Search tips
Search criteria

Results 1-6 (6)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
Document Types
1.  Identification of Potential Prognostic Biomarkers in Patients with Untreated, Advanced Pancreatic Cancer from a Phase III Trial (CALGB 80303) 
Cancer  2011;118(2):571-578.
Patients with advanced stage adenocarcinoma of the pancreas have a poor prognosis. The identification of prognostic and/or predictive biomarkers may help stratify patients so that therapy can be individualized.
Serum samples from patients enrolled in the CALGB 80303 Phase III trial, “Randomized Study of Gemcitabine with Versus without Bevacizumab in Patients with Locally Advanced or Metastatic Adenocarcinoma of the Pancreas” were used to discover novel biomarkers. For the discovery phase, 40 sera were selected based on length of survival and type of therapy, and subjected to LC-MS/MS analysis. The top features (proteins) were then further selected for validation by ELISA.
Quantitation by nano-LC-MS/MS resulted in 1452 peptides mapping to 156 proteins across all 40 samples, 92 of which had 2 or more peptides. After curation of the data we selected one putative prognostic protein, alpha-1 antichymotrypsin (AACT), and two putative predictive proteins, histidine-rich glycoprotein (HRG) and complement factor H (CFH) for validation by ELISA. AACT was found to be negatively correlated with overall survival (τ = −0.30 (−0.38, −0.22); p<0.00001). There was no evidence for interaction with bevacizumab and HRG, but there was some evidence for a weak positive correlation of HRG with overall survival (τ = 0.11 (0.03, 0.19); p<0.01). CFH was found to be neither a predictive nor a prognostic factor for overall survival.
AACT may be a useful prognostic marker in patients with advanced stage pancreatic carcinoma, although additional validation studies are needed.
PMCID: PMC3184330  PMID: 21713765
pancreatic neoplasms; prognosis; biological markers; bevacizumab; alpha 1-antichymotrypsin
2.  Discovery of Novel Cyclophilin A Ligands Using an H/D Exchange- and Mass Spectrometry-Based Strategy 
Journal of biomolecular screening  2010;15(9):1051-1062.
Cyclophilin A (CypA) is an overexpressed protein in lung cancer tumors and as a result is a potential therapeutic and diagnostic target. Here we utilize an H/D exchange- and MALDI mass spectrometry-based assay, termed single-point SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange), to screen two chemical libraries, including the 1280-compound LOPAC library and the 9600 compound DIVERSet library, for binding to CypA. This work represents the first application of single-point SUPREX using a pooled ligand approach, which we demonstrate is capable of screening rates as fast as six seconds/ligand. The false positive and false negative rates determined in the current work using a set of control samples were 0% and 9%, respectively. A false positive rate of 20% was found in screening the actual libraries. Eight novel ligands to CypA were discovered including: 2-(α-naphthoyl)ethyltrimethyl-ammonium iodide, (E)-3-(4-t-Butylphenylsulfonyl)-2-propenenitrile, 3-(N-benzyl-N-isopropyl)amino-1-(naphthalen-2-yl)propan-1-one, cis-diammineplatinum (II) chloride, 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione, N-(3-chloro-1,4-dioxo-1,4-dihydro-2-naphthalenyl)-N-cyclohexylacetamide, 1-[2-(3,4-dimethoxyphenyl)ethyl]-1H-pyrrole-2,5-dione, and 4-(2-methoxy-4-nitrophenyl)-1-methyl-10-oxa-4-azatricyclo[,6~]dec-8-ene-3,5-dione. These compounds, which had moderate binding affinities to CypA (i.e., Kd values in the low micromolar range), provide new molecular scaffolds that might be useful in the development of CypA targeted diagnostic imaging or therapeutic agents for lung cancer.
PMCID: PMC3197229  PMID: 20855564
Cyclophilin A; Matrix-Assisted Laser Desorption/Ionization; amide H/D exchange; high-throughput screening
3.  Complement Factor H Autoantibodies are Associated with Early Stage NSCLC 
In order to discover diagnostic biomarkers associated with early stage non-small cell lung cancer (NSCLC), we searched for autoantibodies preferentially present in stage I patients compared to patients with advanced stage disease. Here we describe an autoantibody against complement factor H (CFH) and its association with early stage NSCLC.
Experimental Design
Immunoblots were used to detect autoantibodies in the sera of stage I NSCLC patients. An autoantibody recognizing a 150 kDa protein was discovered and the protein was identified by mass spectrometry. The association of the autoantibody with early stage disease was suggested by the results of immunoblot analysis with sera from 28 stage I patients and 28 stage III/IV patients. This association was confirmed by protein microarray of sera from 125 NSCLC patients of all stages as well as 125 age, gender, and smoking history matched controls.
The immunoreactive protein was identified as CFH. By immunoblot analysis, anti-CFH autoantibody was found in 50% of stage I NSCLC patients and 11% of late stage NSCLC patients (P=0.003). By protein microarray analysis, patients with stage I NSCLC had a significantly higher incidence of anti-CFH antibody than those with late stage NSCLC (P=0.0051). The percentage of sera with a positive level of CFH autoantibody was 30.4% in stage I, 21.1% in stage II, 12.5% in stage III, 7.4% in stage IV and 8.0% in the control group.
These findings suggest that in patients with NSCLC, CFH autoantibody is a molecular marker associated with early stage disease.
PMCID: PMC2891404  PMID: 20515868
Lung Cancer; Metastasis; Complement Factor H; Autoimmunity
4.  An H/D Exchange- and Protease Digestion-Based Screening Assay for Protein-Ligand Binding Detection 
Analytical chemistry  2009;81(16):6860-6867.
A protease digestion strategy was incorporated into single-point SUPREX (stability of unpurified proteins from rates of H/D exchange), which is an H/D exchange- and mass spectrometry-based assay for the detection of protein-ligand binding. Single-point SUPREX is an abbreviated form of SUPREX in which protein-ligand binding interactions are detected by measuring the increase in a protein’s thermodynamic stability upon ligand binding. The new protease digestion protocol provides a noteworthy increase in the efficiency of single-point SUPREX because peptide masses can be determined with greater precision than intact protein masses in the MALDI readout of single-point SUPREX. The protocol was evaluated in test screens on two model protein systems, including cyclophilin A (CypA) and the minor allele variant of human alanine:glyoxylate aminotransferase (AGTmi). The test screening results obtained on both proteins revealed that the peptide readout of the single-point SUPREX-protease digestion protocol was more efficient than the intact protein readout of the original single-point SUPREX protocol at discriminating hits and non-hits. In addition to this improvement in screening efficiency, the protease digestion strategy described here is expected to significantly increase the generality of the single-point SUPREX assay.
PMCID: PMC2858457  PMID: 20337380
5.  Throughput and Efficiency of a Mass Spectrometry-Based Screening Assay for Protein-Ligand Binding Detection 
An H/D exchange- and MALDI mass spectrometry-based screening assay was applied to search for novel ligands that bind to cyclophilin A, a potential therapeutic and diagnostic target in lung cancer. The assay is based on SUPREX (stability of unpurified proteins from rates of H/D exchange), which exploits the H/D exchange properties of amide protons to measure the increase in a protein's thermodynamic stability upon ligand binding in solution. The current study evaluates the throughput and efficiency with which 880 potential ligands from the Prestwick Chemical Library could be screened for binding to cyclophilin A. Screening was performed at a rate of 3 min/ligand using a conventional MALDI mass spectrometer. False positive and false negative rates, based on a set of control data, were as low as 0% and 9%, respectively. Based on the 880-member library screening, a false positive rate of 0% was observed when a 2-tier selection strategy was implemented. Although novel ligands for cyclophilin A were not discovered, cyclosporin A, a known ligand to CypA and a blind control in the library, was identified as a hit. We also describe a new strategy to eliminate some of the complications related to back exchange that can arise in screening applications of SUPREX.
PMCID: PMC2563801  PMID: 18653356
6.  Characterising phase variations in MALDI-TOF data and correcting them by peak alignment 
Cancer Informatics  2007;1(1):32-40.
The use of MALDI-TOF mass spectrometry as a means of analyzing the proteome has been evaluated extensively in recent years. One of the limitations of this technique that has impeded the development of robust data analysis algorithms is the variability in the location of protein ion signals along the x-axis. We studied technical variations of MALDI-TOF measurements in the context of proteomics profiling. By acquiring a benchmark data set with five replicates, we estimated 76% to 85% of the total variance is due to phase variation. We devised a lobster plot, so named because of the resemblance to a lobster claw, to help detect the phase variation in replicates. We also investigated a peak alignment algorithm to remove the phase variation. This operation is analogous to the normalization step in microarray data analysis. Only after this critical step can features of biological interest be clearly revealed. With the help of principal component analysis, we demonstrated that after peak alignment, the differences among replicates are reduced. We compared this approach to peak alignment with a model-based calibration approach in which there was known information about peaks in common among all spectra. Finally, we examined the potential value at each point in an analysis pipeline of having a set of methods available that includes parametric, semiparametric and nonparametric methods; among such methods are those that benefit from the use of prior information.
PMCID: PMC2657651  PMID: 19305630
variation; amplitude; phase; MALDI-TOF; peak alignment

Results 1-6 (6)