Gut immunoglobulin alpha anti-glycan binding profiles as a research tool for local disease detection.

A promising approach capitalizing on the specific and highly sensitive characteristics of the body's own immune system is demonstrated in the context of revealing pancreatic ductal adenocarcinoma cancer (PDAC). IgA from a local biofluid called gastrointestinal lavage fluid (GLF) is used to investigate glycan reactivity to show the potential of this approach. IgA antibody responses, just as with IgG, result in amplification of a small signal which aids in detecting changes from a healthy state. IgA from GLF was screened against glycan arrays containing 609 glycan structures to investigate differential binding patterns associated with the disease. Samples included PDAC (n = 14) and non-PDAC (n = 6). Non-PDAC conditions included samples from healthy patients and the potentially confounding conditions of colon cancer and its precancerous lesion, colon adenoma. Results demonstrated characteristic reactivity in the PDAC sample group to a glycan structure. Also, IgA non-reactive motifs arose showing remarkable consistency within and between sample groups. While sample sizes are too small to identify putative biomarkers, these data show the use of IgA from GLF to be a promising avenue of research for local disease biomarker discovery.

Rectal effluent as a research tool.

Studies of localized secretions are generally superior to those of blood because they contain higher concentrations of molecules specific to the organ of interest. A common method used to analyze localized secretions is lavage. The flow of fluid over the lining of a cavity picks up both cells and soluble factors, and the effluent can be collected for study. Gastrointestinal (GI) lavage is easily and noninvasively performed by the administration of gut lavage solutions such as those routinely given to patients prior to colonoscopy, with GI lavage fluid being the copious, watery rectal effluent subsequently induced. Residual effluent is currently suctioned from the colon and discarded during colonoscopy. With millions of routine colonoscopies performed per year, GI lavage fluid is a rich and largely untapped resource for basic and clinical research. Rectal effluent can also be easily collected in a toilet receptacle without need for a colonoscopy. Rectal effluent generated in this manner has been used to study diarrheal disease, mucosal immunology, inflammatory bowel disease, celiac disease, and cancer. It is often referred to as gut lavage, colon lavage, GI lavage, or whole gut lavage fluid, which makes it challenging to locate previous studies in the literature and there are currently no comprehensive reviews of its use as a research tool. This review attempts to fill this void by discussing previous applications of rectal effluent in research and the methods that have been developed for its collection, stabilization, and analysis.

Multi-profile Bayesian alignment model for LC-MS data analysis with integration of internal standards.

MOTIVATION: Liquid chromatography-mass spectrometry (LC-MS) has been widely used for profiling expression levels of biomolecules in various '-omic' studies including proteomics, metabolomics and glycomics. Appropriate LC-MS data preprocessing steps are needed to detect true differences between biological groups. Retention time (RT) alignment, which is required to ensure that ion intensity measurements among multiple LC-MS runs are comparable, is one of the most important yet challenging preprocessing steps. Current alignment approaches estimate RT variability using either single chromatograms or detected peaks, but do not simultaneously take into account the complementary information embedded in the entire LC-MS data. RESULTS: We propose a Bayesian alignment model for LC-MS data analysis. The alignment model provides estimates of the RT variability along with uncertainty measures. The model enables integration of multiple sources of information including internal standards and clustered chromatograms in a mathematically rigorous framework. We apply the model to LC-MS metabolomic, proteomic and glycomic data. The performance of the model is evaluated based on ground-truth data, by measuring correlation of variation, RT difference across runs and peak-matching performance. We demonstrate that Bayesian alignment model improves significantly the RT alignment performance through appropriate integration of relevant information. AVAILABILITY AND IMPLEMENTATION: MATLAB code, raw and preprocessed LC-MS data are available at http://omics.georgetown.edu/alignLCMS.html. CONTACT: hwr@georgetown.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Differential proteomic analysis of Rickettsia prowazekii propagated in diverse host backgrounds.

The obligate intracellular growth of Rickettsia prowazekii places severe restrictions on the analysis of rickettsial gene expression. With a small genome, predicted to code for 835 proteins, identifying which proteins are differentially expressed in rickettsiae that are isolated from different hosts or that vary in virulence is critical to an understanding of rickettsial pathogenicity. We employed a liquid chromatography (LC)-linear trap quadrupole (LTQ)-Orbitrap mass spectrometer for simultaneous acquisition of quantitative mass spectrometry (MS)-only data and tandem mass spectrometry (MS-MS) sequence data. With the use of a combination of commercially available algorithms and in-house software, quantitative MS-only data and comprehensive peptide coverage generated from MS-MS were integrated, resulting in the assignment of peptide identities with intensity values, allowing for the differential comparison of complex protein samples. With the use of these protocols, it was possible to directly compare protein abundance and analyze changes in the total proteome profile of R. prowazekii grown in different host backgrounds. Total protein extracted from rickettsiae grown in murine, tick, and insect cell lines or hen egg yolk sacs was analyzed. Here, we report the fold changes, including an upregulation of shock-related proteins, in rickettsiae cultivated in tissue culture compared to the level for rickettsiae harvested from hen yolk sacs. The ability to directly compare, in a complex sample, differential rickettsial protein expression provides a snapshot of host-specific proteomic profiles that will help to identify proteins important in intracellular growth and virulence.

Isolation, structural elucidation, and absolute stereochemistry of enigmazole A, a cytotoxic phosphomacrolide from the Papua New Guinea marine sponge Cinachyrella enigmatica.

Enigmazole A (1), a novel phosphate-containing macrolide, was isolated from a Papua New Guinea collection of the marine sponge Cinachyrella enigmatica. The structure of 1, including the absolute stereochemistry at all eight chiral centers, was determined by a combination of spectroscopic analyses and a series of microscale chemical derivatization studies. Compound 1 is comprised of an 18-membered phosphomacrolide that contains an embedded exomethylene-substituted tetrahydropyran ring and an acyclic portion that spans an embedded oxazole moiety. Two additional analogues, 15-O-methylenigmazole A and 13-hydroxy-15-O-methylenigmazole A, were also isolated and assigned. The enigmazoles are the first phosphomacrolides from a marine source and 1 exhibited significant cytotoxicity in the NCI 60-cell line antitumor screen, with a mean GI(50) of 1.7 microM.

Preparation of nasal secretions for proteome analysis.

The determination of protein patterns in nasal secretions of healthy subjects can help in the early diagnosis of diseases such as acute sinusitis. The comparison of nasal lavage fluid collected from subjects with acute sinusitis before and after pharmacological treatment gives information about the drug effects on glandular secretions. Nasal secretions were stimulated with 1x NS (0.9% Normal Saline) and 24x NS in healthy subjects and in sinusitis subjects before and after pharmacological treatment. The nasal lavage fluid (NLF) proteins are precipitated with a solution of "acid-ethanol." Using this solution, the high molecular weight proteins precipitate and separate from the low molecular weight proteins. The proteins are digested and the peptides are separated using a capillary liquid chromatographic system. Eluted peptides are analyzed on ESI-Q-TOF mass spectrometry instrument.

Comparative Proteomic Analysis of Whole-Gut Lavage Fluid and Pancreatic Juice Reveals a Less Invasive Method of Sampling Pancreatic Secretions.

OBJECTIVES: There are currently no reliable, non-invasive screening tests for pancreatic ductal adenocarcinoma. The fluid secreted from the pancreatic ductal system ("pancreatic juice") has been well-studied as a potential source of cancer biomarkers. However, it is invasive to collect. We recently observed that the proteomic profile of intestinal effluent from the bowel in response to administration of an oral bowel preparation solution (also known as whole-gut lavage fluid, WGLF) contains large amounts of pancreas-derived proteins. We therefore hypothesized that the proteomic profile is similar to that of pancreatic juice. In this study, we compared the proteomic profiles of 77 patients undergoing routine colonoscopy with the profiles of 19 samples of pure pancreatic juice collected during surgery. METHODS: WGLF was collected from patients undergoing routine colonoscopy, and pancreatic juice was collected from patients undergoing pancreatic surgery. Protein was isolated from both samples using an optimized method and analyzed by LC-MS/MS. Identified proteins were compared between samples and groups to determine similarity of the two fluids. We then compared our results with literature reports of pancreatic juice-based studies to determine similarity. RESULTS: We found 104 proteins in our pancreatic juice samples, of which 90% were also found in our WGLF samples. The majority (67%) of the total proteins found in the WGLF were common to pancreatic juice, with intestine-specific proteins making up a smaller proportion. CONCLUSIONS: WGLF and pancreatic juice appear to have similar proteomic profiles. This supports the notion that WGLF is a non-invasive, surrogate bio-fluid for pancreatic juice. Further studies are required to further elucidate its role in the diagnosis of pancreatic cancer.

Dissecting the Rickettsia prowazekii genome: genetic and proteomic approaches.

The obligate nature of Rickettsia prowazekii intracellular growth places severe restrictions on the analysis of rickettsial gene function and gene expression. Fortunately, this situation is improving as methods for the genetic manipulation and proteomic analysis of this fascinating human pathogen become available. In this paper, we review the current status of rickettsial genetics and the isolation of rickettsial mutants using a genetic approach. In addition, the examination of rickettsial gene expression through characterization of the rickettsial proteome will be described. This will include a description of a high-throughput, accurate mass approach that has identified 596 rickettsial proteins in a complex rickettsial protein sample.

Human neuroglobin protein in cerebrospinal fluid.

BACKGROUND: Neuroglobin is a hexacoordinated member of the globin family of proteins. It is predominantly localized to various brain regions and retina where it may play a role in protection against ischemia and nitric oxide-induced neural injury. Cerebrospinal fluid was collected from 12 chronic regional or systemic pain and 5 control subjects. Proteins were precipitated by addition of 50% 0.2 N acetic acid, 50% ethanol, 0.02% sodium bisulfite. The pellet was extensively digested with trypsin. Peptides were separated by capillary liquid chromatography using a gradient from 95% water to 95% acetonitrile in 0.2% formic acid, and eluted through a nanoelectrospray ionization interface into a quadrapole - time-of-flight dual mass spectrometer (QToF2, Waters, Milford, MA). Peptides were sequenced (PepSeq, MassLynx v3.5) and proteins identified using MASCOT (R). RESULTS: Six different neuroglobin peptides were identified in various combinations in 3 of 9 female pain subjects, but none in male pain, or female or male control subjects. CONCLUSION: This is the first description of neuroglobin in cerebrospinal fluid. The mechanism(s) leading to its release in chronic pain states remain to be defined.

A Chronic Fatigue Syndrome - related proteome in human cerebrospinal fluid.

BACKGROUND: Chronic Fatigue Syndrome (CFS), Persian Gulf War Illness (PGI), and fibromyalgia are overlapping symptom complexes without objective markers or known pathophysiology. Neurological dysfunction is common. We assessed cerebrospinal fluid to find proteins that were differentially expressed in this CFS-spectrum of illnesses compared to control subjects. METHODS: Cerebrospinal fluid specimens from 10 CFS, 10 PGI, and 10 control subjects (50 mul/subject) were pooled into one sample per group (cohort 1). Cohort 2 of 12 control and 9 CFS subjects had their fluids (200 mul/subject) assessed individually. After trypsin digestion, peptides were analyzed by capillary chromatography, quadrupole-time-of-flight mass spectrometry, peptide sequencing, bioinformatic protein identification, and statistical analysis. RESULTS: Pooled CFS and PGI samples shared 20 proteins that were not detectable in the pooled control sample (cohort 1 CFS-related proteome). Multilogistic regression analysis (GLM) of cohort 2 detected 10 proteins that were shared by CFS individuals and the cohort 1 CFS-related proteome, but were not detected in control samples. Detection of >or=1 of a select set of 5 CFS-related proteins predicted CFS status with 80% concordance (logistic model). The proteins were alpha-1-macroglobulin, amyloid precursor-like protein 1, keratin 16, orosomucoid 2 and pigment epithelium-derived factor. Overall, 62 of 115 proteins were newly described. CONCLUSION: This pilot study detected an identical set of central nervous system, innate immune and amyloidogenic proteins in cerebrospinal fluids from two independent cohorts of subjects with overlapping CFS, PGI and fibromyalgia. Although syndrome names and definitions were different, the proteome and presumed pathological mechanism(s) may be shared.

Protein networks in induced sputum from smokers and COPD patients.

RATIONALE: Subtypes of cigarette smoke-induced disease affect different lung structures and may have distinct pathophysiological mechanisms. OBJECTIVE: To determine if proteomic classification of the cellular and vascular origins of sputum proteins can characterize these mechanisms and phenotypes. SUBJECTS AND METHODS: Individual sputum specimens from lifelong nonsmokers (n=7) and smokers with normal lung function (n=13), mucous hypersecretion with normal lung function (n=11), obstructed airflow without emphysema (n=15), and obstruction plus emphysema (n=10) were assessed with mass spectrometry. Data reduction, logarithmic transformation of spectral counts, and Cytoscape network-interaction analysis were performed. The original 203 proteins were reduced to the most informative 50. Sources were secretory dimeric IgA, submucosal gland serous and mucous cells, goblet and other epithelial cells, and vascular permeability. RESULTS: Epithelial proteins discriminated nonsmokers from smokers. Mucin 5AC was elevated in healthy smokers and chronic bronchitis, suggesting a continuum with the severity of hypersecretion determined by mechanisms of goblet-cell hyperplasia. Obstructed airflow was correlated with glandular proteins and lower levels of Ig joining chain compared to other groups. Emphysema subjects' sputum was unique, with high plasma proteins and components of neutrophil extracellular traps, such as histones and defensins. In contrast, defensins were correlated with epithelial proteins in all other groups. Protein-network interactions were unique to each group. CONCLUSION: The proteomes were interpreted as complex "biosignatures" that suggest distinct pathophysiological mechanisms for mucin 5AC hypersecretion, airflow obstruction, and inflammatory emphysema phenotypes. Proteomic phenotyping may improve genotyping studies by selecting more homogeneous study groups. Each phenotype may require its own mechanistically based diagnostic, risk-assessment, drug- and other treatment algorithms.

Application of high-field NMR and cryogenic probe technologies in the structural elucidation of poecillastrin a, a new antitumor macrolide lactam from the sponge poecillastra species.

Poecillastrin A (1), a new polyketide-derived macrolide lactam, was isolated from a deep-water collection of the marine sponge Poecillastra species. The structure of poecillastrin A (1) was assigned using NMR data acquired at 500 MHz with an inverse-detection cryogenic probe and at 800 MHz with a room-temperature probe.

Chirality determination of unusual amino acids using precolumn derivatization and liquid chromatography-electrospray ionization mass spectrometry.

Unusual amino acids such as beta-methoxytyrosine (beta-MeOTyr), allo-threonine (allo-Thr) and allo-isoleucine (allo-Ile) were derivatized with N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA), 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester (S-NIFE), or o-phthalaldehyde/isobutyryl-L-cysteine (OPA-IBLC), and then separated via reversed-phase high-performance chromatography followed by UV and electrospray ionization mass spectrometry detection. FDAA generally showed the highest enantioselectivity but the lowest sensitivity among the chiral derivatizing agents (CDAs) investigated. The detection limit of FDAA-derivatized amino acids was in the low picomolar range. Although the enantioselectivity of FDAA derivatives was generally quite high, its selectivity among beta-MeOTyr isomers was poor. The best separation of beta-MeOTyr stereoisomers was achieved with S-NIFE. Due to the complex relationships between the investigated CDAs, stereochemical analyses using a combination of two or more of the CDAs gave the most reliable results for a given separation problem. In general, the methods described are selective and reliable, and are being applied to the analysis of unusual amino acids as they occur in marine peptides.

Receptor tyrosine kinase ErbB2 translocates into mitochondria and regulates cellular metabolism.

It is well known that ErbB2, a receptor tyrosine kinase, localizes to the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and cellular ATP levels were also decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to the ErbB2-targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 has an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics.

Production, safety and antitumor efficacy of recombinant Oncofetal Antigen/immature laminin receptor protein.

We describe here for the first time an efficient high yield production method for clinical grade recombinant human Oncofetal Antigen/immature laminin receptor protein (OFA/iLRP). We also demonstrate significant antitumor activity for this protein when administered in liposomal delivery form in a murine model of syngeneic fibrosarcoma. OFA/iLRP is a therapeutically very promising universal tumor antigen that is expressed in all mammalian solid tumors tested so far. We have cloned the human OFA/iLRP cDNA in a bacterial expression plasmid which incorporates a 6x HIS-tag. Large scale cultures of the plasmid transformed Escherichia coli were performed and the crude HIS-tagged OFA/iLRP was isolated as inclusion bodies and solubilized in guanidine chloride. The protein was then purified by successive passage through three column chromatography steps of immobilized metal affinity, anion exchange, and gel filtration. The resulting protein was 94% pure and practically devoid of endotoxin and host cell protein. The purified OFA/iLRP was tested in mice for safety and efficacy in tumor rejection with satisfactory results. This protein will be used for loading onto autologous dendritic cells in an FDA approved phase I/II human cancer vaccine trial in OFA/iLRP-positive breast cancer patients.

Expression and functional analysis of the WAP four disulfide core domain 1 gene in human melanoma.

The exact cellular and molecular mechanisms involved in melanoma tumorigenesis remain obscure. Previous gene expression profiling analyses performed upon NHEM and human melanoma samples identified WFDC1 as one of the most frequently down-regulated genes. Here we further showed that NHEM readily express WFDC1 but expression is reduced or completely lost in 80% of the patients-derived melanoma cell lines and tissue samples examined. Furthermore, we show that promoter hypermethylation accounts for the silencing of the WFDC1 gene in 20% of the melanoma cell lines examined. The over-expression of WFDC1 in two metastatic melanoma cell lines, A375 and LOX, resulted in a significant delay of tumor growth in a murine xenograft model, despite a non-significant difference in tumor cell growth in vitro. Gene expression microarray analysis and further expression validation suggests that the Dickkopf-1 (Dkk1) gene is up-regulated in WFDC1 over-expressing cell lines, suggesting that the tumor suppressive function of WFDC1 may be partially a result of up-regulated Dkk1 gene expression, which is known to be a potent inhibitor of the Wnt signaling pathway.

Identification of differentially secreted biomarkers using LC-MS/MS in isogenic cell lines representing a progression of breast cancer.

Proteins secreted (the secretome) from cancer cells are potentially useful as biomarkers of the disease. Using LC-MS/MS, the secreted proteomes from a series of isogenic breast cancer cell lines varying in aggressiveness were analyzed by mass spectrometry: nontumorigenic MCF10A, premalignant/tumorigenic MCF10AT, tumorigenic/locally invasive MCF10 DCIS.com, and tumorigenic/metastatic MCF 10CA cl. D. Proteomes were obtained from conditioned serum-free media, partially fractionated using a small reverse phase C2 column, and digested with trypsin for analysis by LC-MS/MS, using a method previously shown to give highly enriched secreted proteomes (Mbeunkui et al. J. Proteome Res. 2006, 5, 899-906). The search files produced from five analyses (three separate preparations) were combined for database searching (Mascot) which produced a list of over 250 proteins from each cell line. The aim was to discover highly secreted proteins which changed significantly in abundance corresponding with aggressiveness. The most apparent changes were observed for alpha-1-antichymotrypsin and galectin-3-binding protein which were highly secreted proteins from MCF10 DCIS.com and MCF10CA cl. D, yet undetected in the MCF10A and MCF10AT cell lines. Other proteins showing increasing abundance in the more aggressive cell lines included alpha-1-antitrypsin, cathepsin D, and lysyl oxidase. The S100 proteins, often associated with metastasis, showed variable changes in abundance. While the cytosolic proteins were low (e.g., actin and tubulin), there was significant secretion of proteins often associated with the cytoplasm. These proteins were all predicted as products of nonclassical secretion (SecretomeP, Center for Biological Sequence Analysis). The LC-MS/MS results were verified for five selected proteins by western blot analysis, and the relevance of other significant proteins is discussed. Comparisons with two other aggressive breast cancer cell lines are included. The protein with consistent association with aggressiveness in all lines, and in unrelated cancer cells, was the galectin-3-binding protein which has been associated with breast, prostate, and colon cancer earlier, supporting the approach and findings. This analysis of an isogenic series of cell lines suggests the potential usefulness of the secretome for identifying prospective markers for the early detection and aggressiveness/progression of cancer.

Protein expression in sputum of smokers and chronic obstructive pulmonary disease patients: a pilot study by CapLC-ESI-Q-TOF.

The current report describes the use of CapLC-ESI-Q/TOF-MS for investigating the proteome profiles of hypertonic saline-induced sputum samples from 56 smokers. The severity of their lung disease ranged from normal (healthy smokers) to chronic bronchitis, chronic obstructive pulmonary disease (COPD), and COPD with emphysema. This pilot study examined the hypothesis that there were distinct differences in protein expression profiles that were related to the phenotype and cigarette smoking illness severity. A total of 203 unique proteins were identified. These may represent the most highly expressed proteins in induced sputum. Our results provide evidence that different proteins are expressed, as the disease progresses from health to more advanced stages, and support our contention that a proteomic approach would be beneficial in discovering selective molecules linked to specific COPD stages.

Secretory protein enrichment and analysis: an optimized approach applied on cancer cell lines using 2D LC-MS/MS.

Reliable methods for profiling secretory proteins are highly desirable for the identification of biomarkers of disease progression. Secreted proteins are often masked by high amounts of protein supplements in the culture medium. We have developed an efficient method for the enrichment and analysis of the secretome of different cancer cell lines, free of essential contaminants. The method is based on the optimization of cell incubation conditions in protein-free medium. Secreted proteins are concentrated and fractionated using a reversed-phase tC2 Sorbent, followed by peptide mass fingerprinting for protein identification. An average of 88 proteins were identified in each cancer cell line, of which more than 76% are known to be secreted, possess a signal peptide or a transmembrane domain. Given the importance of secreted proteins as a source for early detection and diagnosis of disease, this approach may help to discover novel candidate biomarkers with potential clinical significance.