Rapid clearance of Schistosoma mansoni circulating cathodic antigen after treatment shown by urine strip tests in a Ugandan fishing community - Relevance for monitoring treatment efficacy and re-infection.

Schistosomiasis control and elimination has priority in public health agendas in several sub-Saharan countries. However, achieving these goals remains a substantial challenge. In order to assess progress of interventions and treatment efficacy it is pertinent to have accurate, feasible and affordable diagnostic tools. Detection of Schistosoma mansoni infection by circulating cathodic antigen (CCA) in urine is an attractive option as this measure describes live worm infection noninvasively. In order to interpret treatment efficacy and re-infection levels, knowledge about clearance of this antigen is necessary. The current study aims to investigate, whether antigen clearance as a proxy for decreasing worm numbers is reflected in decreasing CCA levels in urine shortly after praziquantel treatment. Here CCA levels are measured 24 hours post treatment in response to both a single and two treatments. The study was designed as a series of cross-sectional urine and stool sample collections from 446 individuals nested in a two-arm randomised single blinded longitudinal clinical trial cohort matched by gender and age (ClinicalTrials.gov Identifier: NCT00215267) receiving one or two praziquantel treatments. CCA levels in urine were determined by carbon-conjugated monoclonal antibody lateral flow strip assay and eggs per gram faeces for S. mansoni and soil-transmitted helminths by Kato-Katz. Significant correlations between CCA levels and S. mansoni egg count at every measured time point were found and confirmed the added beneficial effect of a second treatment at two weeks after baseline. Furthermore, presence of hookworm was found not to be a confounder for CCA test specificity. Twenty-four hours post treatment measures of mean CCA scores showed significant reductions. In conclusion, removal of CCA in response to treatment is detectable as a decline in CCA in urine already after 24 hours. This has relevance for use and interpretation of laboratory based and point-of-care CCA tests in terms of treatment efficacy and re-infection proportions as this measure provides information on the presence of all actively feeding stages of S. mansoni, which conventional faecal microscopy methods do not accurately reflect. TRIAL REGISTRATION: ClinicalTrials.gov NCT00215267.

N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression.

Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.

Developments in FTICR-MS and Its Potential for Body Fluid Signatures.

Fourier transform mass spectrometry (FTMS) is the method of choice for measurements that require ultra-high resolution. The establishment of Fourier transform ion cyclotron resonance (FTICR) MS, the availability of biomolecular ionization techniques and the introduction of the Orbitrap™ mass spectrometer have widened the number of FTMS-applications enormously. One recent example involves clinical proteomics using FTICR-MS to discover and validate protein biomarker signatures in body fluids such as serum or plasma. These biological samples are highly complex in terms of the type and number of components, their concentration range, and the structural identity of each species, and thus require extensive sample cleanup and chromatographic separation procedures. Clearly, such an elaborate and multi-step sample preparation process hampers high-throughput analysis of large clinical cohorts. A final MS read-out at ultra-high resolution enables the analysis of a more complex sample and can thus simplify upfront fractionations. To this end, FTICR-MS offers superior ultra-high resolving power with accurate and precise mass-to-charge ratio (m/z) measurement of a high number of peptides and small proteins (up to 20 kDa) at isotopic resolution over a wide mass range, and furthermore includes a wide variety of fragmentation strategies to characterize protein sequence and structure, including post-translational modifications (PTMs). In our laboratory, we have successfully applied FTICR "next-generation" peptide profiles with the purpose of cancer disease classifications. Here we will review a number of developments and innovations in FTICR-MS that have resulted in robust and routine procedures aiming for ultra-high resolution signatures of clinical samples, exemplified with state-of-the-art examples for serum and saliva.

Skewed Fc glycosylation profiles of anti-proteinase 3 immunoglobulin G1 autoantibodies from granulomatosis with polyangiitis patients show low levels of bisection, galactosylation, and sialylation.

Granulomatosis with polyangiitis (GPA) is associated with circulating immunoglobulin (Ig) G anti-proteinase 3 specific (anti-PR3) anti-neutrophil cytoplasm antibodies (ANCA), which activate cytokine primed neutrophils via Fcgamma receptors. ANCA are class switched IgG antibodies implying T cell help in their production. Glycosylation of IgG Fc, under the control of T cell cytokines, determines the interaction between IgG and its receptors. Previous studies have reported aberrant glycosylation of Ig Fc in GPA patients. We investigated whether aberrant Fc glycosylation was present on anti-PR3 ANCA as well as whole IgG subclass preparations compared to healthy controls and whether this correlated with Birmingham vasculitis activity scores (BVAS), serum cytokines, and time to remission. Here, IgG Fc glycosylation of GPA patients and controls and anti-PR3 ANCA Fc glycosylation were determined by mass spectrometry of glycopeptides. IgG1 and IgG2 subclasses from GPA patients showed reduced galactosylation, sialylation, and bisection compared to healthy controls. Anti-PR3 IgG1 ANCA Fc galactosylation, sialylation, and bisection were reduced compared to total IgG1 in GPA. Galactosylation of anti-PR3 ANCA Fc correlated with inflammatory cytokines and time to remission but not BVAS. Bisection of anti-PR3 ANCA Fc correlated with BVAS. Total IgG1 and anti-PR3 IgG1 Fc galactosylation were weakly correlated, while bisection of IgG1 and anti-PR3 showed no correlation. Our data indicate that aberrant ANCA galactosylation may be driven in an antigen-specific manner.

N-glycomic profiling as a tool to separate rectal adenomas from carcinomas.

All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world's three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I-II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.

De novo discovery of phenotypic intratumour heterogeneity using imaging mass spectrometry.

An essential and so far unresolved factor influencing the evolution of cancer and the clinical management of patients is intratumour clonal and phenotypic heterogeneity. However, the de novo identification of tumour subpopulations is so far both a challenging and an unresolved task. Here we present the first systematic approach for the de novo discovery of clinically detrimental molecular tumour subpopulations. In this proof-of-principle study, spatially resolved, tumour-specific mass spectra were acquired, using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry, from tissues of 63 gastric carcinoma and 32 breast carcinoma patients. The mass spectra, representing the proteomic heterogeneity within tumour areas, were grouped by a corroborated statistical clustering algorithm in order to obtain segmentation maps of molecularly distinct regions. These regions were presumed to represent different phenotypic tumour subpopulations. This was confirmed by linking the presence of these tumour subpopulations to the patients' clinical data. This revealed several of the detected tumour subpopulations to be associated with a different overall survival of the gastric cancer patients (p = 0.025) and the presence of locoregional metastases in patients with breast cancer (p = 0.036). The procedure presented is generic and opens novel options in cancer research, as it reveals microscopically indistinct tumour subpopulations that have an adverse impact on clinical outcome. This enables their further molecular characterization for deeper insights into the biological processes of cancer, which may finally lead to new targeted therapies.

Towards imaging metabolic pathways in tissues.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging using 9-aminoacridine as the matrix leads to the detection of low mass metabolites and lipids directly from cancer tissues. These included lactate and pyruvate for studying the Warburg effect, as well as succinate and fumarate, metabolites whose accumulation is associated with specific syndromes. By using the pathway information present in the human metabolome database, it was possible to identify regions within tumor tissue samples with distinct metabolic signatures that were consistent with known tumor biology. We present a data analysis workflow for assessing metabolic pathways in their histopathological context.

Serum peptide signatures for pancreatic cancer based on mass spectrometry: a comparison to CA19-9 levels and routine imaging techniques.

PURPOSE: The detection of pancreatic tumors lacks a sensitive and specific diagnostic tool. Mass spectrometry (MS)-based profiling of serum proteins is a promising approach for discovery of new clinical biomarkers or biomarker signatures. METHODS: Serum samples from pancreatic cancer (PC) patients and control individuals were collected and processed using a standardized protocol. Samples were divided in a calibration set (n = 49 PC and 110 controls) and a validation set (n = 39 PC and 75 controls). Peptide profiles were obtained using a combination of automated solid-phase extraction with reversed-phase C18 paramagnetic beads and matrix-assisted laser desorption ionization time-of-flight MS. RESULTS: Linear discriminant analysis with double cross-validation resulted in a discriminating peptide signature for PC in the calibration set with a sensitivity of 78 % and a specificity of 91 % [area under the curve (AUC) of 92 %]. Classification was validated with a sensitivity of 93 % and a specificity of 100 % (AUC of 98 %), and the results were compared with carbohydrate antigen 19-9 levels and currently available clinical imaging techniques. The ten most discriminating peptide peaks were identified as fragments of proteins involved in the clotting cascade, acute phase response and immunologic response. CONCLUSIONS: In this study, it is shown that MS-based serum peptide profiles can discriminate between PC and control samples. The approach has great potential for high-throughput analysis in surveillance programs and appears to be most promising for patients with an inherited risk for PC, who benefit from more frequent screening.

Capillary-electrophoresis mass spectrometry for the detection of carbapenemases in (multi-)drug-resistant Gram-negative bacteria.

In a time in which the spread of multidrug resistant microorganisms is ever increasing, there is a need for fast and unequivocal identification of suspect organisms to supplement existing techniques in the clinical laboratory, especially in single bacterial colonies. Mass-spectrometry coupled with efficient peptide separation techniques offer great potential for identification of resistant-related proteins in complex microbiological samples in an unbiased manner. Here, we developed a capillary electrophoresis-electrospray ionization-tandem mass spectrometry CE-ESI-MS/MS bottom-up proteomics workflow for sensitive and specific peptide analysis with the emphasis on the identification of ß-lactamases (carbapenemases OXA-48 and KPC in particular) in bacterial species. For this purpose, tryptic peptides from whole cell lysates were analyzed by sheathless CE-ESI-MS/MS and proteins were identified after searching of the spectral data against bacterial protein databases. The CE-ESI-MS/MS workflow was first evaluated using a recombinant TEM-1 ß-lactamase, resulting in 68% of the amino acid sequence being covered by 20 different unique peptides. Subsequently, a resistant and susceptible Escherichia coli lab strain were analyzed and based on the observed ß-lactamase peptides, the two strains could easily be discriminated. Finally, the method was tested in an unbiased setup using a collection of in-house characterized OXA-48 (n = 17) and KPC (n = 10) clinical isolates. The developed CE-ESI-MS/MS method was able to identify the presence of OXA-48 and KPC in all of the carbapenemase positive samples, independent of species and degree of susceptibility. Four negative controls were tested and classified as negative by this method. Furthermore, a number of extended-spectrum beta-lactamases (ESBL) were identified in the same analyses, confirming the multiresistant character in 19 out of 27 clinical isolates. Importantly, the method performed equally well on protein lysates from single colonies. As such, it demonstrates CE-ESI-MS/MS as a potential next generation mass spectrometry platform within the clinical microbiology laboratory.

Comprehensive gas chromatography-electron ionisation mass spectrometric analysis of fatty acids and sterols using sequential one-pot silylation: quantification and isotopologue analysis.

RATIONALE: Fatty acids and sterol lipids play crucial roles in several biological processes and several biological facts underline the interconnection between these lipid classes. Therefore, it is of interest to develop a comprehensive method analysing both classes in the form of their most favourable derivatives suitable for quantification and isotopologue analysis. METHODS: Lipids were derivatised by a sequential one-pot procedure using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MtBSTFA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). No clean-up or concentration steps were necessary. The prepared samples were directly available for gas chromatography-electron ionisation mass spectrometric (GC-EI-MS) analysis on a standard column. For quantification, the SIM mode was used and for isotopologue analysis scheduled scan mode was applied. RESULTS: Development of a sequential one-pot derivatisation for GC-EI-MS allowing comprehensive analysis of fatty acids and sterols as their most favourable derivatives. Validation carried out using human plasma, comparison with certified NIST plasma. LLOQ of usually 3.3 ng/mL achieved. Isotopologue analysis of 2-[(13)C]-acetate incorporation in HL-60 cells proving feasibility of method. CONCLUSIONS: The presented method successfully combines two consecutive silylation reactions in one pot, enabling the analysis of both fatty acids and sterols in a comprehensive analytical method. The method has great potential for the quantification of lipids as well as the comprehensive study of both biochemical pathways, using [(13)C]-flux analysis.

PeptidePicker: a scientific workflow with web interface for selecting appropriate peptides for targeted proteomics experiments.

One challenge in Multiple Reaction Monitoring (MRM)-based proteomics is to select the most appropriate surrogate peptides to represent a target protein. We present here a software package to automatically generate these most appropriate surrogate peptides for an LC/MRM-MS analysis. Our method integrates information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM which is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our knowledge in choosing the best candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it previously has been observed. The modularity of the workflow allows further extension and additional selection criteria to be incorporated. We have developed a simple Web interface where the researcher provides the protein accession number, the subject organism, and peptide-specific options. Currently, the software is designed for human and mouse proteomes, but additional species can be easily be added. Our software improved the peptide selection by eliminating human error, considering multiple data sources and all of the isoforms of the protein, and resulted in faster peptide selection - approximately 50 proteins per hour compared to 8 per day. BIOLOGICAL SIGNIFICANCE: Compiling a list of optimal surrogate peptides for target proteins to be analyzed by LC/MRM-MS has been a cumbersome process, in which expert researchers retrieved information from different online repositories and used their own reasoning to find the most appropriate peptides. Our scientific workflow automates this process by integrating information from different data sources including UniProt, Global Proteome Machine, NCBI's dbSNP, and PeptideAtlas, simulating the researchers' reasoning, and incorporating their knowledge of how to select the best proteotypic peptides for an MRM analysis. The developed software can help to standardize the selection of peptides, eliminate human error, and increase productivity.

Fibronectin is a serum biomarker for Duchenne muscular dystrophy.

PURPOSE: To identify and validate serum biomarkers for the progression of Duchenne muscular dystrophy (DMD) using a MS-based bottom-up pipeline. EXPERIMENTAL DESIGN: We used a bottom-up proteomics approach, including a protein concentration equalization step, different proteolytic digestions, and MS detection schemes, to identify candidate biomarkers in serum samples from control subjects and DMD patients. Fibronectin was chosen for follow-up based on the differences in peptide spectral counts and sequence coverage observed between the DMD and control groups. Subsequently, fibronectin levels were determined with ELISA in 68 DMD patients, 38 milder Becker muscular dystrophy patients, 33 patients with other neuromuscular disorders, and 15 age-matched adult and child controls. RESULTS: There was a significant increase in fibronectin levels in DMD patients compared to age-matched controls. Fibronectin levels in patients with Becker muscular dystrophy, Bethlem myopathy, or myasthenia gravis were comparable to control levels. Progressive elevation in fibronectin levels was observed in longitudinal samples from 22 DMD patients followed up for a period of 6 months up to 4 years. CONCLUSION AND CLINICAL RELEVANCE: This study suggests that serum fibronectin levels may constitute a promising biomarker to monitor disease progression in DMD patients.

Site-specific N-glycosylation analysis of human immunoglobulin e.

Immunoglobulin E (IgE) is a heterodimeric glycoprotein involved in antiparasitic and allergic immune reactions. IgE glycosylation is known to exhibit significant interindividual variation, and several reports have indicated its relevance in determining IgE activity. Here, we present site-specific glycosylation analysis of IgE from three different sources: IgE from the serum of a hyperimmune donor, from the pooled serum of multiple nondiseased donors, and from the pooled serum of 2 patients with IgE myeloma. The heavy chains were isolated and digested with either trypsin, proteinase K, or chymotrypsin, which permitted coverage of all seven potential N-glycosylation sites. The resulting (glyco-)peptides were analyzed by nano-reversed-phase-LC-MS/MS and MALDI-TOF/TOF-MS/MS. Site Asn264 was shown to be unoccupied. In all three samples, site Asn275 contained exclusively oligomannosidic structures with between 2 and 9 mannoses, whereas sites Asn21, Asn49, Asn99, Asn146, and Asn252 contained exclusively complex-type glycans. For the nonmyeloma IgE, the majority of these glycans were biantennary and core-fucosylated and contained one or two terminal N-acetylneuraminic acids. In contrast, myeloma IgE showed a higher abundance of triantennary and tetraantennary glycan structures and a low abundance of species with a bisecting N-acetylglucosamine. Our approach allows comparison of the glycosylation of IgE samples in a site-specific manner.

A latent Markov modelling approach to the evaluation of circulating cathodic antigen strips for schistosomiasis diagnosis pre- and post-praziquantel treatment in Uganda.

Regular treatment with praziquantel (PZQ) is the strategy for human schistosomiasis control aiming to prevent morbidity in later life. With the recent resolution on schistosomiasis elimination by the 65th World Health Assembly, appropriate diagnostic tools to inform interventions are keys to their success. We present a discrete Markov chains modelling framework that deals with the longitudinal study design and the measurement error in the diagnostic methods under study. A longitudinal detailed dataset from Uganda, in which one or two doses of PZQ treatment were provided, was analyzed through Latent Markov Models (LMMs). The aim was to evaluate the diagnostic accuracy of Circulating Cathodic Antigen (CCA) and of double Kato-Katz (KK) faecal slides over three consecutive days for Schistosoma mansoni infection simultaneously by age group at baseline and at two follow-up times post treatment. Diagnostic test sensitivities and specificities and the true underlying infection prevalence over time as well as the probabilities of transitions between infected and uninfected states are provided. The estimated transition probability matrices provide parsimonious yet important insights into the re-infection and cure rates in the two age groups. We show that the CCA diagnostic performance remained constant after PZQ treatment and that this test was overall more sensitive but less specific than single-day double KK for the diagnosis of S. mansoni infection. The probability of clearing infection from baseline to 9 weeks was higher among those who received two PZQ doses compared to one PZQ dose for both age groups, with much higher re-infection rates among children compared to adolescents and adults. We recommend LMMs as a useful methodology for monitoring and evaluation and treatment decision research as well as CCA for mapping surveys of S. mansoni infection, although additional diagnostic tools should be incorporated in schistosomiasis elimination programs.

Investigations on aberrant glycosylation of glycosphingolipids in colorectal cancer tissues using liquid chromatography and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS).

Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment.

An automated RP-SCX solid-phase extraction procedure for urinary peptidomics biomarker discovery studies.

Urine represents the most easily obtainable body fluid and consequently one of the most common samples in clinical chemistry. The majority of pathological changes in human organs may well be reflected in urine. In this way, urine analysis can aid in disease diagnosis, treatment monitoring, and prognosis. Currently, the most commonly used method for identification of new urine biomarkers involves centrifugation of the urine sample to collect either the soluble urine proteins or the urinary exosomes followed by 1 or 2 protein purification and separation steps before visualization and finally identification of potential biomarkers, usually by mass spectrometry. Here we present a generally applicable, rapid, and robust method for screening large number of urine samples, resulting in a broad spectrum of native peptides, as a tool to be used for biomarker discovery. The method combines online sample pretreatment with a well-established mass spectrometric technique. Native peptides are extracted from urine samples on a miniaturized reverse-phase-strong cation exchange cartridge system. As the proper identification of native peptides often requires combination of data acquired on different mass analyzers, we have aimed at a procedure providing us with sufficient material to identify and characterize the differentially expressed markers.

Use of expressed sequence tags as an alternative approach for the identification of Taenia solium metacestode excretion/secretion proteins.

BACKGROUND: Taenia solium taeniasis/cysticercosis is a zoonotic helminth infection mainly found in rural regions of Africa, Asia and Latin America. In endemic areas, diagnosis of cysticercosis largely depends on serology, but these methods have their drawbacks and require improvement. This implies better knowledge of the proteins secreted and excreted by the parasite. In a previous study, we used a custom protein database containing protein sequences from related helminths to identify T. solium metacestode excretion/secretion proteins. An alternative or complementary approach would be to use expressed sequence tags combined with BLAST and protein mapping to supercontigs of Echinococcus granulosus, a closely related cestode. In this study, we evaluate this approach and compare the results to those obtained in the previous study. FINDINGS: We report 297 proteins organized in 106 protein groups based on homology. Additional classification was done using Gene Ontology information on biological process and molecular function. Of the 106 protein groups, 58 groups were newly identified, while 48 groups confirmed previous findings. Blast2GO analysis revealed that the majority of the proteins were involved in catalytic activities and binding. CONCLUSIONS: In this study, we used translated expressed sequence tags combined with BLAST and mapping strategies to both confirm and complement previous research. Our findings are comparable to recent studies on other helminth genera like Echinococcus, Schistosoma and Clonorchis, indicating similarities between helminth excretion/secretion proteomes.

Detection of pancreatic cancer using serum protein profiling.

BACKGROUND: Currently, no suitable biomarkers for the early detection of pancreatic cancer (PC) are available. Proteins present in the serum could reflect a state of the disease. In this study, these profiles as a diagnostic marker for PC were evaluated. METHODS: Serum samples were obtained from PC patients (n = 50 calibration set, n = 39 validation set) and healthy volunteers (n = 110 and n = 75 respectively) according to a uniform standardized collection and processing protocol. For peptide and protein isolation, automated solid-phase extraction (SPE) with Weak Cation Exchange (WCX) magnetic beads (MB) was performed using a 96-channel liquid handling platform. Protein profiles were obtained by mass spectrometry (MS) and evaluated by linear discriminant analysis with double cross-validation. RESULTS: A discriminating profile for PC has been identified, with a sensitivity of 78% and a specificity of 89% in the calibration set with an area under the curve (AUC) of 90%. These results were validated with a sensitivity of 74% and a specificity of 91% (AUC 90%). CONCLUSION: Serum profiles of healthy controls and PC can be discrimated between. Further research is warranted to evaluate specificity and whether this biosignature can be used for early detection in a high risk population.

Imaging mass spectrometry-based molecular histology differentiates microscopically identical and heterogeneous tumors.

Many tumors display significant cellular heterogeneity as well as molecular heterogeneity. Sensitive biomarkers that differentiate between diagnostically challenging tumors must contend with this heterogeneity. Mass spectrometry-based molecular histology of a patient series of heterogeneous, microscopically identical bone tumors highlighted the tumor cell types that could be characterized by a single profile and led to the identification of specific peptides that differentiate between the tumors.

Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers.

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative measurements of N-glycosylation using ultra-performance liquid chromatography (UPLC) in 2,247 individuals from four European discovery populations. In parallel, we measured IgG N-glycans using MALDI-TOF mass spectrometry (MS) in a replication cohort of 1,848 Europeans. Meta-analysis of genome-wide association study (GWAS) results identified 9 genome-wide significant loci (P<2.27 × 10(-9)) in the discovery analysis and two of the same loci (B4GALT1 and MGAT3) in the replication cohort. Four loci contained genes encoding glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3), while the remaining 5 contained genes that have not been previously implicated in protein glycosylation (IKZF1, IL6ST-ANKRD55, ABCF2-SMARCD3, SUV420H1, and SMARCB1-DERL3). However, most of them have been strongly associated with autoimmune and inflammatory conditions (e.g., systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, Crohn's disease, diabetes type 1, multiple sclerosis, Graves' disease, celiac disease, nodular sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma). Follow-up functional experiments in haplodeficient Ikzf1 knock-out mice showed the same general pattern of changes in IgG glycosylation as identified in the meta-analysis. As IKZF1 was associated with multiple IgG N-glycan traits, we explored biomarker potential of affected N-glycans in 101 cases with SLE and 183 matched controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve = 0.842). Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. The results may also provide an explanation for the reported pleiotropy and antagonistic effects of loci involved in autoimmune diseases and haematological cancer.