6 research outputs found
Immunoassay with Single-Walled Carbon Nanotubes as Near-Infrared Fluorescent Labels
The
intrinsic photoluminescence of single-walled carbon nanotubes (CNTs)
in the near-infrared (NIR) above 1000 nm makes them promising candidates
for biological probes owing to low interference by bioorganic molecules
and deep tissue penetration. We here demonstrate an immunoassay by
using a NIR CNT labels conjugated to immunoglobulin G (IgG) antibodies.
Most of the CNT-conjugated IgG was successfully immunoprecipitated
with protein G-attached magnetic beads and eluted from them, which
was confirmed by the NIR emission of the conjugated CNTs at 1000–1200
nm. The photoluminescence intensity of the CNT labels was strong enough
to detect antigens at 600 pM by our simple procedures
Glycobiomarker, Fucosylated Short-Form Secretogranin III Levels Are Increased in Serum of Patients with Small Cell Lung Carcinoma
Secretogranin
III (SgIII) is a member of the chromogranin/secretogranin
family of neuroendocrine secretory proteins. Granins are expressed
in endocrine and neuroendocrine cells and subsequently processed into
bioactive hormones. Although granin-derived peptide expression is
correlated with neuroendocrine carcinomas, little is known about SgIII.
We previously identified SgIII by a comparative glycoproteomics approach
for elucidation of glycobiomarker candidates in lung carcinoma. Here,
we examined the expression, secretion, and glycosylation of SgIII
to identify novel biomarkers of small cell lung carcinoma (SCLC).
In comparative immunohistochemical analysis and secretion profiling,
SgIII was observed in all types of lung cancer. However, low-molecular-weight
SgIII (short-form SgIII) was specifically found in SCLC culture medium.
Glycoproteomics analysis showed that a fucosylated glycan was attached
to the first of three potential <i>N</i>-glycosylation sites
and an unfucosylated glycan was detected on the second site; however,
the third site was not glycosylated. Next, we performed lectin capture
with a fucose-binding lectin and detected short-form SgIII specifically
in the sera of patients with SCLC. The results suggested an association
between the fucosylated glycoform of short-form SgIII and SCLC. Thus,
fucosylated short-form SgIII may be a valuable biomarker for SCLC
and could be used to monitor development of the disease. All MS data
are available via ProteomeXchange and jPOST with identifiers PXD007626
and JPST000313, respectively
Glycoproteomics Approach for Identifying Glycobiomarker Candidate Molecules for Tissue Type Classification of Non-small Cell Lung Carcinoma
Histopathological
classification of lung cancer has important implications
in the application of clinical practice guidelines and the prediction
of patient prognosis. Thus, we focused on discovering glycobiomarker
candidates to classify the types of lung cancer tissue. First, we
performed lectin microarray analysis of lung cancer tissue specimens
and cell lines and identified Aleuria aurantia lectin (AAL), <i>Hippeastrum</i> hybrid lectin (HHL),
and Concanavalia ensiformis agglutinin
(ConA) as lectin probes specific to non-small cell lung carcinoma
(NSCLC). LC–MS-based analysis was performed for the comprehensive
identification of glycoproteins and N-linked glycosylation sites using
lectin affinity capture of NSCLC-specific glycoforms of glycoproteins.
This analysis identified 1092 AAL-bound glycoproteins (316 gene symbols)
and 948 HHL/ConA-bound glycoproteins (279 gene symbols). The lectin
microarray-assisted verification using 15 lung cancer cell lines revealed
the NSCLC-specific expression of fibronectin. The glycosylation profiling
of fibronectin indicated that the peanut agglutinin (PNA) signal appeared
to differentiate two NSCLC types, adenocarcinoma and large cell carcinoma,
whereas the protein expression level was similar between these types.
Our glycoproteomics approach together with the concurrent use of an
antibody and lectin is applicable to the quantitative and qualitative
monitoring of variations in glycosylation of fibronectin specific
to certain types of lung cancer tissue
Novel Glycobiomarker for Ovarian Cancer That Detects Clear Cell Carcinoma
Epithelial
ovarian cancer (EOC) is often asymptomatic and thus
diagnosed at advanced stages with a poor prognosis. False-negative
results for the conventional marker CA125 frequently occur in cases
of clear cell carcinoma (CCC), a type of EOC; therefore, it is necessary
to develop biomarkers with greater sensitivity. We previously reported
a strategy to discover glycobiomarker candidates by combined lectin
microarray and IGOT–LC/MS analysis. We have now optimized this
strategy for discovering EOC biomarkers. Glycopeptides possessing
cancerous glycans were enriched from the ascites fluids and culture
supernatants of cancer cell lines with a fucose-binding lectin, AAL.
IGOT–LC/MS analysis of CCC samples yielded 144 candidate glycoproteins.
We selected WFA by lectin microarray as the optimal lectin to distinguish
EOC from gastric and colon cancer. The candidates were narrowed by
Western
analysis of the WFA-bound fraction of ascites fluids. One of the final
candidates, WFA-reactive ceruloplasmin, produced higher signals in
the ascites fluids of EOC patients, including CCC, in comparison with
the benign samples, while CA125 levels were comparable in the sandwich
ELISA. Thus, our glycoproteomic strategy featuring efficient enrichment
of glycans with disease-related alterations is applicable to various
diseases
Glycoproteomic Discovery of Serological Biomarker Candidates for HCV/HBV Infection-Associated Liver Fibrosis and Hepatocellular Carcinoma
We
previously proposed a high-throughput strategy to discover serological
biomarker candidates of cancer. This strategy focuses on a series
of candidate glycoproteins that are specifically expressed in the
original tissues (cells) of the target cancer and that carry glycan
structures associated with carcinogenesis [Narimatsu, H., et al. <i>FEBS J.</i> <b>2010</b>, <i>277</i>(1), 95–105].
Here, we examined the effectiveness of our strategy in identifying
biomarkers to assess progression of liver fibrosis and for the early
detection of hepatocellular carcinoma (HCC). On the basis of the results
of lectin array analyses in culture media of hepatoma cell lines,
we captured glycopeptides carrying AAL-ligands (fucosylated glycans)
or DSA-ligands (branched glycans) from digests of culture media proteins
and sera from HCC patients with a background of liver cirrhosis (LC).
Glycoproteins were identified by the IGOT-LC–MS method. In
all, 21 candidates were selected from 744 AAL-bound glycoproteins
for further verification according to (i) their abundance in serum,
(ii) their specific expression in liver, and (iii) the availability
of antibodies to the glycoproteins. All selected candidates showed
enhancement of AAL-reactivity in sera of HCC patients compared with
that of healthy volunteers (HV). These results indicate that our glycoproteomic
strategy is effective for identifying multiple glyco-biomarker candidates
in a high-throughput manner
Lectin Microarray-Based Sero-Biomarker Verification Targeting Aberrant <i>O</i>‑Linked Glycosylation on Mucin 1
Glycoform
of mucin 1 (MUC1) in cancerous cells changes markedly
with cell differentiation, and thus, qualitative detection and verification
of the MUC1 glycosylation changes have potential diagnostic value.
We have developed an ultrasensitive method to detect the changes in
cholangiocarcinoma (CC), which produces MUC1, and applied it in the
diagnostics development. The focused glycan analysis using 43-lectin-immobilized
microarray could obtain the glycan profiles of sialylated MUC1 in
5 ÎĽL of sera. The high-throughput analysis detected disease-specific
alterations of glycosylation, and the statistical analysis confirmed
that use of Wisteria floribunda agglutinin
(WFA) alone produced a diagnostic score sufficient for discriminating
33 CC cases from 40 hepatolithiasis patients and 48 normal controls
(<i>p</i> < 0.0001). The CC-related glycosylation change
was verified by the lectin–antibody sandwich ELISA with WFA
in two cohorts: (1) 78 Opisthorchis viverrini infected patients without CC and 78 with CC, (2) 33 CC patients
and 40 hepatolithiasis patients (the same cohort used for the above
lectin microarray). The WFA positivity distinguished patients with
CC (opisthorchiasis: <i>p</i> < 0.0001, odds ratio =
1.047; hepatolithiasis: <i>p</i> = 0.0002, odds ratio =
1.018). Sensitive detection of qualitative alterations of sialylated
MUC1 glycosylation is indispensable for the development of our glycodiagnostic
test for CC