Guoquan Yan

Highly specific revelation of rat serum glycopeptidome by boronic acid-functionalized mesoporous silica.

Although the specific profiling of endogenous glycopeptides in serum is highly inclined towards the discovery of disease biomarkers, studies on the endogenous glycopeptides (glycopeptidome) have never been conducted because of several factors. These factors include the high dynamic range of serum proteins, the inadequacy of traditional sample preparation techniques in proteomics for low-molecular-weight (LMW) proteins, and the relatively low abundances of glycopeptides. Boronic acid-functionalized mesoporous silica was synthesized in this study to overcome the limitations of the state-of-the-art methods for glycopeptidome research. The boronic acid-functionalized mesoporous silica exhibited excellent selectivity by analyzing glycopeptides in the mixture of glycopeptides/non-glycopeptides at molar ratio of 1:100, extreme sensitivity (the limit of detection was at the fmol level), good binding capacity (40 mg g(-1)), as well as the high post-enrichment recovery of glycopeptides (up to 88.10%). The as-prepared material possessing both glycopeptide-suitable pore size and glycopeptide-specific selectivity has shown special capability for enriching the endogenous glycopeptides. Fifteen unique glycosylation sites mapped to 15 different endogenous glycopeptides were identified in rat serum. The established protocol revealed for the first time the rat serum glycopeptidome.

Identification of N-Glycosylation Sites on Secreted Proteins of Human Hepatocellular Carcinoma Cells with a Complementary Proteomics Approach

N-linked glycosylation is prevalent in proteins destined for extracellular environments; nearly all secreted proteins are glycosylated. However, with respect to their glycosylation sites, little attention has been paid. Here, we report the analysis of N-glycosylation sites on secreted proteins of human hepatocellular carcinoma cells. For the enrichment of glycopeptides, capture methods with hydrophilic affinity (HA) and hydrazide chemistry (HC) were used complementarily. With the use of both methods in combination with nano-LC-ESI-MS/MS analysis, 300 different glycosylation sites within 194 unique glycoproteins were identified, and 172 glycosites have not been determined experimentally previously. A direct comparison between HA and HC methods was also investigated for the first time. In brief, in terms of selectivity for glycopeptides, HC is superior to HA (92.9% vs 51.3%); however, based on the number of glycosites identified, HA outweighs HC (265 vs 159). Furthermore, unavoidable contaminants such as actin and bovine serum albumin which are not N-glycosylated could be easily depleted by using this glycoproteomic strategy. As a consequence, more low-abundance and genuinely secreted proteins were identified. Among the glycoproteins identified, alpha-fetoprotein, CD44 and laminin have been reported to be implicated in HCC and its metastasis.

Concanavalin A-immobilized magnetic nanoparticles for selective enrichment of glycoproteins and application to glycoproteomics in hepatocelluar carcinoma cell line.

Protein glycosylation is one of the most important PTMs in biological organism. Lectins such as concanavalin A (Con A) have been widely applied to N‐glycosylated protein investigation. In this study, we developed Con A‐immobilized magnetic nanoparticles for selective separation of glycoproteins. At first, a facile immobilization of Con A on aminophenylboronic acid‐functionalized magnetic nanoparticles was performed by forming boronic acid‐sugar‐Con A bond in sandwich structure using methyl α‐D‐mannopyranoside as an intermedium. The selective capture ability of Con A‐modified magnetic nanoparticles for glycoproteins was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line 7703. In total 184 glycosylated sites were detected within 172 different glycopeptides corresponding to 101 glycoproteins. Also, the regeneration of the protein‐immobilized nanoparticles can easily be performed taking advantage of the reversible binding mechanism between boronic acid and sugar chain. The experiment results demonstrated that Con A‐modified magnetic nanoparticles by the facile and low‐cost synthesis provided a convenient and efficient enrichment approach for glycoproteins, and are promising candidates for large‐scale glycoproteomic research in complicated biological samples.

iTRAQ-2DLC-ESI-MS/MS based identification of a new set of immunohistochemical biomarkers for classification of dysplastic nodules and small hepatocellular carcinoma.

The study aims to develop novel clinical immunohistochemical biomarkers for distinguishing small hepatocellular carcinoma (sHCC) from dysplastic nodules (DN). iTRAQ-2DLC-ESI-MS/MS technique was used to screen immunohistochemical biomarkers between precancerous lesions (liver cirrhosis and DN) and sHCC. A total of 1951 proteins were quantified, including 52 proteins upregulated in sHCC and 95 proteins downregulated in sHCC by at least 1.25- or 0.8-fold at p < 0.05. The selected biomarker candidates were further verified using Western blotting and immunohistochemistry. Furthermore, receiver operation characteristics (ROC) curves and logistic regression model were carried out to evaluate the diagnostic values of the biomarkers. Finally, aminoacylase-1 (ACY1) and sequestosome-1 (SQSTM1) were chosen as novel candidate biomarkers for distinction of sHCC from DN. A constructed logistic regression model included ACY1, SQSTM1, and CD34. The sensitivity and specificity of this model for distinguishing sHCC from DN was 96.1% and 96.7%. In conclusion, ACY1 and SQSTM1 were identified as novel immunohistochemical biomarkers distinguishing sHCC from DN. In conclusion, expression levels of CD34, ACY1, and SQSTM1 can be used to establish an accurate diagnostic model for distinction of sHCC from DN.

Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia

Besides the conventional carbon sources, acetyl-CoA has recently been shown to be generated from acetate in various types of cancers, where it promotes lipid synthesis and tumour growth. The underlying mechanism, however, remains largely unknown. We find that acetate induces a hyperacetylated state of histone H3 in hypoxic cells. Acetate predominately activates lipogenic genes ACACA and FASN expression by increasing H3K9, H3K27 and H3K56 acetylation levels at their promoter regions, thus enhancing de novo lipid synthesis, which combines with its function as the metabolic precursor for fatty acid synthesis. Acetyl-CoA synthetases (ACSS1, ACSS2) are involved in this acetate-mediated epigenetic regulation. More importantly, human hepatocellular carcinoma with high ACSS1/2 expression exhibit increased histone H3 acetylation and FASN expression. Taken together, this study demonstrates that acetate, in addition to its ability to induce fatty acid synthesis as an immediate metabolic precursor, also functions as an epigenetic metabolite to promote cancer cell survival under hypoxic stress.

LC-MS/MS analysis of ovarian cancer metastasis-related proteins using a nude mouse model: 14-3-3 zeta as a candidate biomarker.

Peritoneal implantation is the most common metastatic pattern of epithelial ovarian cancer, and the five-year survival rate of patients is dramatically decreased when large-scale peritoneal metastasis occurs. This study aimed to determine serum proteins that could be used to detect early peritoneal metastasis of ovarian cancer. The secreted (or shed) proteins of the ovarian cancer cell line SKOV-3 were analyzed using LC-MS/MS, and 97 proteins were identified in the SKOV-3 culture supernatant. After the SKOV-3 cells were xenografted into the peritoneal cavities of nude mice, 3 of the 97 proteins were detected in animal sera. Following enzyme-linked immunosorbent assay (ELISA)-based screening of clinical blood samples, one of the three proteins, 14-3-3 zeta, was identified as a candidate biomarker. The average serum levels of 14-3-3 zeta in patients with epithelial ovarian cancer and benign gynecological diseases were significantly different. The expression of 14-3-3 zeta was associated with the degree of cancer peritoneal metastasis, the emergence of ascites, bilateral involvement, and the clinical stage and substage. Using 14-3-3 zeta, the overall diagnostic accuracy for ovarian cancer was greatly improved. Furthermore, siRNA-based experiments demonstrated that 14-3-3 zeta was responsible for approximately 62, 65, and 30% of the migratory, invasive, and implantation abilities of SKOV-3 cells, respectively. The present results demonstrated that the nude mouse xenograft model is an efficient system for performing function-oriented biomarker discovery, which can be used for a variety of research tasks in future molecular diagnoses, targeted therapies, and ovarian cancer vaccine development.

Up-regulation of type I collagen during tumorigenesis of colorectal cancer revealed by quantitative proteomic analysis.

UNLABELLED Colorectal cancer (CRC) is one of the most prevalent cancers worldwide. The discovery of non-invasive biomarker candidates for diagnosis and prognosis is important for the management of CRC. In this study, we performed proteomic profiling of serum from patients with different stages of CRC using a 2D-LC-MS/MS based approach combined with the APEX quantitative method. 917 proteins were identified and 93 were differentially expressed in normal and three patient groups (stages I, II and III). These proteins were predominantly involved in cell adhesion, immune response, coagulation process and metabolism. Importantly, we found collagen I dynamically changed from stages I to IV, with maximum expression in stage II, as detected in serum by MS analysis. Expression of collagen I was also validated in tumor tissues from the same group of CRC patients by real-time PCR and western blotting. Furthermore, we demonstrate that serum levels of collagen I degradation telopeptide (CTx) are correlated with staging and poor disease-free survival of CRC patients by ELISA analysis. These results suggest (1) serum proteomics may reflect biological changes in colorectal tumor tissues and (2) altered collagen I expression may be an early event in CRC tumorigenesis and CTx may provide additional information for prognosis of CRC. BIOLOGICAL SIGNIFICANCE In this work, we performed a systematic characterization of serum proteomic alterations in colorectal cancer (CRC) with different stages using a LC-MS based approach combined with the APEX quantitative method, attempting to gain overview of relevant proteins in tumorigenesis and discover non-invasive CRC-derived markers. We found a significant up-regulation of collagen I based on the proteomics data, and confirmed its expression in tissue and serum of the same group of patients. In addition, we also demonstrated that serum levels of collagen I degradation telopeptide (CTx) are correlated with the staging and poor survival rate of CRC. Those findings imply that alternation of collagen I might be an early event during tumorigenesis of CRC, and might contribute to the metastasis of CRC under the degradation regulated by some specific proteases. This work provides evidence for the clinical application of serum proteomics, and would aid the understanding of the role of the ECM in the clinical progression of CRC.

pGlyco: a pipeline for the identification of intact N-glycopeptides by using HCD- and CID-MS/MS and MS3

Confident characterization of the microheterogeneity of protein glycosylation through identification of intact glycopeptides remains one of the toughest analytical challenges for glycoproteomics. Recently proposed mass spectrometry (MS)-based methods still have some defects such as lack of the false discovery rate (FDR) analysis for the glycan identification and lack of sufficient fragmentation information for the peptide identification. Here we proposed pGlyco, a novel pipeline for the identification of intact glycopeptides by using complementary MS techniques: 1) HCD-MS/MS followed by product-dependent CID-MS/MS was used to provide complementary fragments to identify the glycans, and a novel target-decoy method was developed to estimate the false discovery rate of the glycan identification; 2) data-dependent acquisition of MS3 for some most intense peaks of HCD-MS/MS was used to provide fragments to identify the peptide backbones. By integrating HCD-MS/MS, CID-MS/MS and MS3, intact glycopeptides could be confidently identified. With pGlyco, a standard glycoprotein mixture was analyzed in the Orbitrap Fusion, and 309 non-redundant intact glycopeptides were identified with detailed spectral information of both glycans and peptides.

A novel quantitative proteomics workflow by isobaric terminal labeling.

Quantification by series of b, y fragment ion pairs generated from isobaric-labeled peptides in MS2 spectra has recently been considered an accurate strategy in quantitative proteomics. Here we developed a novel MS2 quantification approach named quantitation by isobaric terminal labeling (QITL) by coupling (18)O labeling with dimethylation. Trypsin-digested peptides were labeled with two (16)O or (18)O atoms at their C-termini in H(2)(16)O or H(2)(18)O. After blocking all ε-amino groups of lysines through guanidination, the N-termini of the peptides were accordingly labeled with formaldehyde-d(2) or formaldehyde. These indistinguishable, isobaric-labeled peptides in MS1 spectra produce b, y fragment ion pairs in the whole mass range of MS2 spectra that can be used for quantification. In this study, the feasibility of QITL was first demonstrated using standard proteins. An accurate and reproducible quantification over a wide dynamic range was achieved. Then, complex rat liver samples were used to verify the applicability of QITL for large-scale quantitative analysis. Finally, QITL was applied to profile the quantitative proteome of hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues. Given its simplicity, low-cost, and accuracy, QITL can be widely applied in biological samples (cell lines, tissues, and body fluids, etc.) for quantitative proteomic research.

First proteomic exploration of protein-encoding genes on chromosome 1 in human liver, stomach, and colon.

The launch of the Chromosome-Centric Human Proteome Project provides an opportunity to gain insight into the human proteome. The Chinese Human Chromosome Proteome Consortium has initiated proteomic exploration of protein-encoding genes on human chromosomes 1, 8, and 20. Collaboration within the consortium has generated a comprehensive proteome data set using normal and carcinomatous tissues from human liver, stomach, and colon and 13 cell lines originating in these organs. We identified 12,101 proteins (59.8% coverage against Swiss-Prot human entries) with a protein false discovery rate of less than 1%. On chromosome 1, 1,252 proteins mapping to 1,227 genes, representing 60.9% of Swiss-Prot entries, were identified; however, 805 proteins remain unidentified, suggesting that analysis of more diverse samples using more advanced proteomic technologies is required. Genes encoding the unidentified proteins were concentrated in seven blocks, located at p36, q12-21, and q42-44, partly consistent with correlation of these blocks with cancers of the liver, stomach, and colon. Combined transcriptome, proteome, and cofunctionality analyses confirmed 23 coexpression clusters containing 165 genes. Biological information, including chromosome structure, GC content, and protein coexpression pattern was analyzed using multilayered, circular visualization and tabular visualization. Details of data analysis and updates are available in the Chinese Chromosome-Centric Human Proteome Database ( http://proteomeview.hupo.org.cn/chromosome/ ).