A serum glycomics approach to breast cancer biomarkers

Because the glycosylation of proteins is known to change in tumor cells during the development of breast cancer, a glycomics approach is used here to find relevant biomarkers of breast cancer. These glycosylation changes are known to correlate with increasing tumor burden and poor prognosis. Current antibody-based immunochemical tests for cancer biomarkers of ovarian (CA125), breast (CA27.29 or CA15-3), pancreatic, gastric, colonic, and carcinoma (CA19-9) target highly glycosylated mucin proteins. However, these tests lack the specificity and sensitivity for use in early detection. This glycomics approach to find glycan biomarkers of breast cancer involves chemically cleaving oligosaccharides (glycans) from glycosylated proteins that are shed or secreted by breast cancer tumor cell lines. The resulting free glycan species are analyzed by MALDI-FT-ICR MS. Further structural analysis of the glycans can be performed in FTMS through the use of tandem mass spectrometry with infrared multiphoton dissociation. Glycan profiles were generated for each cell line and compared. These methods were then used to analyze sera obtained from a mouse model of breast cancer and a small number of serum samples obtained from human patients diagnosed with breast cancer or patients with no known history of breast cancer. In addition to the glycosylation changes detected in mice as mouse mammary tumors developed, glycosylation profiles were found to be sufficiently different to distinguish patients with cancer from those without. Although the small number of patient samples analyzed so far is inadequate to make any legitimate claims at this time, these promising but very preliminary results suggest that glycan profiles may contain distinct glycan biomarkers that may correspond to glycan "signatures of cancer."     

Liquid Chromatography/Mass Spectrometry (LC/MS)-Based Glycoproteomics Technologies for Cancer Biomarker Discovery

Introduction

Biomarker discovery is a major objective of clinical proteomics; molecular biomarkers allow for detection of early-stage human diseases, especially cancer, and for monitoring their progression and/or regression after treatment. Biomarkers also help to elucidate the pathology of disease and its diagnosis, drug discovery, and toxicology. Glycans are ideal candidates for biomarkers because (1) glycoconjugates are localized on the cell surface and in the secretions such as plasma, (2) their structures are frequently and drastically changed during normal and aberrant cell differentiation, and (3) different cell types express different glycan signatures. Certain serodiagnostic glycoconjugate markers, such as carcinoembryonic antigen (CEA), are currently available; however, comprehensive glycome analysis has yet to be performed, mainly because of the difficulties of isolating and structurally analyzing complex glycans. Large-scale glycoprotein analysis, termed glycoproteomics, has the potential to effectively trace cellular glycoproteins and therefore to search for new serodiagnostic biomarkers.

Conclusions

In this review, we describe current mass spectrometry-based glycoproteomics technologies. Quantitative “shotgun” proteomics analyses of glycopeptides captured from complex biological mixtures such as plasma, coupled with advanced glycome technologies, enhance our knowledge of protein glycosylation and facilitate discovery of new biomarkers for human diseases.     

Glycans as cancer biomarkers

Background

Non-invasive biomarkers, such as those from serum, are ideal for disease prognosis, staging and monitoring. In the past decade, our understanding of the importance of glycosylation changes with disease has evolved.

Scope of review

We describe potential biomarkers derived from serum glycoproteins for liver, pancreatic, prostate, ovarian, breast, lung and stomach cancers. Methods for glycan analysis have progressed and newly developed high-throughput platform technologies have enabled the analysis of large cohorts of samples in an efficient manner. We also describe this evolution and trends to follow in the future.

Major conclusions

Many convincing examples of aberrant glycans associated with cancer have come about from glycosylation analyses. Most studies have been carried out to identify changes in serum glycan profiles or through the isolation and identification of glycoproteins that contain these irregular glycan structures. In a majority of cancers the fucosylation and sialylation expression are found to be significantly modified. Therefore, these aberrations in glycan structures can be utilized as targets to improve existing cancer biomarkers.

General significance

The ability to distinguish differences in the glycosylation of proteins between cancer and control patients emphasizes glycobiology as a promising field for potential biomarker identification. Furthermore, the high-throughput and reproducible nature of the chromatography platform have highlighted extensive applications in biomarker discovery and allowed integration of glycomics with other -omics fields, such as proteomics and genomics, making systems glycobiology a reality. This article is part of a Special Issue entitled Glycoproteomics.     

The GlycanBuilder: a fast, intuitive and flexible software tool for building and displaying glycan structures

Background  

Carbohydrates play a critical role in human diseases and their potential utility as biomarkers for pathological conditions is a major driver for characterization of the glycome. However, the additional complexity of glycans compared to proteins and nucleic acids has slowed the advancement of glycomics in comparison to genomics and proteomics. The branched nature of carbohydrates, the great diversity of their constituents and the numerous alternative symbolic notations, make the input and display of glycans not as straightforward as for example the amino-acid sequence of a protein. Every glycoinformatic tool providing a user interface would benefit from a fast, intuitive, appealing mechanism for input and output of glycan structures in a computer readable format.     

Evaluation of the serum N-linked glycome for the diagnosis of cancer and chronic inflammation

The identification of serum biomarkers has lead to improvements in the detection and diagnosis of cancer, and combinations of these biomarkers have increased further their sensitivity and specificity. Glycosylation is the most common PTM of secreted proteins and the identification of novel serum glyco-biomarkers has become a topic of increasing interest because the glycan processing pathways are frequently disturbed in cancer cells. A future goal is to combine current biomarkers with glyco-biomarkers to yield further improvements. Well characterised N-glycosylation changes in the serum glycome of cancer patients include changes in the levels of tri- and tetra-antennary glycan structures, sialyl Lewis X epitopes and agalactosylated bi-antennary glycans. Several of these glycosylated markers have been linked to chronic inflammatory diseases, promoting questions about the links between inflammation and cancer. In this review, the glycoproteins which display these glycan epitopes, the glycosyl transferases which can generate them, their potential functions and their use as biomarkers are evaluated.     

Serum Glycan Markers for Evaluation of Disease Activity and Prediction of Clinical Course in Patients with Ulcerative Colitis

Background

The aims of this study were to determine the change of whole-serum N-glycan profile in ulcerative colitis (UC) patients and to investigate its clinical utility.

Methods

We collected serum from 75 UC patients at the time of admission and the same number of age/sex-matched healthy volunteers. Serum glycan profile was measured by comprehensive quantitative high-throughput glycome analysis and was compared with disease activity and prognosis.

Results

Out of 61 glycans detected, 24 were differentially expressed in UC patients. Pathway analysis demonstrated that highly sialylated multi-branched glycans and agalactosyl bi-antennary glycans were elevated in UC patients; in addition, the glycan ratio m/z 2378/1914, which also increased in UC, showed the highest Area under Receiver Operating Characteristic curve (0.923) for the diagnosis of UC. Highly sialylated multi-branched glycans and the glycan ratio m/z 2378/1914 were higher in the patients with total colitis, Clinical Activity Index >10, Mayo endoscopic score 3, or a steroid-refractory status. In particular, the glycan ratio m/z 2378/1914 (above median) was an independent prognostic factor for the need for an operation (hazard ratio, 2.67; 95% confidence interval, 1.04–7.84).

Conclusions

Whole-serum glycan profiles revealed that the glycan ratio m/z 2378/1914 and highly sialylated multi-branched glycans increase in UC patients, and are correlated with disease activity. The glycan ratio m/z 2378/1914 was an independent predictive factor of the prognosis of UC.     

N-glycomic Profiling as a Tool to Separate Rectal Adenomas from Carcinomas

All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world''s three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I–II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.Glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans, that cover all human cells. Around 1% of the human genome participates in the biosynthesis of glycans(1). This biosynthesis is the most complex post-translational modification of proteins, and the great variability in glycan structures contains a tremendous ability to fine-tune the chemical and biological properties of glycoproteins. The glycosylation process occurs most abundantly in the Golgi apparatus and the endoplasmic reticulum, but also occurs in the cytoplasm and nucleus (2). Most glycoconjugates are localized to cell surfaces, where glycans participate in events essential for cell viability and function, such as cell adhesion, motility, and intracellular signaling (2). Changes in these functions are key steps seen when normal cells transform to malignant ones, and these are also reflected in changes of a cell''s glycan profile, observed in many cancers (3, 4). Specific structural changes in glycans may serve as cancer biomarkers (5, 6), and changes in glycosylation profiles are related to aggressive behavior in tumor cells (7–9).Cancer-associated asparagine-linked glycan (N-glycan) structures may play specific roles in supporting tumor progression; growth (10, 11), invasion (12, 13), and angiogenesis (14). Changes in the N-glycan profile emerge in numerous cancers, including lung (15, 16), breast (17), and colorectal cancer (CRC)¹ (16, 18). Balog et al. (18) comparing the N-glycomic profile of CRC tissue to adjacent normal mucosa, reported differences in specific glycan structures. Moreover, serum N-glycosylation profile from patients with CRC differ from those of healthy controls (19).Colorectal cancer is the third most common cause of cancer-related death worldwide and its incidence is rising; 40% of CRCs are of rectal origin. Roughly 40% of patients have localized disease (stage I–II; Dukes A–B), another 40% loco regional disease (stage III; Dukes C), and 20% metastasized disease (stage IV; Dukes D) (20). Although stage at diagnosis is the most important factor determining prognosis, clinical outcome, and response to adjuvant treatment can markedly vary within each stage. Adjuvant therapy routinely goes to stage III patients, but the benefit of adjuvant treatment for stage II patients is unclear. Of stage II patients, 80% are cured by radical surgery alone. To identify patients who will benefit from postoperative treatment, we need novel biomarkers. The glycan profile of the tumor tissue could provide new biomarkers for diagnosis and prognosis of cancer.In this study, we characterized the N-glycomic profiles of rectal adenomas and carcinomas by MALDI-TOF mass spectrometric (MS) profiling of asparagine-linked glycans. Our aim was to identify differences between adenomas and carcinomas, and also between cancers of different stages. Based on glycan profiling, we also chose, for immunohistochemical expression studies of a series of 220 CRC patients, two glycan markers: sialyl Lewis a and pauci-mannose.     

Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach

Introduction

Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression.

Method

We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20).

Results

Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively.

Conclusions

Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.     

The Potentials of Glycomics in Biomarker Discovery

Introduction

Glycans have unique characteristics that are significantly different from nucleic acids and proteins in terms of biosynthesis, structures, and functions. Moreover, their isomeric nature and the complex linkages between residues have made glycan analysis a challenging task. Disease development and progression are usually associated with alternations in glycosylation on tissue proteins and/or blood proteins. Glycans released from tissue/blood proteins hence provide a valuable source of biomarkers. In this postgenome era, glycomics is an emerging research field. Glycome refers to a repertoire of glycans in a tissue/cell type, while glycomics is the study of glycome. In the past few years, attempts have been made to develop novel methodologies for quantitative glycomic profiling and to identify potential glycobiomarkers. It can be foreseen that glycomics holds the promise for biomarker discovery. This review provides an overview of the unique features of glycans and the historical applications of such features to biomarker discovery.

Future Prospective

The concept of glycomics and its recent advancement and future prospective in biomarker research are reviewed. Above all, there is no doubt that glycomics is gaining momentum in biomarker research.     

基于FFPE组织切片的膀胱癌N-连接糖链原位酶解及分析

目的 研究膀胱癌FFPE组织切片的N-连接糖链,发现膀胱癌FFPE肿瘤组织的异常N-连接糖链修饰情况。方法 发展基于FFPE组织切片原位提取N-连接糖链的实验流程。通过PNGase F酶切FFPE组织解释放N-连接糖链。对N-连接糖链自由端进行全甲基化修饰。通过MALDI-TOF/TOF-MS检测N-连接糖链的相对含量。进行数据库匹配,确定N-连接糖链的可能糖型。ROC分析用于预测显著差异N-连接糖链作为预测膀胱癌生物标志物的准确度。结果 MALDI-TOF/TOF-MS检测泛甲基化修饰N-连接糖链的数据显示,在16例膀胱癌患者的肿瘤和癌旁组织的3次重复实验中,肿瘤组织中蛋白质高甘露糖型N2H6、N2H7、N2H8、N2H9和复杂型N5H6F1糖链修饰水平显著上升,同时高甘露糖型N2H5、杂合型N3H5以及复杂型N3H4、N4H4、N5H6F1S2糖链修饰水平显著下降。ROC分析显示,双天线型N-连接糖链N3H4（AUC=0.90）和N4H4（AUC=0.91）在单独或者共同区分膀胱癌患者肿瘤组织和癌旁组织中都具有很好的可靠性,可能成为膀胱癌的潜在生物标志物。结论 膀胱癌FFPE肿瘤组织中存在蛋白质异常N-糖基化修饰,N-连接糖链N3H4和N4H4或可成为膀胱癌的潜在生物标志物。     

Differential Anti-Glycan Antibody Responses in Schistosoma mansoni-Infected Children and Adults Studied by Shotgun Glycan Microarray

Background

Schistosomiasis (bilharzia) is a chronic and potentially deadly parasitic disease that affects millions of people in (sub)tropical areas. An important partial immunity to Schistosoma infections does develop in disease endemic areas, but this takes many years of exposure and maturation of the immune system. Therefore, children are far more susceptible to re-infection after treatment than older children and adults. This age-dependent immunity or susceptibility to re-infection has been shown to be associated with specific antibody and T cell responses. Many antibodies generated during Schistosoma infection are directed against the numerous glycans expressed by Schistosoma. The nature of glycan epitopes recognized by antibodies in natural schistosomiasis infection serum is largely unknown.

Methodology/Principal Findings

The binding of serum antibodies to glycans can be analyzed efficiently and quantitatively using glycan microarray approaches. Very small amounts of a large number of glycans are presented on a solid surface allowing binding properties of various glycan binding proteins to be tested. We have generated a so-called shotgun glycan microarray containing natural N-glycan and lipid-glycan fractions derived from 4 different life stages of S. mansoni and applied this array to the analysis of IgG and IgM antibodies in sera from children and adults living in an endemic area. This resulted in the identification of differential glycan recognition profiles characteristic for the two different age groups, possibly reflecting differences in age or differences in length of exposure or infection.

Conclusions/Significance

Using the shotgun glycan microarray approach to study antibody response profiles against schistosome-derived glycan elements, we have defined groups of infected individuals as well as glycan element clusters to which antibody responses are directed in S. mansoni infections. These findings are significant for further exploration of Schistosoma glycan antigens in relation to immunity.     

A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression

Aberrant glycosylation on glycoproteins that are either presented on the surface or secreted by cancer cells is a potential source of disease biomarkers and provides insights into disease pathogenesis. N-Glycans of the total serum glycoproteins from advanced breast cancer patients and healthy individuals were sequenced by HPLC with fluorescence detection coupled with exoglycosidase digestions and mass spectrometry. We observed a significant increase in a trisialylated triantennary glycan containing alpha1,3-linked fucose which forms part of the sialyl Lewis x epitope. Following digestion of the total glycan pool with a combination of sialidase and beta-galactosidase, we segregated and quantified a digestion product, a monogalactosylated triantennary structure containing alpha1,3-linked fucose. We compared breast cancer patients and controls and detected a 2-fold increase in this glycan marker in patients. In 10 patients monitored longitudinally, we showed a positive correlation between this glycan marker and disease progression and also demonstrated its potential as a better indicator of metastasis compared to the currently used biomarkers, CA 15-3 and carcinoembryonic antigen (CEA). A pilot glycoproteomic study of advanced breast cancer serum highlighted acute-phase proteins alpha1-acid glycoprotein, alpha1-antichymotrypsin, and haptoglobin beta-chain as contributors to the increase in the glycan marker which, when quantified from each of these proteins, marked the onset of metastasis in advance of the CA 15-3 marker. These preliminary findings suggest that specific glycans and glycoforms of proteins may be candidates for improved markers in the monitoring of breast cancer progression.     

The Prevalence and Nature of Glycan Alterations on Specific Proteins in Pancreatic Cancer Patients Revealed Using Antibody-Lectin Sandwich Arrays

Changes to the glycan structures of proteins secreted by cancer cells are known to be functionally important and to have potential diagnostic value. However, an exploration of the population variation and prevalence of glycan alterations on specific proteins has been lacking because of limitations in conventional glycobiology methods. Here we report the use of a previously developed antibody-lectin sandwich array method to characterize both the protein and glycan levels of specific mucins and carcinoembryonic antigen-related proteins captured from the sera of pancreatic cancer patients (n = 23) and control subjects (n = 23). The MUC16 protein was frequently elevated in the cancer patients (65% of the patients) but showed no glycan alterations, whereas the MUC1 and MUC5AC proteins were less frequently elevated (30 and 35%, respectively) and showed highly prevalent (up to 65%) and distinct glycan alterations. The most frequent glycan elevations involved the Thomsen-Friedenreich antigen, fucose, and Lewis antigens. An unexpected increase in the exposure of α-linked mannose also was observed on MUC1 and MUC5ac, indicating possible N-glycan modifications. Because glycan alterations occurred independently from the protein levels, improved identification of the cancer samples was achieved using glycan measurements on specific proteins relative to using the core protein measurements. The most significant elevation was the cancer antigen 19-9 on MUC1, occurring in 19 of 23 (87%) of the cancer patients and one of 23 (4%) of the control subjects. This work gives insight into the prevalence and protein carriers of glycan alterations in pancreatic cancer and points to the potential of using glycan measurements on specific proteins for highly effective biomarkers.Alterations to the glycan structures on extracellular proteins are a common feature of many types of epithelial cancer such as pancreatic, colon, and breast cancers (1, 2). Cancer-associated glycan structures are thought to be functionally involved in many of the phenotypes characterizing cancer cells, including the ability to migrate, avoid apoptosis, evade immune destruction, and enter and exit the vasculature (3). Because proteins bearing cancer-associated glycans can be shed by tumor cells into the circulation, blood-based diagnostic tests using glycan detection may be possible. A potential advantage of using glycans for diagnostics is that carbohydrate modifications of particular proteins may be altered more frequently or more specifically in certain disease states than their underlying core protein concentrations. However, to evaluate and use such a strategy, the prevalence with which various structures appear and the specific proteins on which they appear must be better characterized.Previous studies of cancer-associated glycosylation using enzymatic, chromatographic, and mass spectrometry methods have been very effective for providing detailed information about the glycan structures produced by cancer cells, but because of the requirements for large amounts of material and the time involved to analyze each sample, these studies generally used either cell culture material or a small number of patient samples. Therefore, while many cancer-associated glycans have been identified, much remains unknown about these glycans, including how often they appear, how closely they are associated with particular disease states, and the distribution of protein carriers on which they appear.Affinity-based methods, using reagents such as lectins or glycan-binding antibodies, are a valuable complement to the above mentioned methods. Using antibodies or lectins that bind specific glycans, one may reproducibly measure the levels of those glycans over multiple samples. Although affinity-based glycosylation studies do not provide the structural detail provided by mass spectrometry and enzymatic methods, they can provide information about the biological variation of a particular motif.Lectins and glycan-binding antibodies have been used extensively in immunohistochemistry, for example in studies to examine the tissue distribution in pancreatic tumors of certain blood group carbohydrates (4, 5). Lectins have been valuable in immunoaffinity electrophoresis and blotting methods to identify cancer-associated glycan variants on major serum proteins such as α-fetoprotein (6), haptoglobin (7, 8), α₁-acid glycoprotein (9), and α₁-antitrypsin (10). Antibodies raised against particular glycan groups, such as the Thomsen-Friedenreich antigens (11), the Lewis blood group structures (12), and underglycosylated MUC1¹ (13) also have been used to study the roles of glycans in cancer. As a means of quantifying glycans on specific proteins, lectins have been used in the capture or detection of proteins in microtiter plates (14).We previously demonstrated an antibody-lectin sandwich array method (15) that is a valuable complement to the above methods and is ideal for profiling the prevalence of multiple glycans on multiple proteins. Glycan levels can be probed directly from biological samples, and many samples or detection conditions can be processed efficiently in a low volume, high throughput format (16). This method is complementary to lectin microarrays (17–19), which are useful for measuring glycan levels on individual, purified proteins; glycan microarrays (20, 21), which are used to measure the recognition of carbohydrate structures by various glycan-binding reagents; and glycoprotein arrays (22) for examining glycosylation on proteins isolated from biological samples.We applied this method to the study of glycan alterations on proteins in the circulation of pancreatic cancer patients. We sought to define the prevalence of various glycan alterations on particular protein carriers and to investigate whether those measurements have advantages for cancer diagnostics relative to measurements of core proteins. We designed antibody microarrays to target members of the mucin and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) families because some of those proteins are known to carry cancer-associated glycans. Mucins are extracellular, long-chain glycoproteins involved in the control and protection of epithelial surfaces, and the expression and glycosylation of several mucins are often altered and functionally involved in cancer (23, 24). The CEACAM family of proteins also is functionally involved in cancer, and they carry cancer-associated glycans (25, 26), but the glycans on CEACAMs are less well studied than those on mucins. By measuring both glycan levels and the core protein levels of several of these molecules, we were able to investigate whether alterations to glycans can appear at a higher rate than changes to core protein abundances. The ability to test the presence of glycan structures on multiple protein carriers in multiple samples was critical to investigating these questions.     

Volatile metabolomic signature of bladder cancer cell lines based on gas chromatography–mass spectrometry

Introduction

Recent studies provide a convincing support that the presence of cancer cells in the body leads to the alteration of volatile organic compounds (VOCs) emanating from biological samples, particularly of those closely related with tumoral tissues. Thus, a great interest emerged for the study of cancer volatilome and subsequent attempts to confirm VOCs as potential diagnostic biomarkers.

Objectives

The aim of this study was to determine the volatile metabolomic signature of bladder cancer (BC) cell lines and provide an in vitro proof-of-principle that VOCs emanated into the extracellular medium may discriminate BC cells from normal bladder epithelial cells.

Methods

VOCs in the culture media of three BC cell lines (Scaber, J82, 5637) and one normal bladder cell line (SV-HUC-1) were extracted by headspace-solid phase microextraction and analysed by gas chromatography-mass spectrometry (HS-SPME/GC–MS). Two different pH (pH 2 and 7) were used for VOCs extraction to infer the best pH to be used in in vitro metabolomic studies.

Results

Multivariate analysis revealed a panel of volatile metabolites that discriminated cancerous from normal bladder cells, at both pHs, although a higher number of discriminative VOCs was obtained at neutral pH. Most of the altered metabolites were ketones and alkanes, which were generally increased in BC compared to normal cells, and alcohols, which were significantly decreased in BC cells. Among them, three metabolites, namely 2-pentadecanone, dodecanal and γ-dodecalactone (the latter only tentatively identified), stood out as particularly important metabolites and promising volatile biomarkers for BC detection. Furthermore, our results also showed the potential of VOCs in discriminating BC cell lines according to tumour grade and histological subtype.

Conclusions

We demonstrate that a GC–MS metabolomics-based approach for analysis of VOCs is a valuable strategy for identifying new and specific biomarkers that may improve BC diagnosis. Future studies should entail the validation of volatile signature found for BC cell lines in biofluids from BC patients.

     

Proteins from formalin-fixed paraffin-embedded prostate cancer sections that predict the risk of metastatic disease

Background

Prostate cancer is the most frequently diagnosed cancer in men and the third leading cause of cancer related deaths among men living in developed countries. Biomarkers that predict disease outcome at the time of initial diagnosis would substantially aid disease management.

Results

Proteins extracted from formalin-fixed paraffin-embedded tissue were identified using nanoflow liquid chromatography-MALDI MS/MS or after separation by one- or two-dimensional electrophoresis. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000963. A list of potential biomarker candidates, based on proposed associations with prostate cancer, was derived from the 320 identified proteins. Candidate biomarkers were then examined by multiplexed Western blotting of archival specimens from men with premetastatic disease and subsequent disease outcome data. Annexin A2 provided the best prediction of risk of metastatic disease (log-rank Chi squared p = 0. 025). A tumor/control tissue >2-fold relative abundance increase predicted early biochemical failure, while <2-fold change predicted late or no biochemical failure.

Conclusions

This study confirms the potential for use of archival FFPE specimens in the search for prognostic biomarkers for prostate cancer and suggests that annexin A2 abundance in diagnostic biopsies is predictive for metastatic potential. Protein profiling each cancer may lead to an overall reduction in mortality from metastatic prostate cancer as well as reduced treatment associated morbidity.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9096-3) contains supplementary material, which is available to authorized users.     

Insight into the Regulation of Glycan Synthesis in Drosophila Chaoptin Based on Mass Spectrometry

Background

A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown.

Methodology/Principal Findings

In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate α-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type.

Conclusions/Significance

These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins.     

Altered glycosylation in cancer: A promising target for biomarkers and therapeutics

Glycosylation is a well-regulated cell and microenvironment specific post-translational modification. Several glycosyltransferases and glycosidases orchestrate the addition of defined glycan structures on the proteins and lipids. Recent advances and systemic approaches in glycomics have significantly contributed to a better understanding of instrumental roles of glycans in health and diseases. Emerging research evidence recognized aberrantly glycosylated proteins as the modulators of the malignant phenotype of cancer cells. The Cancer Genome Atlas has identified alterations in the expressions of glycosylation-specific genes that are correlated with cancer progression. However, the mechanistic basis remains poorly explored. Recent researches have shown that specific changes in the glycan structures are associated with 'stemness' and epithelial-to-mesenchymal transition of cancer cells. Moreover, epigenetic changes in the glycosylation pattern make the tumor cells capable of escaping immunosurveillance mechanisms. The deciphering roles of glycans in cancer emphasize that glycans can serve as a source for the development of novel clinical biomarkers. The ability of glycans in intervening various stages of tumor progression and the biosynthetic pathways involved in glycan structures constitute a promising target for cancer therapy. Advances in the knowledge of innovative strategies for identifying the mechanisms of glycan-binding proteins are hoped to hold great potential in cancer therapy. This review discusses the fundamental role of glycans in regulating tumorigenesis and tumor progression and provides insights into the influence of glycans in the current tactics of targeted therapies in the clinical setting.     

Clinical application of quantitative glycomics

ABSTRACT

Introduction: Aberrant glycosylation has been associated with many diseases. Decades of research activities have reported many reliable glycan biomarkers of different diseases which enable effective disease diagnostics and prognostics. However, none of the glycan markers have been approved for clinical diagnosis. Thus, a review of these studies is needed to guide the successful clinical translation.

Area covered: In this review, we describe and discuss advances in analytical methods enabling clinical glycan biomarker discovery, focusing only on studies of released glycans. This review also summarizes the different glycobiomarkers identified for cancers, Alzheimer’s disease, diabetes, hepatitis B and C, and other diseases.

Expert commentary: Along with the development of techniques in quantitative glycomics, more glycans or glycan patterns have been reported as better potential biomarkers of different diseases and proved to have greater diagnostic/diagnostic sensitivity and specificity than existing markers. However, to successfully apply glycan markers in clinical diagnosis, more studies and verifications on large biological cohorts need to be performed. In addition, faster and more efficient glycomic strategies need to be developed to shorten the turnaround time. Thus, glycan biomarkers have an immense chance to be used in clinical prognosis and diagnosis of many diseases in the near future.