Tyramide Signal Amplification for Antibody-Overlay Lectin Microarray: A Strategy to Improve the Sensitivity of Targeted Glycan Profiling

Antibody-overlay lectin microarray (ALM) has been used for targeted glycan profiling to identify disease-related protein glycoforms. In this context, high sensitivity is desired because it allows for the identification of disease-related glycoforms that are often present at low concentrations. We describe a new tyramide signal amplification (TSA) for the antibody-overlay lectin microarray procedure for sensitive profiling of glycosylation patterns. We demonstrate that TSA increased the sensitivity of the microarray over 100 times for glycan profiling using the model protein prostate specific antigen (PSA). The glycan profile of PSA enriched from LNCAP cells, obtained at a subnanogram level with the aid of TSA, was consistent with the previous reports. We also established the glycan profile of prostate specific membrane antigen (PSMA) using the TSA and ALM. Thus, the TSA for antibody-overlay lectin microarray is a sensitive, rapid, comprehensive, and high-throughput method for targeted glycan profiling and can potentially be used for the identification of disease-related protein glycoforms

Supplementary Figures S1-S2 from Evaluation of Colon Cancer–Specific Antigen 2 as a Potential Serum Marker for Colorectal Cancer

Supplementary Figures S1-S2 from Evaluation of Colon Cancer–Specific Antigen 2 as a Potential Serum Marker for Colorectal Cance

Predicting Ovarian Cancer Patients’ Clinical Response to Platinum-Based Chemotherapy by Their Tumor Proteomic Signatures

Ovarian cancer is the deadliest gynecologic malignancy in the United States with most patients diagnosed in the advanced stage of the disease. Platinum-based antineoplastic therapeutics is indispensable to treating advanced ovarian serous carcinoma. However, patients have heterogeneous responses to platinum drugs, and it is difficult to predict these interindividual differences before administering medication. In this study, we investigated the tumor proteomic profiles and clinical characteristics of 130 ovarian serous carcinoma patients analyzed by the Clinical Proteomic Tumor Analysis Consortium (CPTAC), predicted the platinum drug response using supervised machine learning methods, and evaluated our prediction models through leave-one-out cross-validation. Our data-driven feature selection approach indicated that tumor proteomics profiles contain information for predicting binarized platinum response (<i>P</i> < 0.0001). We further built a least absolute shrinkage and selection operator (LASSO)-Cox proportional hazards model that stratified patients into early relapse and late relapse groups (<i>P</i> = 0.00013). The top proteomic features indicative of platinum response were involved in ATP synthesis pathways and Ran GTPase binding. Overall, we demonstrated that proteomic profiles of ovarian serous carcinoma patients predicted platinum drug responses as well as provided insights into the biological processes influencing the efficacy of platinum-based therapeutics. Our analytical approach is also extensible to predicting response to other antineoplastic agents or treatment modalities for both ovarian and other cancers

Detection and Verification of Glycosylation Patterns of Glycoproteins from Clinical Specimens Using Lectin Microarrays and Lectin-Based Immunosorbent Assays

Aberrant glycosylation is a fundamental characteristic of progression of diseases such as cancer. Therefore, characterization of glycosylation patterns of proteins from disease tissues may identify changes specific to the disease development and improve diagnostic performance. Thus, analysis strategies with sufficient sensitivity for evaluation of glycosylation patterns in clinical specimens are needed. Here, we describe an analytical strategy for detection and verification of glycosylation patterns. It is based on a two-phase platform including a pattern discovery phase to identify the glycosylation changes using high-density lectin microarrays and a verification phase by developing lectin-based immunosorbent assays using the identified lectins. We evaluated the analytical performance of the platform using the glycoprotein standard and found that the lectin microarray could detect specific bindings of glycoprotein to lectins at the nanogram level and the lectin-based immunosorbent assay could be used for verification of protein glycosylation. We then applied the approach to the analysis of glycosylation patterns of two glycoproteins, which are highly expressed in prostate cancer in our prior studies, prostate specific antigen (PSA) and membrane metallo-endopeptidase (MME), from aggressive (AC) and nonaggressive prostate cancer (NAC) tissues. The observed differences in glycosylation patterns of PSA and MME may represent a significant clinical importance and could be used to develop multiplex assays for diagnosis of aggressive prostate cancer

An Integrated Workflow for Global, Glyco‑, and Phospho-proteomic Analysis of Tumor Tissues

Recently, the rapid development and application of mass spectrometry (MS)-based technologies have markedly improved the comprehensive proteomic characterization of global proteome and protein post-translational modifications (PTMs). However, the current conventional approach for global proteomic analysis is often carried out separately from PTM analysis. In our study, we developed an integrated workflow for multiplex analysis of global, glyco-, and phospho-proteomics using breast cancer patient-derived xenograft (PDX) tumor samples. Our approach included the following steps: trypsin-digested tumor samples were enriched for phosphopeptides through immobilized metal ion affinity chromatography (IMAC), followed by enrichment of glycopeptides through mixed anion exchange (MAX) method, and then the flow-through peptides were analyzed for global proteomics. Our workflow demonstrated an increased identification of peptides and associated proteins in global proteome, as compared to those using the peptides without PTM depletion. In addition to global proteome, the workflow identified phosphopeptides and glycopeptides from the PTM enrichment. We also found a subset of glycans with unique distribution profiles in the IMAC flow-through, as compared to those enriched directly using the MAX method. Our integrated workflow provided an effective platform for simultaneous global proteomic and PTM analysis of biospecimens

An Integrated Workflow for Global, Glyco‑, and Phospho-proteomic Analysis of Tumor Tissues

Recently, the rapid development and application of mass spectrometry (MS)-based technologies have markedly improved the comprehensive proteomic characterization of global proteome and protein post-translational modifications (PTMs). However, the current conventional approach for global proteomic analysis is often carried out separately from PTM analysis. In our study, we developed an integrated workflow for multiplex analysis of global, glyco-, and phospho-proteomics using breast cancer patient-derived xenograft (PDX) tumor samples. Our approach included the following steps: trypsin-digested tumor samples were enriched for phosphopeptides through immobilized metal ion affinity chromatography (IMAC), followed by enrichment of glycopeptides through mixed anion exchange (MAX) method, and then the flow-through peptides were analyzed for global proteomics. Our workflow demonstrated an increased identification of peptides and associated proteins in global proteome, as compared to those using the peptides without PTM depletion. In addition to global proteome, the workflow identified phosphopeptides and glycopeptides from the PTM enrichment. We also found a subset of glycans with unique distribution profiles in the IMAC flow-through, as compared to those enriched directly using the MAX method. Our integrated workflow provided an effective platform for simultaneous global proteomic and PTM analysis of biospecimens

Identification of Glycoproteins Containing Specific Glycans Using a Lectin-Chemical Method

Glycosylation is one of the most common protein modifications. Each glycoprotein can be glycosylated at multiple glycosites, and each glycosites can be modified by different glycans. Due to this heterogeneity of glycosylation, it has proven difficult to study the structure–function relationship of specific glycans and their affected glycoproteins. Here, we report a novel method for rapid and quantitative identification of glycoproteins containing specific glycans. Lectin affinity isolations are followed by chemical immobilization of the captured glycopeptides, allowing the identification of glycoproteins containing specific glycans by subsequent mass spectrometry. The application of the method should be useful to facilitate our understanding of how changes in glycan associate with diseases, and to discover novel glycoproteins with certain glycans that could serve as biomarkers or therapeutic targets

An Integrated Workflow for Global, Glyco‑, and Phospho-proteomic Analysis of Tumor Tissues

Recently, the rapid development and application of mass spectrometry (MS)-based technologies have markedly improved the comprehensive proteomic characterization of global proteome and protein post-translational modifications (PTMs). However, the current conventional approach for global proteomic analysis is often carried out separately from PTM analysis. In our study, we developed an integrated workflow for multiplex analysis of global, glyco-, and phospho-proteomics using breast cancer patient-derived xenograft (PDX) tumor samples. Our approach included the following steps: trypsin-digested tumor samples were enriched for phosphopeptides through immobilized metal ion affinity chromatography (IMAC), followed by enrichment of glycopeptides through mixed anion exchange (MAX) method, and then the flow-through peptides were analyzed for global proteomics. Our workflow demonstrated an increased identification of peptides and associated proteins in global proteome, as compared to those using the peptides without PTM depletion. In addition to global proteome, the workflow identified phosphopeptides and glycopeptides from the PTM enrichment. We also found a subset of glycans with unique distribution profiles in the IMAC flow-through, as compared to those enriched directly using the MAX method. Our integrated workflow provided an effective platform for simultaneous global proteomic and PTM analysis of biospecimens

Identification of Glycoproteins Containing Specific Glycans Using a Lectin-Chemical Method

Glycosylation is one of the most common protein modifications. Each glycoprotein can be glycosylated at multiple glycosites, and each glycosites can be modified by different glycans. Due to this heterogeneity of glycosylation, it has proven difficult to study the structure–function relationship of specific glycans and their affected glycoproteins. Here, we report a novel method for rapid and quantitative identification of glycoproteins containing specific glycans. Lectin affinity isolations are followed by chemical immobilization of the captured glycopeptides, allowing the identification of glycoproteins containing specific glycans by subsequent mass spectrometry. The application of the method should be useful to facilitate our understanding of how changes in glycan associate with diseases, and to discover novel glycoproteins with certain glycans that could serve as biomarkers or therapeutic targets

An Integrated Workflow for Global, Glyco‑, and Phospho-proteomic Analysis of Tumor Tissues

Recently, the rapid development and application of mass spectrometry (MS)-based technologies have markedly improved the comprehensive proteomic characterization of global proteome and protein post-translational modifications (PTMs). However, the current conventional approach for global proteomic analysis is often carried out separately from PTM analysis. In our study, we developed an integrated workflow for multiplex analysis of global, glyco-, and phospho-proteomics using breast cancer patient-derived xenograft (PDX) tumor samples. Our approach included the following steps: trypsin-digested tumor samples were enriched for phosphopeptides through immobilized metal ion affinity chromatography (IMAC), followed by enrichment of glycopeptides through mixed anion exchange (MAX) method, and then the flow-through peptides were analyzed for global proteomics. Our workflow demonstrated an increased identification of peptides and associated proteins in global proteome, as compared to those using the peptides without PTM depletion. In addition to global proteome, the workflow identified phosphopeptides and glycopeptides from the PTM enrichment. We also found a subset of glycans with unique distribution profiles in the IMAC flow-through, as compared to those enriched directly using the MAX method. Our integrated workflow provided an effective platform for simultaneous global proteomic and PTM analysis of biospecimens