Discovery of lipid profiles in plasma-derived extracellular vesicles as biomarkers for breast cancer diagnosis.

Lipids are a major component of extracellular vesicles; however, their significance in tumorigenesis and progression has not been well elucidated. As we previously found that lipid profiles drastically changed in breast tumors upon progression, we hypothesized that lipid profiles of plasma-derived extracellular vesicles could be utilized as breast cancer biomarkers. Here, we adopted modified sucrose cushion ultracentrifugation to isolate plasma-derived extracellular vesicles from breast cancer (n = 105), benign (n = 11), and healthy individuals (n = 43) in two independent cohorts (n = 126 and n = 33) and conducted targeted lipidomic analysis. We established a breast cancer diagnostic model comprising three lipids that showed favorable performance with the area under the receiver operating characteristic curve of 0.759, 0.743, and 0.804 in the training, internal validation, and external test sets, respectively. Moreover, we identified several lipids that could effectively discriminate breast cancer progression and subtypes: phosphatidylethanolamines and phosphatidylserines were relatively higher in Stage III, whereas phosphatidylcholines and sphingomyelins were higher in Stage IV; phosphatidylcholines and ceramides were correspondingly concentrated in HER2-positive patients, while lysophosphatidylcholines and polyunsaturated triglycerides were concentrated in the triple-negative breast cancer subtype. Lipid profiling of plasma-derived extracellular vesicles is a non-invasive and promising approach for diagnosing, staging, and subtyping breast cancer.

ELOVL2 promotes cancer progression by inhibiting cell apoptosis in renal cell carcinoma.

Renal cell carcinoma (RCC) is an aggressive genitourinary malignancy which has been associated with a poor prognosis, particularly in patients with metastasis, its major subtypes being clear cell RCC (ccRCC), papillary PCC (pRCC) and chromophobe RCC (chRCC). The presence of intracellular lipid droplets (LDs) is considered to be a hallmark of ccRCC. The importance of an altered lipid metabolism in ccRCC has been widely recognized. The elongation of very­long­chain fatty acid (ELOVL) catalyzes the elongation of fatty acids (FAs), modulating lipid composition, and is required for normal bodily functions. However, the involvement of elongases in RCC remains unclear. In the present study, the expression of ELOVL2 in ccRCC was examined; in particular, high levels of seven ELOVL isozymes were observed in primary tumors. Of note, elevated ELOVL2 expression levels were observed in ccRCC, as well as in pRCC and chRCC. Furthermore, a higher level of ELOVL2 was significantly associated with the increased incidence of a poor prognosis of patients with ccRCC and pRCC. The CRISPR/Cas9­mediated knockdown of ELOVL2 resulted in the suppression of the elongation of long­chain polyunsaturated FAs and increased LD production in renal cancer cells. Moreover, ELOVL2 ablation resulted in the suppression of cellular proliferation via the induction of apoptosis in vitro and the attenuation of tumor growth in vivo. On the whole, the present study provides new insight into the tumor proliferation mechanisms involving lipid metabolism, and suggests that ELOVL2 may be an attractive novel target for RCC therapy.

Molecular classification and diagnostics of upper urinary tract urothelial carcinoma.

Upper urinary tract urothelial carcinoma (UTUC) is one of the common urothelial cancers. Its molecular pathogenesis, however, is poorly understood, with no useful biomarkers available for accurate diagnosis and molecular classification. Through an integrated genetic study involving 199 UTUC samples, we delineate the landscape of genetic alterations in UTUC enabling genetic/molecular classification. According to the mutational status of TP53, MDM2, RAS, and FGFR3, UTUC is classified into five subtypes having discrete profiles of gene expression, tumor location/histology, and clinical outcome, which is largely recapitulated in an independent UTUC cohort. Sequencing of urine sediment-derived DNA has a high diagnostic value for UTUC with 82.2% sensitivity and 100% specificity. These results provide a solid basis for better diagnosis and management of UTUC.

The liposome of trehalose dimycolate extracted from M. bovis BCG induces antitumor immunity via the activation of dendritic cells and CD8+ T cells.

Intravesical Bovis bacillus Calmette-Guérin (BCG) therapy is the most effective immunotherapy for bladder cancer, but it sometime causes serious side effects because of its inclusion of live bacteria. It is necessary to develop a more active but less toxic immunotherapeutic agent. Trehalose 6,6'-dimycolate (TDM), the most abundant hydrophobic glycolipid of the BCG cell wall, has been reported to show various immunostimulatory activities such as granulomagenesis and adjuvant activity. Here, we developed cationic liposomes incorporating TDM purified from Mycobacterium bovis BCG Connaught, and we investigated the antitumor effect of the cationic liposome TDM (Lip-TDM). Lip-TDM exerted an antitumor effect in bladder cancer, colon cancer, and melanoma-bearing mouse models that was comparable or even superior to that of BCG, with no body weight loss or granuloma formation. The antitumor effect of Lip-TDM disappeared in two types of mice: those with depletion of CD8+ T cells, and those with knockout of macrophage-inducible C-type lectin (Mincle) which recognize TDM. Lip-TDM treatment enhanced the maturation and migration of dendritic cells in the tumor microenvironment in a Mincle-dependent manner. Our results elucidate mechanisms that underlie Lip-TDM treatment and suggest that Lip-TDM has potential as a safe and effective treatment for various cancers.

Case Report: Molecular Characterization of Aggressive Malignant Retroperitoneal Solitary Fibrous Tumor: A Case Study.

Solitary fibrous tumors (SFT) are mesenchymal neoplasms with a favorable prognosis usually originating from the visceral pleura. Rarely, they may occur at various extrapleural sites and show malignant behavior coupled with dedifferentiation. NAB2-STAT6 fusion gene and STAT6 nuclear expression are biomarkers for diagnosis of SFT in addition to CD34, Bcl-2, and CD99. Furthermore, several reports have shown specific NAB2-STAT6 fusion variants and loss of STAT6 protein expression are associated with malignancy. We report a rare case of retroperitoneal SFT which rapidly progressed to death within 35 days after admission. Autopsy found a primary tumor containing both benign and malignant histologies, with multiple metastatic sites similar to the malignant, dedifferentiated tumor. STAT6 was detected in the primary differentiated tumor but not in the primary dedifferentiated tumor or lung/liver metastases. However, the NAB2-STAT6 fusion gene (NAB2ex6/STAT6ex16 variant) was detected in the primary tumor and lung/liver metastases. Intriguingly, fusion gene expression at the transcriptional level was downregulated in the dedifferentiated tumors compared to the differentiated tumor. We further performed target DNA sequencing and found gene mutations in TP53, FLT3, and AR in the dedifferentiated tumors, with TP53 mutations especially found among them. We demonstrate that downregulation of NAB2-STAT6 fusion gene at the transcriptional level is associated with malignant SFT for the first time. Moreover, the present study supports the idea that TP53 mutations promote malignancy in SFTs.

The Inhibitor of Apoptosis Protein Livin Confers Resistance to Fas-Mediated Immune Cytotoxicity in Refractory Lymphoma.

Death receptor Fas-mediated apoptosis not only eliminates nonspecific and autoreactive B cells but also plays a major role in antitumor immunity. However, the possible mechanisms underlying impairment of Fas-mediated induction of apoptosis during lymphomagenesis remain unknown. In this study, we employed our developed syngeneic lymphoma model to demonstrate that downregulation of Fas is required for both lymphoma development and lymphoma cell survival to evade immune cytotoxicity. CD40 signal activation significantly restored Fas expression and thereby induced apoptosis after Fas ligand treatment in both mouse and human lymphoma cells. Nevertheless, certain human lymphoma cell lines were found to be resistant to Fas-mediated apoptosis, with Livin (melanoma inhibitor of apoptosis protein; ML-IAP) identified as a driver of such resistance. High expression of Livin and low expression of Fas were associated with poor prognosis in patients with aggressive non-Hodgkin's lymphoma. Livin expression was tightly driven by bromodomain and extraterminal (BET) proteins BRD4 and BRD2, suggesting that Livin expression is epigenetically regulated in refractory lymphoma cells to protect them from Fas-mediated apoptosis. Accordingly, the combination of CD40-mediated Fas restoration with targeting of the BET proteins-Livin axis may serve as a promising immunotherapeutic strategy for refractory B-cell lymphoma. SIGNIFICANCE: These findings yield insights into identifying risk factors in refractory lymphoma and provide a promising therapy for tumors resistant to Fas-mediated antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/20/4439/F1.large.jpg.

Combined Cohesin-RUNX1 Deficiency Synergistically Perturbs Chromatin Looping and Causes Myelodysplastic Syndromes.

STAG2 encodes a cohesin component and is frequently mutated in myeloid neoplasms, showing highly significant comutation patterns with other drivers, including RUNX1. However, the molecular basis of cohesin-mutated leukemogenesis remains poorly understood. Here we show a critical role of an interplay between STAG2 and RUNX1 in the regulation of enhancer-promoter looping and transcription in hematopoiesis. Combined loss of STAG2 and RUNX1, which colocalize at enhancer-rich, CTCF-deficient sites, synergistically attenuates enhancer-promoter loops, particularly at sites enriched for RNA polymerase II and Mediator, and deregulates gene expression, leading to myeloid-skewed expansion of hematopoietic stem/progenitor cells (HSPC) and myelodysplastic syndromes (MDS) in mice. Attenuated enhancer-promoter loops in STAG2/RUNX1-deficient cells are associated with downregulation of genes with high basal transcriptional pausing, which are important for regulation of HSPCs. Downregulation of high-pausing genes is also confirmed in STAG2-cohesin-mutated primary leukemia samples. Our results highlight a unique STAG2-RUNX1 interplay in gene regulation and provide insights into cohesin-mutated leukemogenesis. SIGNIFICANCE: We demonstrate a critical role of an interplay between STAG2 and a master transcription factor of hematopoiesis, RUNX1, in MDS development, and further reveal their contribution to regulation of high-order chromatin structures, particularly enhancer-promoter looping, and the link between transcriptional pausing and selective gene dysregulation caused by cohesin deficiency.This article is highlighted in the In This Issue feature, p. 747.

Frequent mutations that converge on the NFKBIZ pathway in ulcerative colitis.

Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer1-3. However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer.

Extracellular vesicles mediate the horizontal transfer of an active LINE-1 retrotransposon.

Long interspersed element-1 (LINE-1 or L1) retrotransposons replicate through a copy-and-paste mechanism using an RNA intermediate. However, little is known about the physical transmission of retrotransposon RNA between cells. To examine the horizontal transfer of an active human L1 retrotransposon mediated by extracellular vesicles (EVs), human cancer cells were transfected with an expression construct containing a retrotransposition-competent human L1 tagged with a reporter gene. Using this model, active retrotransposition events were detected by screening for the expression of the reporter gene inserted into the host genome by retrotransposition. EVs including exosomes and microvesicles were isolated from cells by differential centrifugation. The enrichment of L1-derived reporter RNA transcripts were detected in EVs isolated from cells expressing active L1 retrotransposition. The delivery of reporter RNA was confirmed in recipient cells, and reporter genes were detected in the genome of recipient cells. Additionally, employing qRT-PCR, we found that host-encoded factors are activated in response to increased exposure to L1-derived RNA transcripts in recipient cells. Our results suggest that the horizontal transfer of retrotransposons can occur through the incorporation of RNA intermediates delivered via EVs and may have important implications for the intercellular regulation of gene expression and gene function.

Extracellular vesicles as trans-genomic agents: Emerging roles in disease and evolution.

The composition of genetic material in extracellular vesicles (EV) has sparked interest particularly in the potential for horizontal gene transfer by EV. Although the RNA content of EV has been studied extensively, few reports have examined the DNA content of EV. It is still unclear how DNA is packaged inside EV, and whether they are functional in recipient cells. In this review, we describe the biological significance of genetic material in EV and their possible impacts in recipient cells, with focus on DNA from cancer cell-derived EV and the potential roles they may play in the cancer microenvironment. Another important feature of the genetic content of EV is the presence of retrotransposon elements. In this review, we discuss the possibility of an EV-mediated mechanism for the dispersal of retrotransposon elements, and their potential involvement in the development of genetically influenced diseases. In addition to this, we discuss the potential involvement of EV in the transfer of genetic material across species, and their possible impacts in modulating genome evolution.

Plasma low-molecular-weight proteome profiling identified neuropeptide-Y as a prostate cancer biomarker polypeptide.

In prostate cancer diagnosis, PSA test has greatly contributed to the early detection of prostate cancer; however, expanding overdiagnosis and unnecessary biopsies have emerged as serious issues. To explore plasma biomarkers complementing the specificity of PSA test, we developed a unique proteomic technology QUEST-MS (Quick Enrichment of Small Targets for Mass Spectrometry). The QUEST-MS method based on 96-well formatted sequential reversed-phase chromatography allowing efficient enrichment of <20 kDa proteins quickly and reproducibly. Plasma from 24 healthy controls, 19 benign prostate hypertrophy patients, and 73 prostate cancer patients were purified with QUEST-MS and analyzed by LC/MS/MS. Among 153 057 nonredundant peptides, 189 peptides showed prostate cancer specific detection pattern, which included a neurotransmitter polypeptide neuropeptide-Y (NPY). We further validated the screening results by targeted multiple reaction monitoring technology using independent sample set (n = 110). The ROC curve analysis revealed that logistic regression-based combination of NPY, and PSA showed 81.5% sensitivity and 82.2% specificity for prostate cancer diagnosis. Thus QUEST-MS technology allowed comprehensive and high-throughput profiling of plasma polypeptides and had potential to effectively uncover very low abundant tumor-derived small molecules, such as neurotransmitters, peptide hormones, or cytokines.

High-resolution imaging mass spectrometry reveals detailed spatial distribution of phosphatidylinositols in human breast cancer.

High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is an emerging application for lipid research that provides a comprehensive and detailed spatial distribution of ionized molecules. Recent lipidomic approach has identified several phospholipids and phosphatidylinositols (PIs) are accumulated in breast cancer tissues and are therefore novel biomarker candidates. Because their distribution and significance remain unclear, we investigated the precise spatial distribution of PIs in human breast cancer tissues using high-resolution MALDI IMS. We evaluated tissues from nine human breast cancers and one normal mammary gland by negative ion MALDI IMS at a resolution of 10 µm. We detected 10 PIs with different fatty acid compositions, and their proportions were remarkably variable in the malignant epithelial regions. High-resolution imaging enabled us to discriminate cancer cell clusters from the adjacent stromal tissue within epithelial regions; moreover, this technique revealed that several PIs were specifically localized to cancer cell clusters. These PIs were heterogeneously distributed within cancer cell clusters, allowing us to identify two different populations of cancer cells that predominantly expressed either PI(18:0/18:1) or PI(18:0/20:3). Tracing the expression level of PIs during cancer cell progression suggested that the latter population is associated with the invasion. Our study documents a novel model for phospholipid analysis of breast cancer tissues by using high-resolution MALDI IMS and identifies candidate PIs that can describe a specific phenotype of cancer cells.

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid.

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.

Rapid quantitative profiling of N-glycan by the glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid.

Protein glycosylation is a crucial phenomenon for understanding protein functions, since its patterns and degree are associated with many biological processes, such as intercellular signaling and immune response. We previously reported a novel glycan-labeling method using a 3-ainoquinoline/α-cyano-4-hydroxycinnamic acid (3-AQ/CHCA) liquid matrix for highly sensitive detection by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). In the present study, we examined the practicality of this method for qualitative and quantitative glycan profile analysis. We first investigated the reproducibility of the data for 16 N-glycans prepared from human epidermal growth factor receptor type 2 (HER2). All of the data obtained in intra-assays and interassays were highly correlated with statistical significance (R(2) > 0.9, p < 0.05). In addition, the HER2 glycosylation pattern differed significantly between different breast cancer cell lines SK-BR-3 and BT474 in a comparative analysis of profile data. Finally, the quantitative capability of this method was examined by using PA-labeled monosialylated N-glycan as an internal standard (IS). Using IS for AQ-labeled neutral and sialylated standard glycans, the ion peak intensity was highly linear (R(2) > 0.9) from 0.5 to 5000 fmol. Furthermore, using IS for HER2 N-glycans, all of the N-glycans were highly linear with their dilution factors (R(2) > 0.9). These results suggest that our developed AQ labeling method enabled rapid qualitative and quantitative analyses of glycans. This glycan analysis method should contribute to the field of biomarker discovery and biomedicine in applications such as quality control of biotechnology-based drugs.

Proteomic characterization of ovarian cancers identifying annexin-A4, phosphoserine aminotransferase, cellular retinoic acid-binding protein 2, and serpin B5 as histology-specific biomarkers.

Numerous studies have suggested that the different histological subtypes of ovarian carcinoma (i.e. clear cell, endometrioid, mucinous, and serous) have distinct clinical histories and characteristics; however, most studies that have aimed to determine biomarker have not performed comprehensive analyses based on subtype specificity. In the present study, we performed two-dimensional gel electrophoresis-based differential proteomic analysis of the different histological subtypes of ovarian carcinoma using tissue specimens from 39 patients. Seventy-seven protein spots (55 unique proteins) were found to be up- or downregulated in a subtype-specific manner. The most significant difference was observed for: (i) annexin-A4 (ANXA4) and phosphoserine aminotransferase (PSAT1), which are expressed strongly in clear cell carcinoma; (ii) cellular retinoic acid-binding protein 2 (CRABP2), which is expressed specifically in serous carcinoma; and (iii) serpin B5 (SPB5), which is upregulated in mucinous carcinoma. Validation of these candidates by western blotting using a 34 additional test sample set resulted in an expression pattern that was consistent with the screening and revealed that differential expression was independent of cancer stage or tumor grade within each subtype. Thus, the present study reinforces the notion that ovarian cancer subtypes can be clearly delineated on a molecular basis into four histopathological groups, and we propose that ANXA4, PSAT1, CRABP2, and SPB5 are candidate subtype-specific biomarkers that can help define the basis of tumor histology at a molecular level.

Development of iodoacetic acid-based cysteine mass tags: detection enhancement for cysteine-containing peptide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We developed and characterized 6 new cysteine mass tags for high-sensitivity peptide analysis. The structural features are: (1) iodoacetyl group for thiol tagging, (2) hydrophilic character for reducing sample loss, (3) tertiary amino, quaternary ammonium, or guanidino group for high proton affinity, and (4) no amide bonding for minimizing fragmentation of tag moiety in collision-induced dissociation. By using these tags, 2- to 200-fold MS sensitivity was achieved, compared to control peptide with carbamydomethylation.

Flexible antibodies with nonprotein hinges.

There is a significant need for antibodies that can bind targets with greater affinity. Here we describe a novel strategy employing chemical semisynthesis to produce symmetroadhesins: antibody-like molecules having nonprotein hinge regions that are more flexible and extendible and are capable of two-handed binding. Native chemical ligation was carried out under mild, non-denaturing conditions to join a ligand binding domain (Aß peptide) to an IgG1 Fc dimer via discrete oxyethylene oligomers of various lengths. Two-handed Aß-Fc fusion proteins were obtained in quantitative yield and shown by surface plasmon resonance to bind an anti-Aß antibody with a K(D) at least two orders of magnitude greater than the cognate Aß peptide. MALDI-TOF MS analysis confirmed the protein/nonprotein/protein structure of the two-handed molecules, demonstrating its power to characterize complex protein-nonprotein hybrids by virtue of desorption/ionization mediated by peptide sequences contained therein. We anticipate many applications for symmetroadhesins that combine the target specificity of antibodies with the novel physical, chemical and biological properties of nonprotein hinges.

A comprehensive peptidome profiling technology for the identification of early detection biomarkers for lung adenocarcinoma.

The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000-5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273-283, FIBA 5-16, and LBN 306-313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens.

Deglycosylation and label-free quantitative LC-MALDI MS applied to efficient serum biomarker discovery of lung cancer.

BACKGROUND: Serum is an ideal source of biomarker discovery and proteomic profiling studies are continuously pursued on serum samples. However, serum is featured by high level of protein glycosylations that often cause ionization suppression and confound accurate quantification analysis by mass spectrometry. Here we investigated the effect of N-glycan and sialic acid removal from serum proteins on the performance of label-free quantification results. RESULTS: Serum tryptic digests with or without deglycosylation treatment were analyzed by LC-MALDI MS and quantitatively compared on the Expressionist Refiner MS module. As a result, 345 out of 2,984 peaks (11.6%) showed the specific detection or the significantly improved intensities in deglycosylated serum samples (P < 0.01). We then applied this deglycosylation-based sample preparation to the identification of lung cancer biomarkers. In comparison between 10 healthy controls and 20 lung cancer patients, 40 peptides were identified to be differentially presented (P < 0.01). Their quantitative accuracies were further verified by multiple reaction monitoring. The result showed that deglycosylation was needed for the identification of some unique candidates, including previously unreported O-linked glycopeptide of complement component C9. CONCLUSIONS: We demonstrated here that sample deglycosylation improves the quantitative performance of shotgun proteomics, which can be effectively applied to any samples with high glycoprotein contents.

Development of serum glycoproteomic profiling technique; simultaneous identification of glycosylation sites and site-specific quantification of glycan structure changes.

Characterization and interpretation of disease-associated alterations of protein glycosylation are the central aims of the emerging glycoproteomics projects, which are expected to lead to more sensitive and specific diagnosis and improve therapeutic outcomes for various diseases. Here we report a new approach to identify carbohydrate-targeting serum biomarkers, termed isotopic glycosidase elution and labeling on lectin-column chromatography (IGEL). This technology is based on glycan structure-specific enrichment of glycopeptides by lectin-column chromatography and site-directed tagging of N-glycosylation sites by (18)O during the elution with N-glycosidase. The combination of IGEL with 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) stable isotope labeling enabled us not only to identify N-glycosylation sites effectively but also to compare glycan structures on each glycosylation site quantitatively in a single LC/MS/MS analysis. We applied this method to eight sera from lung cancer patients and controls, and finally identified 107 glycopeptides in their sera, including A2GL_Asn151, A2GL_Asn290, CD14_Asn132, CO8A_Asn417, C163A_Asn64, TIMP1_Asn30, and TSP1_Asn1049 which showed the significant change of the affinity to Concanavalin A (ConA) lectin between the lung cancer samples and the controls (p < 0.05 and more than twofold change). These screening results were further confirmed by the conventional lectin-column chromatography and immunoblot analysis using additional serum samples. Our novel methodology, which should be valuable for diverse biomarker discoveries, can provide high-throughput and quantitative profiling of glycan structure alterations.