Biotin labeling in combination with mass spectrometry has been widely applied in large-scale biological studies, such as determination of protein partners, protein subcellular localization, and protein post-translational modifications. Previous studies have shown that immunoaffinity enrichment is a better method than streptavidin/avidin purification for site-specific studies of biotinylated molecules. In this study, we made a crucial improvement to the elution phase of the immunoaffinity enrichment step for biotinylated peptides, which involves the addition of a highly organic solvent, and developed a monoclonal anti-biotin antibody that improved the identification number for biotinylated peptides. We then demonstrated its application in the characterization of protein interaction sites for the β2 adrenergic receptor (β2AR) by proximity labeling in living cells. Our research provides an improved and reproducible immunoaffinity enrichment method for site-specific biotin-related research.

The aim of this study was to clarify the epigenetic regulation of ten eleven translocation protein (TET) family genes, which can provide insights into the mechanisms of tumorigenesis and the risk of disease recurrence in head and neck squamous cell carcinoma (HNSCC). We generated methylation profiles of TET1, TET2 and TET3 genes in tumor samples obtained from 233 patients with HNSCC; these included 57 hypopharynx, 44 larynx, 69 oral cavity, and 63 oropharynx tumor samples. The mRNA expression and promoter DNA methylation of TET family genes were examined via quantitative RT-PCR and methylation-specific PCR, respectively. Promoter methylation was compared with various clinical characteristics and the TET methylation index (TE-MI). The TE-MI, representing the number of methylation events in TET family genes, was positively correlated with alcohol consumption (P = 0.004), high-risk human papilloma virus (HPV) status (P = 0.004) and disease recurrence (P = 0.002). The simultaneous methylation analysis of TET family genes was correlated with reduced disease-free survival in unfavorable event groups (log-rank test, P = 0.026). In the multivariate Cox proportional hazards analysis, TET3 methylation in T1 and T2 tumor stages, oropharyngeal cancer, and oral cancer patients exhibited high association with poor survival (hazard ratio: 2.64, P = 0.014; 3.55, P = 0.048; 2.63, P = 0.028, respectively). A joint analysis of the tumor suppressor gene methylation index showed a significant trend toward a higher TE-MI. The methylation status of TET3 was independently associated with aggressive tumor behavior and a global effect on DNA methylation status in HNSCC.

Staging and pathological grading systems are convenient but imperfect predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). Identifying biomarkers for HNSCC that will progress and cause death is a critical research area, particularly if the biomarker can be linked to selection of patients. Therefore, to identify potential alternative prognostic markers, we investigated the methylation status of five neuropeptide gene promoters. The promoter methylation status was determined by quantitative methylation-specific PCR in 230 cases of HNSCC; 58 hypopharynx, 45 larynx, 56 oropharynx, and 71 oral cavity tumor samples were studied.
The somatostatin (SST), tachykinin precursor 1 (TAC1), hypocretin neuropeptide precursor (HCRT), neuropeptide Y (NPY), and galanin (GAL) promoters were methylated in 84.3, 63.5, 32.6, 28.3, and 20.0%, respectively, of the samples. The mean number of methylated genes per sample was 2.29 (range, 0-5). Disease-free survival was lower in patients with 3-5 methylated genes than in those with 0-2 methylated genes (log-rank test, P = 0.007). In multivariate Cox proportional hazards analysis, TAC1 and GAL promoter methylation independently predicted recurrence (odds ratios 1.620, 95% confidence interval [CI] 1.018-2.578, P = 0.042, and odds ratios 1.692, 95% CI 1.063-2.694, P = 0.027, respectively). In patients with oral cancer, TAC1 methylation showed the best correlation with poor survival (odds ratio 4.427, 95% CI 1.634-12.00, P = 0.003). Similar findings were observed for HCRT and GAL in patients with laryngeal cancer and oropharyngeal cancer, respectively.
In this study, we demonstrated the methylation status of the neuropeptide-encoding genes SST, TAC1, HCRT, NPY, and GAL and its relationship with recurrence and survival in HNSCC. These methylation changes may serve as potential molecular markers for defining the risk and prognosis of HNSCC.

Clarifying the epigenetic regulation of tumor-related genes (TRGs) can provide insights into the mechanisms of tumorigenesis and the risk for disease recurrence in HPV-negative head and neck cancers, originating in the hypopharynx, larynx, and oral cavity. We analyzed the methylation status of the promoters of 30 TRGs in 178 HPV-negative head and neck cancer patients using a quantitative methylation-specific PCR. Promoter methylation was correlated with various clinical characteristics and patient survival. The mean number of methylated TRGs was 14.2 (range, 2-25). In the multivariate Cox proportional hazards analysis, the methylation of COL1A2 and VEGFR1 was associated with poor survival for hypopharyngeal cancer, with hazard ratios: 3.19; p = 0.009 and 3.07; p = 0.014, respectively. The methylation of p16 and COL1A2 were independent prognostic factors for poor survival in laryngeal cancer (hazard ratio: 4.55; p = 0.013 and 3.12; p = 0.035, respectively). In patients with oral cancer, the methylation of TAC1 and SSTR1 best correlated with poor survival (hazard ratio: 4.29; p = 0.005 and 5.38; p = 0.029, respectively). Our findings suggest that methylation status of TRGs could serve as important site-specific biomarkers for prediction of clinical outcomes in patients with HPV-negative head and neck cancer.

Alterations of site-specific gene expression profiles in disease-relevant networks within the different layers of the intestinal wall may contribute to the onset and clinical course of gastrointestinal disorders. To date, no systematic analysis has assessed and compared sub-regional gene expression patterns in all distinct layers of the gut using fresh frozen human samples. Our aim was to establish an optimized protocol for site-specific RNA isolation in order to achieve maximum RNA quality and amount for subsequent gene expression analysis combining laser-capture microdissection (LCM) with a probe-based technology, the NanoString nCounter Analysis system.
Four full-thickness colon samples from patients who underwent surgery due to pathological conditions were processed and separated into epithelium, lamina propria, myenteric plexus, submucosa, and tunica muscularis by LCM. Site-specific marker expression by nCounter technology was performed on total RNA from each sub-region, respectively.
Collecting ~10 mm² (~100 000-250 000 cells) of tissue from the epithelial layer, lamina propria, and myenteric plexus provided sufficient amounts of RNA of appropriate quality for subsequent analyses. In contrast, ~40 mm² (~250 000-650 000 cells) of tissue were dissected from the less cell-rich submucosal and tunica muscularis layer. nCounter analysis revealed a site-specific expression pattern of marker genes in the different layers of the colonic wall which were highly correlating (r > .9).
LCM in combination with nCounter expression analysis enables site-specific, sensitive, reliable detection, and quantification of mRNA from histologically heterogeneous tissues.

Fucosylation is an important type of glycosylation involved in cancer, and fucosylated proteins could be employed as cancer biomarkers. Previously, we reported that fucosylated N-glycans on haptoglobin in the sera of patients with pancreatic cancer were increased by lectin-ELISA and mass spectrometry analyses. However, an increase in fucosylated haptoglobin has been reported in various types of cancer. To ascertain if characteristic fucosylation is observed in each cancer type, we undertook site-specific analyses of N-glycans on haptoglobin in the sera of patients with five types of operable gastroenterological cancer (esophageal, gastric, colon, gallbladder, pancreatic), a non-gastroenterological cancer (prostate cancer) and normal controls using ODS column LC-ESI MS. Haptoglobin has four potential glycosylation sites (Asn184, Asn207, Asn211, Asn241). In all cancer samples, monofucosylated N-glycans were significantly increased at all glycosylation sites. Moreover, difucosylated N-glycans were detected at Asn 184, Asn207 and Asn241 only in cancer samples. Remarkable differences in N-glycan structure among cancer types were not observed. We next analyzed N-glycan alditols released from haptoglobin using graphitized carbon column LC-ESI MS to identify the linkage of fucosylation. Lewis-type and core-type fucosylated N-glycans were increased in gastroenterological cancer samples, but only core-type fucosylated N-glycan was relatively increased in prostate cancer samples. In metastatic prostate cancer, Lewis-type fucosylated N-glycan was also increased. These data suggest that the original tissue/cell producing fucosylated haptoglobin is different in each cancer type and linkage of fucosylation might be a clue of primary lesion, thereby enabling a differential diagnosis between gastroenterological cancers and non-gastroenterological cancers.

Cancer-related alterations in protein glycosylation may serve as diagnostic or prognostic biomarkers or may be used for monitoring disease progression. Clusterin is a medium abundance, yet heavily glycosylated, glycoprotein that is upregulated in clear cell renal cell carcinoma (ccRCC) tumors. We recently reported that the N-glycan profile of clusterin is altered in the plasma of ccRCC patients. Here, we characterized the occupancy and the degree of heterogeneity of individual N-glycosylation sites of clusterin in the plasma of patients diagnosed with localized ccRCC, before and after curative nephrectomy (n = 40). To this end, we used tandem mass spectrometry of immunoaffinity-enriched plasma samples to analyze the individual glycosylation sites in clusterin. We determined the levels of targeted clusterin glycoforms containing either a biantennary digalactosylated disialylated (A2G2S2) glycan or a core fucosylated biantennary digalactosylated disialylated (FA2G2S2) glycan at N-glycosite N374. We showed that the presence of these two clusterin glycoforms differed significantly in the plasma of patients prior to and after curative nephrectomy for localized ccRCC. Removal of ccRCC led to a significant increase in the levels of both FA2G2S2 and A2G2S2 glycans in plasma clusterin. These changes were further confirmed by lectin blotting of plasma clusterin. It is envisioned that these identified glycan alterations may provide an additional level of therapeutic or biomarker sensitivity than levels currently achievable by monitoring expression differences alone.