David M. Lubman

Multiple Sclerosis An Important Role for Post-Translational Modifications of Myelin Basic Protein in Pathogenesis

Myelin basic protein (MBP) represents a candidate autoantigen in multiple sclerosis (MS). We isolated MBP from normal and MS human white matter and purified six components (charge isomers) to compare the post-translational modifications on each. The sites and extent of methylation, deimination, and phosphorylation were documented for all tryptic peptides by mass spectrometry. We found that mono and dimethylated arginine 107 was increased in MS samples; deimination of arginine occurred at a number of sites and was elevated in MS; phosphorylation was observed in 10 peptides in normal samples but was greatly reduced or absent in most peptides from MS samples. Data obtained with MBP isolated from fresh brain obtained from a spontaneously demyelinating mouse model supported the view that the changes observed in human brain were probably related to pathogenesis of demyelination, i.e. we found decreased phosphorylation and decreased amounts of glycogen synthesis kinase in brain homogenates using specific antibodies. This study represents the first to define post-translational modifications in demyelinating disease and suggest an important role in pathogenesis.

Plasma glycoprotein profiling for colorectal cancer biomarker identification by lectin glycoarray and lectin blot

Colorectal cancer (CRC) remains a major worldwide cause of cancer-related morbidity and mortality largely due to the insidious onset of the disease. The current clinical procedures utilized for disease diagnosis are invasive, unpleasant, and inconvenient; hence, the need for simple blood tests that could be used for the early detection of CRC. In this work, we have developed methods for glycoproteomics analysis to identify plasma markers with utility to assist in the detection of colorectal cancer (CRC). Following immunodepletion of the most abundant plasma proteins, the plasma N -linked glycoproteins were enriched using lectin affinity chromatography and subsequently further separated by nonporous silica reversed-phase (NPS-RP)-HPLC. Individual RP-HPLC fractions were printed on nitrocellulose coated slides which were then probed with lectins to determine glycan patterns in plasma samples from 9 normal, 5 adenoma, and 6 colorectal cancer patients. Statistical tools, including principal component analysis, hierarchical clustering, and Z-statistics analysis, were employed to identify distinctive glycosylation patterns. Patients diagnosed with colorectal cancer or adenomas were shown to have dramatically higher levels of sialylation and fucosylation as compared to normal controls. Plasma glycoproteins with aberrant glycosylation were identified by nano-LC-MS/MS, while a lectin blotting methodology was used to validate proteins with significantly altered glycosylation as a function of cancer progression. The potential markers identified in this study for diagnosis to distinguish colorectal cancer from adenoma and normal include elevated sialylation and fucosylation in complement C3, histidine-rich glycoprotein, and kininogen-1. These potential markers of colorectal cancer were subsequently validated by lectin blotting in an independent set of plasma samples obtained from 10 CRC patients, 10 patients with adenomas, and 10 normal subjects. These results demonstrate the utility of this strategy for the identification of N -linked glycan patterns as potential markers of CRC in human plasma, and may have the utility to distinguish different disease states.

Pancreatic Cancer Serum Detection Using A Lectin/Glyco-Antibody Array Method

Pancreatic cancer is a formidable disease and early detection biomarkers are needed to make inroads into improving the outcomes in these patients. In this work, lectin antibody microarrays were utilized to detect unique glycosylation patterns of proteins from serum. Antibodies to four potential glycoprotein markers that were found in previous studies were printed on nitrocellulose coated glass slides and these microarrays were hybridized against patient serum to extract the target glycoproteins. Lectins were then used to detect different glycan structural units on the captured glycoproteins in a sandwich assay format. The biotinylated lectins used to assess differential glycosylation patterns were Aleuria aurentia lectin (AAL), Sambucus nigra bark lectin (SNA), Maackia amurensis lectin II (MAL), Lens culinaris agglutinin (LCA), and Concanavalin A (ConA). Captured glycoproteins were evaluated on the microarray in situ by on-plate digestion and direct analysis using MALDI QIT-TOF mass spectroscopy. Analysis was performed using serum from 89 normal controls, 35 chronic pancreatitis samples, 37 diabetic samples and 22 pancreatic cancer samples. We found that this method had excellent reproducibility as measured by the signal deviation of control blocks as on-slide standard and 41 pairs of pure technical replicates. It was possible to discriminate cancer from the other disease groups and normal samples with high sensitivity and specificity where the response of Alpha-1-beta glycoprotein to lectin SNA increased by 69% in the cancer sample compared to the other noncancer groups (95% confidence interval 53-86%). These data suggest that differential glycosylation patterns detected on high-throughput lectin glyco-antibody microarrays are a promising biomarker approach for the early detection of pancreatic cancer.

An ion trap storage/time‐of‐flight mass spectrometer

An ion trap/time‐of‐flight (TOF) mass spectrometer combination has been developed in order to combine the storage capabilities of an ion trap with the speed and resolution of a time‐of‐flight device. The ion trap is an rf quadrupole trap which operates in the total storage mode, i.e., with the dc voltage=0 on the end caps and rf voltage on the ring electrode. The trap has an ion storage time of ≥2 s at an rf potential of 310 Vpp. The stored ions are ejected into the time‐of‐flight device using a −150 V dc pulse on the exit end cap which causes the ion trajectories in the trap to become unstable. The ions are mass analyzed using either a linear or reflectron TOF. In the linear mode the resolution is 240 while in the reflectron mode a resolution of 1300 at m/z 93 is achieved. The storage capabilities of this device may have important applications towards enhancing sensitivity, the study of very slow metastable decay, and photodissociation mass spectrometry and spectroscopy.

Two-dimensional liquid separations-mass mapping of proteins from human cancer cell lysates.

A review of two-dimensional (2D) liquid separation methods used in our laboratory to map the protein content of human cancer cells is presented herein. The methods discussed include various means of fractionating proteins according to isoelectric point (pI) in the first dimension. The proteins in each pI fraction are subsequently separated using nonporous (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC). The liquid eluent of the RP-HPLC separation is directed on-line into an electrospray ionization time-of-flight (ESI-TOF) mass spectrometer where an accurate value of the protein intact M(r) can be obtained. The result is a 2D map of pI versus M(r) analogous to 2D gel electrophoresis; however the highly accurate and reproducible M(r) serves as the basis for interlysate comparisons. In addition, the use of liquid separations allows for the collection of hundreds of purified proteins in the liquid phase for further analysis via peptide mass mapping using matrix assisted laser desorption ionization TOF MS. A description of the methodology used and its applications to analysis of several types of human cancer cell lines is described. The potential of the method for differential proteomic analysis for the identification of biomarkers of disease is discussed.

Urinary Glycoprotein Biomarker Discovery for Bladder Cancer Detection Using LC/MS-MS and Label-Free Quantification

Background: Cancers of the urinary bladder are the fifth most commonly diagnosed malignancy in the United States. Early clinical diagnosis of bladder cancer remains a major challenge, and the development of noninvasive methods for detection and surveillance is desirable for both patients and health care providers. Approach: To identify urinary proteins with potential clinical utility, we enriched and profiled the glycoprotein component of urine samples by using a dual-lectin affinity chromatography and liquid chromatography/tandem mass spectrometry platform. Results: From a primary sample set obtained from 54 cancer patients and 46 controls, a total of 265 distinct glycoproteins were identified with high confidence, and changes in glycoprotein abundance between groups were quantified by a label-free spectral counting method. Validation of candidate biomarker alpha-1-antitrypsin (A1AT) for disease association was done on an independent set of 70 samples (35 cancer cases) by using an ELISA. Increased levels of urinary A1AT glycoprotein were indicative of the presence of bladder cancer (P < 0.0001) and augmented voided urine cytology results. A1AT detection classified bladder cancer patients with a sensitivity of 74% and specificity of 80%. Summary: The described strategy can enable higher resolution profiling of the proteome in biological fluids by reducing complexity. Application of glycoprotein enrichment provided novel candidates for further investigation as biomarkers for the noninvasive detection of bladder cancer. Clin Cancer Res; 17(10); 3349–59. ©2011 AACR.

CD90 is Identified as a Candidate Marker for Cancer Stem Cells in Primary High-Grade Gliomas Using Tissue Microarrays

Although CD90 has been identified as a marker for various kinds of stem cells including liver cancer stem cells (CSCs) that are responsible for tumorigenesis, the potential role of CD90 as a marker for CSCs in gliomas has not been characterized. To address the issue, we investigated the expression of CD90 in tissue microarrays containing 15 glioblastoma multiformes (GBMs), 19 WHO grade III astrocytomas, 13 WHO grade II astrocytomas, 3 WHO grade I astrocytomas and 8 normal brain tissues. Immunohistochemical analysis showed that CD90 was expressed at a medium to high level in all tested high-grade gliomas (grade III and GBM) whereas it was barely detectable in low-grade gliomas (grade I and grade II) and normal brains. Double immunofluorescence staining for CD90 and CD133 in GBM tissues revealed that CD133+ CSCs are a subpopulation of CD90+ cells in GBMs in vivo. Flow cytometry analysis of the expression of CD90 and CD133 in GBM-derived stem-like neurospheres further confirmed the conclusion in vitro. The expression levels of both CD90 and CD133 were reduced along with the loss of stem cells after differentiation. Furthermore, the limiting dilution assay demonstrated that the sphere formation ability was comparable between the CD90+/CD133+ and the CD90+/CD133− populations of GBM neurospheres, which is much higher than that of the CD90−/CD133− population. We also performed double staining for CD90 and a vascular endothelial cell marker CD31 in tissue microarrays which revealed that the CD90+ cells were clustered around the tumor vasculatures in high-grade glioma tissues. These findings suggest that CD90 is not only a potential prognostic marker for high-grade gliomas but also a marker for CSCs within gliomas, and it resides within endothelial niche and may also play a critical role in the generation of tumor vasculatures via differentiation into endothelial cells.

Detection of Explosives Using Laser Desorption in Ion Mobility Spectrometry/Mass Spectrometry

Laser desorption is used to volatilize explosive materials for analysis by ion mobility/mass spectrometry measurements. The laser desorption is performed at relatively low power (< 107 W/cm2) so that mainly neutrals are produced, which are subsequently ionized by 63Ni-β-source induced ion-molecule reactions at atmospheric pressure. Atmospheric pressure ionization, together with the soft vaporization method, produces relatively simple spectra of the explosives investigated, yielding ions that are very characteristic of each molecule for identification. The technique has great potential for high sensitivity based upon the combination of complete laser vaporization of the sample and the API method, which is an extremely efficient form of ionization for these molecules with high electron affinities.

Glycoprotein biomarker panel for pancreatic cancer discovered by quantitative proteomics analysis.

Pancreatic cancer is a lethal disease where specific early detection biomarkers would be very valuable to improve outcomes in patients. Many previous studies have compared biosamples from pancreatic cancer patients with healthy controls to find potential biomarkers. However, a range of related disease conditions can influence the performance of these putative biomarkers, including pancreatitis and diabetes. In this study, quantitative proteomics methods were applied to discover potential serum glycoprotein biomarkers that distinguish pancreatic cancer from other pancreas related conditions (diabetes, cyst, chronic pancreatitis, obstructive jaundice) and healthy controls. Aleuria aurantia lectin (AAL) was used to extract fucosylated glycoproteins and then both TMT protein-level labeling and label-free quantitative analysis were performed to analyze glycoprotein differences from 179 serum samples across the six different conditions. A total of 243 and 354 serum proteins were identified and quantified by label-free and TMT protein-level quantitative strategies, respectively. Nineteen and 25 proteins were found to show significant differences in samples between the pancreatic cancer and other conditions using the label-free and TMT strategies, respectively, with 7 proteins considered significant in both methods. Significantly different glycoproteins were further validated by lectin-ELISA and ELISA assays. Four candidates were identified as potential markers with profiles found to be highly complementary with CA 19–9 (p < 0.001). Obstructive jaundice (OJ) was found to have a significant impact on the performance of every marker protein, including CA 19–9. The combination of α-1-antichymotrypsin (AACT), thrombospondin-1 (THBS1), and haptoglobin (HPT) outperformed CA 19–9 in distinguishing pancreatic cancer from normal controls (AUC = 0.95), diabetes (AUC = 0.89), cyst (AUC = 0.82), and chronic pancreatitis (AUC = 0.90). A marker panel of AACT, THBS1, HPT, and CA 19–9 showed a high diagnostic potential in distinguishing pancreatic cancer from other conditions with OJ (AUC = 0.92) or without OJ (AUC = 0.95).

Analytical Jet Spectroscopy of Tyrosine and its Analogs Using a Pulsed Laser Desorption Volatilization Method

Pulsed laser desorption is used as a means of volatilizing nonvolatile and thermally labile molecules for entrainment into a supersonic jet expansion. The jet expansion provides ultracold molecules whose sharp spectral features are probed by resonant two-photon ionization spectroscopy in a time-of-flight mass spectrometer. Such jet-cooled spectra are demonstrated for tyrosine and related structural analogs. Despite the similarity between these tyrosine-based compounds, electronic spectroscopy is shown to be a sensitive probe of small structural changes in these related biological compounds.