have been associated with the onset of numerous diseases including cancer. In the present study, we used liquid chromatography coupled with electrospray ionization mass spectrometry (LC–ESI-MS) to analyze N-glycans in sera from 183 participants recruited in Egypt and the U.S. and identified candidate biomarkers that distinguish HCC cases from cirrhotic controls. N-Glycans were released from serum proteins and permethylated prior to the LC–ESI-MS analysis. Through two complementary LC–ESI-MS quantitation approaches, global profiling and targeted quantitation, we identified 11 N-glycans with statistically significant differences between HCC cases and cirrhotic controls. These glycans can further be categorized into four structurally related clusters, matching closely with the implications of important glycosyltransferases in cancer progression and metastasis. The results of this study illustrate the power of the integrative approach combining complementary LC–ESI-MS based quantitation approaches to investigate changes in N-glycan levels between HCC cases and patients with liver cirrhosis.

GPA: An algorithm for LC/MS based glycan profile annotation

Glycomics helps investigate the role glycosylation plays in complex diseases. Liquid chromatography (LC) coupled with mass spectrometry (MS) is routinely used to profile the glycans released from proteins in a biological sample. This enables us to compare observed glycans and their abundances among different biological samples to discover candidate biomarkers. One of the challenges in label-free LC/MS-based glycan profiling is the presence of various charge states and derived adduct ions. We propose a novel Glycan Profile Annotation (GPA) algorithm to automatically cluster and annotate these ions using a graphical model. Specifically, GPA aims to generate a list of unique neutral masses representing putative glycan composition derived from various charge states and multiple adducts. We demonstrate the performance of GPA in recognizing ions derived from the same glycan through analysis of LC/MS data from a serum biomarker discovery study. In addition, a simulation study is carried out to evaluate GPAs performance against existing tools in handling ambiguous cases.

Metabolomic Characterization of Hepatocellular Carcinoma in Patients with Liver Cirrhosis for Biomarker Discovery

Background: Metabolomics plays an important role in providing insight into the etiology and mechanisms of hepatocellular carcinoma (HCC). This is accomplished by a comprehensive analysis of patterns involved in metabolic alterations in human specimens. This study compares the levels of plasma metabolites in HCC cases versus cirrhotic patients and evaluates the ability of candidate metabolites in distinguishing the two groups. Also, it investigates the combined use of metabolites and clinical covariates for detection of HCC in patients with liver cirrhosis. Methods: Untargeted analysis of metabolites in plasma from 128 subjects (63 HCC cases and 65 cirrhotic controls) was conducted using gas chromatography coupled to mass spectrometry (GC-MS). This was followed by targeted evaluation of selected metabolites. LASSO regression was used to select a set of metabolites and clinical covariates that are associated with HCC. The performance of candidate biomarkers in distinguishing HCC from cirrhosis was evaluated through a leave-one-out cross-validation based on area under the receiver operating characteristics (ROC) curve. Results: We identified 11 metabolites and three clinical covariates that differentiated HCC cases from cirrhotic controls. Combining these features in a panel for disease classification using support vector machines (SVM) yielded better area under the ROC curve compared with alpha-fetoprotein (AFP). Conclusions: This study demonstrates the combination of metabolites and clinical covariates as an effective approach for early detection of HCC in patients with liver cirrhosis. Impact: Further investigation of these findings may improve understanding of HCC pathophysiology and possible implication of the metabolites in HCC prevention and diagnosis. Cancer Epidemiol Biomarkers Prev; 26(5); 675–83. ©2016 AACR.

Topic model-based mass spectrometric data analysis in cancer biomarker discovery studies

BackgroundA fundamental challenge in quantitation of biomolecules for cancer biomarker discovery is owing to the heterogeneous nature of human biospecimens. Although this issue has been a subject of discussion in cancer genomic studies, it has not yet been rigorously investigated in mass spectrometry based proteomic and metabolomic studies. Purification of mass spectometric data is highly desired prior to subsequent analysis, e.g., quantitative comparison of the abundance of biomolecules in biological samples.MethodsWe investigated topic models to computationally analyze mass spectrometric data considering both integrated peak intensities and scan-level features, i.e., extracted ion chromatograms (EICs). Probabilistic generative models enable flexible representation in data structure and infer sample-specific pure resources. Scan-level modeling helps alleviate information loss during data preprocessing. We evaluated the capability of the proposed models in capturing mixture proportions of contaminants and cancer profiles on LC-MS based serum proteomic and GC-MS based tissue metabolomic datasets acquired from patients with hepatocellular carcinoma (HCC) and liver cirrhosis as well as synthetic data we generated based on the serum proteomic data.ResultsThe results we obtained by analysis of the synthetic data demonstrated that both intensity-level and scan-level purification models can accurately infer the mixture proportions and the underlying true cancerous sources with small average error ratios (<7 %) between estimation and ground truth. By applying the topic model-based purification to mass spectrometric data, we found more proteins and metabolites with significant changes between HCC cases and cirrhotic controls. Candidate biomarkers selected after purification yielded biologically meaningful pathway analysis results and improved disease discrimination power in terms of the area under ROC curve compared to the results found prior to purification.ConclusionsWe investigated topic model-based inference methods to computationally address the heterogeneity issue in samples analyzed by LC/GC-MS. We observed that incorporation of scan-level features have the potential to lead to more accurate purification results by alleviating the loss in information as a result of integrating peaks. We believe cancer biomarker discovery studies that use mass spectrometric analysis of human biospecimens can greatly benefit from topic model-based purification of the data prior to statistical and pathway analyses.

Integrative analysis of LC-MS based glycomic and proteomic data.

Studies associating changes in the levels of glycans and proteins with the onset of cancer have been widely investigated to identify clinically relevant diagnostic biomarkers. Advances in liquid chromatography mass spectrometry (LC-MS) have enabled high-throughput identification and quantitative analysis of these biomolecules. While results from separate analyses of glycans and proteins have been reported widely, the mutual information obtained by combining the two has been relatively unexplored. In this study, we investigate integrative analysis of glycans and proteins to take advantage complementary information to improve the ability to distinguish cancer cases from controls. Specifically, SVM-RFE algorithm is utilized to select a panel of N-glycans and proteins from LC-MS data previously acquired by analysis of sera from two cohorts in a liver cancer study. Improved performances are observed by integrative analysis compared to separate glycomic and proteomic studies in distinguishing liver cancer cases from patients with liver cirrhosis.

Purification of LC/GC-MS based biomolecular expression profiles using a topic model

Liquid (or gas) chromatography coupled with mass spectrometry (LC/GC-MS) allows quantitative comparison of biomolecular abundance in clinical samples to help with the discovery of candidate biomarkers for complex diseases such as cancer. A fundamental challenge in quantitation of biomolecules for cancer biomarker discovery is owing to the heterogeneous nature of clinical samples. Various contaminations from related disease tissues or adjacent non-cancerous constituents in a sample confound the characterization of molecular expression profiles and thus hinder the discovery of reliable biomarkers. This issue has been raised and discussed in analysis of microarray and RNA-seq data in cancer genomics studies. To the best of our knowledge, the issue has not yet been rigorously addressed in analyzing LC/GC-MS data that are generated in a variety of omic studies including proteomics and metabolomics. Purification of LC/GC-MS based biomolecular expression profiles is highly desired prior to subsequent analysis, e.g., quantitative comparison of the abundance of biomolecules in clinical samples. In this study, we applied a topic model to computationally deconvolute each of LC/GC-MS based cancer expression profiles and infer the underlying sample-specific pure cancer profiles. We demonstrated the capability of the model in capturing mixture proportions of contaminants and cancer profiles on a synthetic LC-MS dataset. Improved performances were also achieved on experimental LC-MS based serum proteomic and GC-MS based tissue metabolomic datasets acquired from patients with hepatocellular carcinoma (HCC).

Multi-omic approaches for characterization of hepatocellular carcinoma

Multi-omic approaches offer the opportunity to characterize complex diseases such as cancer at various molecular levels. In this paper, we present transcriptomic, proteomic/glycoproteomic, glycomic, and metabolomic (TPGM) data we acquired by analysis of liver tissues from hepatocellular carcinoma (HCC) cases and patients with liver cirrhosis. We evaluated changes in the levels of transcripts, proteins, glycans, and metabolites between tumor and cirrhotic tissues by statistical methods. We demonstrated the potential of multi-omic approaches and network analysis to investigate the interactions among these biomolecules in the progression of liver cirrhosis to HCC. Also, we showed the significance of multi-omic approaches to identify pathways altered in HCC.Multi-omic approaches offer the opportunity to characterize complex diseases such as cancer at various molecular levels. In this paper, we present transcriptomic, proteomic/glycoproteomic, glycomic, and metabolomic (TPGM) data we acquired by analysis of liver tissues from hepatocellular carcinoma (HCC) cases and patients with liver cirrhosis. We evaluated changes in the levels of transcripts, proteins, glycans, and metabolites between tumor and cirrhotic tissues by statistical methods. We demonstrated the potential of multi-omic approaches and network analysis to investigate the interactions among these biomolecules in the progression of liver cirrhosis to HCC. Also, we showed the significance of multi-omic approaches to identify pathways altered in HCC.

Metabolomic data deconvolution using probabilistic purification models

Liquid (or gas) chromatography coupled with mass spectrometry (LC-MS or GC-MS) allows quantitative comparison of biomolecular abundance in biological samples to help with the discovery of candidate biomarkers for complex diseases such as cancer. A fundamental challenge in using quantitative analysis of biomolecules by LC-MS or GC-MS for cancer biomarker discovery is owing to the heterogeneous nature of human biospecimens. Various contaminations present in cancerous tissues or adjacent non-cancerous constituents confound the characterization of molecular expression profiles and thus hinder the discovery of reliable biomarkers. We previously applied probabilistic purification model on a relatively small sample-size metabolomic data. In this study, we further apply probabilistic purification models on larger sample-size and multi-group metabolomic datasets acquired by analysis of liver tissues using both LC-MS and GC-MS. We demonstrate the advantages of incorporating purification models in retrieving underlying sources and reduce noise in metabolomic data. Furthermore, we investigate the benefit of the proposed models in improving our ability to detect changes in the level of metabolites among liver tissue from multiple groups (tumor, liver cirrhosis, and normal).

Preprocessing and analysis of LC-MS-based proteomic data

Liquid chromatography coupled with mass spectrometry (LC-MS) has been widely used for profiling protein expression levels. This chapter is focused on LC-MS data preprocessing, which is a crucial step in the analysis of LC-MS based proteomics. We provide a high-level overview, highlight associated challenges, and present a step-by-step example for analysis of data from LC-MS based untargeted proteomic study. Furthermore, key procedures and relevant issues with the subsequent analysis by multiple reaction monitoring (MRM) are discussed.