Discrimination of delays of reinforcement in aversive conditioning.

<p>(a) The protein level changes of CA125 and LRG1 were confirmed in the larger sample set 2. (b) Marker distributions for the EOC patients with different histological subtypes.</p

Analysis of Glycan Variation on Glycoproteins from Serum by the Reverse Lectin-Based ELISA Assay

Altered glycosylation in glycoproteins is associated with carcinogenesis, and certain glycan structures and glycoproteins are well-known markers for tumor progression. To identify potential diagnostic candidate markers, we have developed a novel method for analysis of glycosylation changes of glycoproteins from crude serum samples using lectin-based glycoprotein capture followed by detection with biotin/HRP-conjugated antibodies. The amount of lectin coated on the microplate well was optimized to achieve low background and improved S/N compared with current lectin ELISA methods. In the presence of competing sugars of lectin AAL or with sialic acid removed from the glycoproteins, we confirmed that this method specifically detects glycosylation changes of proteins rather than protein abundance variation. Using our reverse lectin-based ELISA assay, increased fucosylated haptoglobin was observed in sera of patients with ovarian cancer, while the protein level of haptoglobin remained the same between cancers and noncases. The combination of fucosylated haptoglobin and CA125 (AUC = 0.88) showed improved performance for distinguishing stage-III ovarian cancer from noncases compared with CA125 alone (AUC = 0.86). In differentiating early-stage ovarian cancer from noncases, fucosylated haptoglobin showed comparable performance to CA125. The combination of CA125 and fucosylated haptoglobin resulted in an AUC of 0.855, which outperforms CA125 to distinguish early-stage cancer from noncases. Our study provides an alternative method to quantify glycosylation changes of proteins from serum samples, which will be essential for biomarker discovery and validation studies

Mass-Selected Site-Specific Core-Fucosylation of Serum Proteins in Hepatocellular Carcinoma

A mass spectrometry-based methodology has been developed to screen for changes in site-specific core-fucosylation (CF) of serum proteins in early stage HCC with different etiologies. The methods involve depletion of high-abundance proteins, trypsin digestion of medium-to-low-abundance proteins into peptides, iTRAQ labeling, and Lens culinaris Agglutinin (LCA) enrichment of CF peptides, followed by endoglycosidase F3 digestion before mass spectrometry analysis. 1300 CF peptides from 613 CF proteins were identified from patients sera, where 20 CF peptides were differentially expressed in alcohol (ALC)-related HCC samples compared with ALC-related cirrhosis samples and 26 CF peptides changed in hepatitis C virus (HCV)-related HCC samples compared with HCV-related cirrhosis samples. Among these, we found three CF peptides from fibronectin upregulated in ALC-related HCC samples compared with ALC-related cirrhosis samples with an AUC (area under the curve) value of 0.89 at site 1007 with a specificity of 85.7% at a sensitivity of 92.9% (generated with 10-fold cross-validation). When combined with the AFP index, the AUC value reached to 0.92 with a specificity of 92.9% at a sensitivity of 100%, significantly improved compared to that with AFP alone (LR test <i>p</i> < 0.001). In HCV-related samples, the CF level of cadherin-5 at site 61 showed the best AUC value of 0.75 but was not as promising as that of fibronectin site 1007 for ALC-related samples. The CF peptides of fibronectin may serve as potential biomarkers for early stage HCC screening in ALC-related cirrhosis patients

Quantitative Analysis of Single Amino Acid Variant Peptides Associated with Pancreatic Cancer in Serum by an Isobaric Labeling Quantitative Method

Single amino acid variations are highly associated with many human diseases. The direct detection of peptides containing single amino acid variants (SAAVs) derived from nonsynonymous single nucleotide polymorphisms (SNPs) in serum can provide unique opportunities for SAAV associated biomarker discovery. In the present study, an isobaric labeling quantitative strategy was applied to identify and quantify variant peptides in serum samples of pancreatic cancer patients and other benign controls. The largest number of SAAV peptides to date in serum including 96 unique variant peptides were quantified in this quantitative analysis, of which five variant peptides showed a statistically significant difference between pancreatic cancer and other controls (<i>p</i>-value < 0.05). Significant differences in the variant peptide SDNCEDTPEAGYFA<i><u>V</u></i>AVVK from serotransferrin were detected between pancreatic cancer and controls, which was further validated by selected reaction monitoring (SRM) analysis. The novel biomarker panel obtained by combining α-1-antichymotrypsin (AACT), Thrombospondin-1 (THBS1) and this variant peptide showed an excellent diagnostic performance in discriminating pancreatic cancer from healthy controls (AUC = 0.98) and chronic pancreatitis (AUC = 0.90). These results suggest that large-scale analysis of SAAV peptides in serum may provide a new direction for biomarker discovery research

Analysis of Serum Haptoglobin Fucosylation in Hepatocellular Carcinoma and Liver Cirrhosis of Different Etiologies

We have developed herein a quantitative mass spectrometry-based approach to analyze the etiology-related alterations in fucosylation degree of serum haptoglobin in patients with liver cirrhosis and hepatocellular carcinoma (HCC). The three most common etiologies, including infection with hepatitis B virus (HBV), infection with hepatitis C virus (HCV), and heavy alcohol consumption (ALC), were investigated. Only 10 μL of serum was used in this assay in which haptoglobin was immunoprecipitated using a monoclonal antibody. The <i>N</i>-glycans of haptoglobin were released with PNGase F, desialylated, and permethylated prior to MALDI-QIT-TOF MS analysis. In total, <i>N</i>-glycan profiles derived from 104 individual patient samples were quantified (14 healthy controls, 40 cirrhosis, and 50 HCCs). A unique pattern of bifucosylated tetra-antennary glycan, with both core and antennary fucosylation, was identified in HCC patients. Quantitative analysis indicated that the increased fucosylation degree was highly associated with HBV- and ALC-related HCC patients compared to that of the corresponding cirrhosis patients. Notably, the bifucosylation degree was distinctly increased in HCC patients versus that in cirrhosis of all etiologies. The elevated bifucosylation degree of haptoglobin can discriminate early stage HCC patients from cirrhosis in each etiologic category, which may be used to provide a potential marker for early detection and to predict HCC in patients with cirrhosis

Validation of LRG1 as a Potential Biomarker for Detection of Epithelial Ovarian Cancer by a Blinded Study

<div><p>Background</p><p>Leucine-rich alpha-2-glycoprotein (LRG1) was found to be differentially expressed in sera from patients with Epithelial Ovarian Cancer (EOC). The aim of this study is to investigate the performance of LRG1 for detection of EOC, including early stage EOC, and to evaluate if LRG1 can complement CA125 in order to improve EOC detection using two independent blinded sample sets.</p><p>Methods and Results</p><p>Serum LRG1 and CA125 were measured by immunoassays. All assays were performed blinded to clinical data. Using the two independent sample sets (156 participants for sample set 1, and 233 for sample set 2), LRG1 was differentially expressed in EOC cases as compared to healthy, surgical, and benign controls, and its performance was not affected by the conditions of blood collection. The areas under the ROC curve (AUC) for LRG1 in differentiating EOC cases from non-cases were 0.797 and 0.786 for sample set 1 and 2. For differentiating EOC cases from healthy controls, the AUC values for LRG1 were 0.792 and 0.794. At a fixed specificity of 95%, LRG1 detects 52%, and 53.5% of EOC cases from healthy controls for sample set 1 and 2. When combining LRG1 and CA125, the AUC value increased to 0.927, which was improved compared to CA125 (AUC=0.916) (<i>p</i>=0.008) alone in distinguishing EOC cases from non-cases. More importantly, LRG1 also showed potential performance in differentiating early stage EOC from non-cases with an AUC of 0.715 for sample set 1, and 0.690 for sample set 2. The combination of LRG1 and CA125 resulted in an AUC of 0.838, which outperforms CA125 (AUC=0.785) (<i>p</i>=0.018) in detecting early stage EOC cases from non-cases using the larger sample set.</p><p>Conclusions</p><p>LRG1 could be a useful biomarker alone or in combination with CA125 for the diagnosis of ovarian cancer.</p></div

ROC curves comparing marker concentrations in cases to non-cases for sample set 1.

<p>(a) ROC analyses for CA125 and LRG1 to differentiate EOC from non-cases. (b) ROC analyses for CA125 and LRG1 to differentiate early stage EOC from non-cases.</p

ROC curves comparing marker concentrations in cases to healthy controls for sample set 1.

<p>(a) ROC analyses for CA125 and LRG1 to differentiate EOC from healthy controls. (b) ROC analyses for CA125 and LRG1 to differentiate early stage EOC from healthy controls.</p

Multimarker panel analysis.

<p>The performance of multimarker panels in distinguishing EOC/ early stage EOC from non-cases using sample set 1and sample set 2.</p

Development of an Integrated Pipeline for Profiling Microbial Proteins from Mouse Fecal Samples by LC–MS/MS

Metaproteomics is one approach to analyze the functional capacity of the gut microbiome but is limited by the ability to evenly extract proteins from diverse organisms within the gut. Herein, we have developed a pipeline to optimize sample preparation of stool obtained from germ-free (GF) mice that were gavaged a defined community of 11 bacterial strains isolated from the human gut. With 64% more proteins identified, bead-beating was confirmed to be an indispensable step for the extraction of bacterial proteins, especially for Gram-positive bacteria. Bacterial enrichment from mouse fecal samples was further optimized by evaluating three different methods: (1) a high-speed differential centrifugation (HCE) or (2) a low-speed differential centrifugation (LCE) and (3) a filter-aided method (FA). The HCE method was associated with dramatic loss of bacteria and 71% less recovery of bacterial proteins than the LCE method. Compared with LCE, the FA method also showed dramatic loss of the amount of bacteria recovered and decreased protein identifications from Gram-positive bacteria in the stool samples. Ultimately, LCE may provide an alternative and complementary method for enriching bacteria from small amounts of mouse fecal samples, which could aid in investigating bacterial function in health and disease