Human Dendritic Cells with Th2-Polarizing Capacity: Analysis Using Label-Free Quantitative Proteomics.

BACKGROUND: Dendritic cells (DCs) are the sentinels of the immune system. Upon recognition of a pathogen, they mature and migrate to draining lymph nodes to prime and polarize T cell responses. Although it is known that helminths and helminth-derived molecules condition DCs to polarize T helper (Th) cells towards Th2, the underlying mechanisms remain incompletely understood. OBJECTIVES: The aim of this study was to conduct a proteome analysis of helminth antigen-stimulated DCs in order to gain more insight into the cellular processes associated with their ability to polarize immune responses. METHODS: We analyzed the maturation and polarization of monocyte-derived DCs from 9 donors at 2 different time points after stimulation with different Th1- and Th2-polarizing pathogen-derived molecules. The samples were measured using liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for relative quantitation. RESULTS: Lipopolysaccharide-induced maturation promoted the expression of proteins related to metabolic, cellular, and immune system processes. Th1-polarizing DCs, conditioned by IFN-γ during maturation, displayed accelerated maturation by differentially expressing cytoskeletal proteins and proteins involved in immune regulation. The stimulation of DCs with soluble egg antigens and omega-1 derived from Schistosoma mansoni, which are both Th2-inducing stimuli, increased 60S acidic ribosomal protein P2, and vesicle amine transferase 1 while decreasing the expression of proteins related to antigen processing and presentation. CONCLUSION: Our data indicate that not only proteins involved in the interaction between T cells and DCs at the level of the immunological synapse, but also those related to cellular metabolism and stress, may promote Th2 polarization.

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.

Differentiating samples and experimental protocols by direct comparison of tandem mass spectra.

RATIONALE: Peptide tandem mass spectra can be analyzed by a number of means. They can be compared against predicted spectra of peptides derived from genome sequences, compared against previously acquired and identified spectra, or - sometimes - sequenced de novo. We recently introduced another method which compares spectra between liquid chromatography/tandem mass spectrometry (LC/MS/MS) datasets to determine the shared spectral content, and demonstrated how this can be applied in a molecular phylogenetic study using sera from human and non-human primates. We will here explore if such a method have other, serendipitous uses. METHODS: We used the existing compareMS2 algorithm without modification on a diverse set of experiments. RESULTS: First we conducted a small phylogenetic study, using (mammalian) bone samples to study old material, and human pathogens aiming to distinguish clinically important strains. Although not as straightforward as primate sera analysis, the method shows significant promise for all these applications. We also used the algorithm to compare 24 different protocols for extraction of proteins from muscle tissue. The results provided useful information in comparing protocols. Finally, we applied compareMS2 aiming for quality control of two traceable protein reference standards (troponin) used in clinical chemistry assays, by analysing the effect of storage conditions. CONCLUSIONS: The results illustrate a broad applicability of the metric based on shared tandem mass spectra between LC/MS/MS datasets for analysing protein digests in different types of experiments. There is no reason to assume that our instance of this method is optimal in any of these situations, as it makes limited or no use of accurate mass and chromatographic retention time. We propose that with further improvement and refinement, this type of analysis can be applied as a simple but informative first step in many pipelines for bottom-up tandem mass spectrometry data analysis in proteomics and other fields, comparing or analysing large numbers of samples or datasets.

Extensive glycosylation of ACPA-IgG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis.

OBJECTIVES: To understand the molecular features distinguishing anti-citrullinated protein antibodies (ACPA) from 'conventional' antibodies in rheumatoid arthritis (RA). METHODS: Serum of ACPA-positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum, plasma and/or synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and analysed by mass spectrometry. Binding affinity to citrullinated antigens was measured by ELISA and imaging surface plasmon resonance using recombinant monoclonal ACPA with molecular modifications. RESULTS: In all donor samples studied (n=24), ACPA-IgG exhibited a 10-20 kDa higher molecular weight compared with non-autoreactive IgG. This feature also distinguished ACPA-IgG from antibodies against recall antigens or other disease-specific autoantibodies. Structural analysis revealed that a high frequency of N-glycans in the (hyper)variable domains of ACPA is responsible for this observation. In line with their localisation, these N-glycans were found to modulate binding avidity of ACPA to citrullinated antigens. CONCLUSIONS: The vast majority of ACPA-IgG harbour N-glycans in their variable domains. As N-linked glycosylation requires glycosylation consensus sites in the protein sequence and as these are lacking in the 'germline-counterparts' of identified variable domains, our data indicate that the N-glycosylation sites in ACPA variable domains have been introduced by somatic hypermutation. This finding also suggests that ACPA-hyperglycosylation confers a selective advantage to ACPA-producing B cells. This unique and completely novel feature of the citrulline-specific immune response in RA elucidates our understanding of the underlying B cell response.

CE-ESI-MS for bottom-up proteomics: Advances in separation, interfacing and applications.

With the development of more sensitive hyphenation strategies for capillary electrophoresis-electrospray-mass spectrometry the technique has reemerged as technique with high separation power combined with high sensitivity in the analysis of peptides and protein digests. This review will discuss the newly developed hyphenation strategies for CE-ESI-MS and their application in bottom-up proteomics as well as the applications in the same time span, 2009 to present, using co-axial sheathliquid. Subsequently all separate aspects in the development of a CE-ESI-MS method for bottom-up proteomics shall be discussed, highlighting certain applications and discussing pros and cons of the various choices. The separation of peptides in a capillary electrophoresis system is discussed including the great potential for modeling of this migration of peptides due to the simple electrophoretic separation process. Furthermore, the technical aspects of method development are discussed, namely; background electrolyte choice, coating of the separation capillary and chosen loading method. Finally, conclusions and an outlook on future developments in the field of bottom-up proteomics by CE-ESI-MS will be provided.

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.

Ethanol contamination of cerebrospinal fluid during standardized sampling and its effect on (1)H-NMR metabolomics.

Standardization of body fluid sampling, processing and storage procedures is pivotal to ensure data quality in metabolomics studies. Yet, despite strict adherence to standard sampling guidelines, we detected variable levels of ethanol in the (1)H-NMR spectra of human cerebrospinal fluid (CSF) samples (range 9.2 × 10(-3)-10.0 mM). The presence of ethanol in all samples and the wide range of concentrations clearly indicated contamination of the samples of some sort, which affected the (1)H-NMR spectra quality and the interpretation. To determine where in the sampling protocol the ethanol contamination occurs, we performed a CSF sampling protocol simulation with 0.9 % NaCl (saline) instead of CSF and detected ethanol in all simulation samples. Ethanol diffusion through air during sampling and preparation stages appeared the only logical explanation. With a bench study, we showed that ethanol easily diffuses into ex vivo CSF samples via air transmission. Ethanol originated from routinely used skin disinfectants containing ethanol and from laboratory procedures. Ethanol affected the CSF sample matrix at concentrations above ~9.4 mM and obscured a significant part of the (1)H-NMR spectrum. CSF sample preparation for (1)H-NMR-based metabolomics analyses should therefore be carried out in a well-ventilated atmosphere with laminar flow, and use of ethanol should be avoided.

Glycomic Analysis of Life Stages of the Human Parasite Schistosoma mansoni Reveals Developmental Expression Profiles of Functional and Antigenic Glycan Motifs.

Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galß1-4(Fucα1-3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcß1-4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with α3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galß1-3(Galß1-6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated stretches enriched in mature eggs and miracidia. This global analysis of the developing schistosome's glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions.

Quantification of serum apolipoproteins A-I and B-100 in clinical samples using an automated SISCAPA-MALDI-TOF-MS workflow.

A fully automated workflow was developed and validated for simultaneous quantification of the cardiovascular disease risk markers apolipoproteins A-I (apoA-I) and B-100 (apoB-100) in clinical sera. By coupling of stable-isotope standards and capture by anti-peptide antibodies (SISCAPA) for enrichment of proteotypic peptides from serum digests to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS detection, the standardized platform enabled rapid, liquid chromatography-free quantification at a relatively high throughput of 96 samples in 12h. The average imprecision in normo- and triglyceridemic serum pools was 3.8% for apoA-I and 4.2% for apoB-100 (4 replicates over 5 days). If stored properly, the MALDI target containing enriched apoA-1 and apoB-100 peptides could be re-analyzed without any effect on bias or imprecision for at least 7 days after initial analysis. Validation of the workflow revealed excellent linearity for daily calibration with external, serum-based calibrators (R(2) of 0.984 for apoA-I and 0.976 for apoB-100 as average over five days), and absence of matrix effects or interference from triglycerides, protein content, hemolysates, or bilirubins. Quantification of apoA-I in 93 normo- and hypertriglyceridemic clinical sera showed good agreement with immunoturbidimetric analysis (slope = 1.01, R(2) = 0.95, mean bias = 4.0%). Measurement of apoB-100 in the same clinical sera using both methods, however, revealed several outliers in SISCAPA-MALDI-TOF-MS measurements, possibly as a result of the lower MALDI-TOF-MS signal intensity (slope = 1.09, R(2) = 0.91, mean bias = 2.0%). The combination of analytical performance, rapid cycle time and automation potential validate the SISCAPA-MALDI-TOF-MS platform as a valuable approach for standardized and high-throughput quantification of apoA-I and apoB-100 in large sample cohorts.

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.

Top-down MALDI-in-source decay-FTICR mass spectrometry of isotopically resolved proteins.

An accurate mass measurement of a known protein provides information on potential amino acid deletions and post-translational modifications. Although this field is dominated by strategies based on electrospray ionization, mass spectrometry (MS) methods using matrix-assisted laser desorption/ionization (MALDI) have the advantage of yielding predominantly singly charged precursor ions, thus avoiding peak overlap from different charge states of multiple species. Such MALDI-MS methods require mass measurement at ultrahigh resolution, which is provided by Fourier transform ion cyclotron resonance (FTICR) mass analyzers. Recently, using a MALDI-FTICR-MS platform equipped with a 15 T magnet, we reported on the mass analysis of intact human serum peptides and small proteins with isotopic resolution up to ∼15 kDa and identified new proteoforms from an accurate measurement of mass distances. In the current study, we have used this FTICR system after an upgrade with a novel dynamically harmonized ICR cell, i.e., ParaCell, for mapping isotopically resolved intact proteins up to about 17 kDa and performed top-down MALDI in-source decay (ISD) analysis. Standard proteins myoglobin (m/z-value 16,950) and ribonuclease B (m/z-value 14,900) were measured with resolving powers of 62,000 and 61,000, respectively. Furthermore, it will be shown that (singly charged) MALDI-ISD fragment ions can be measured at isotopic resolution up to m/z-value 12,000 (e.g., resolving power 39,000 at m/z-value 12,000) providing more reliable identifications. Moreover, examples are presented of pseudo-MS(3) experiments on ISD fragment ions from RNase B by collisional-induced dissociation (CID).

Insight in genome-wide association of metabolite quantitative traits by exome sequence analyses.

Metabolite quantitative traits carry great promise for epidemiological studies, and their genetic background has been addressed using Genome-Wide Association Studies (GWAS). Thus far, the role of less common variants has not been exhaustively studied. Here, we set out a GWAS for metabolite quantitative traits in serum, followed by exome sequence analysis to zoom in on putative causal variants in the associated genes. 1H Nuclear Magnetic Resonance (1H-NMR) spectroscopy experiments yielded successful quantification of 42 unique metabolites in 2,482 individuals from The Erasmus Rucphen Family (ERF) study. Heritability of metabolites were estimated by SOLAR. GWAS was performed by linear mixed models, using HapMap imputations. Based on physical vicinity and pathway analyses, candidate genes were screened for coding region variation using exome sequence data. Heritability estimates for metabolites ranged between 10% and 52%. GWAS replicated three known loci in the metabolome wide significance: CPS1 with glycine (P-value  = 1.27×10-32), PRODH with proline (P-value  = 1.11×10-19), SLC16A9 with carnitine level (P-value  = 4.81×10-14) and uncovered a novel association between DMGDH and dimethyl-glycine (P-value  = 1.65×10-19) level. In addition, we found three novel, suggestively significant loci: TNP1 with pyruvate (P-value  = 1.26×10-8), KCNJ16 with 3-hydroxybutyrate (P-value  = 1.65×10-8) and 2p12 locus with valine (P-value  = 3.49×10-8). Exome sequence analysis identified potentially causal coding and regulatory variants located in the genes CPS1, KCNJ2 and PRODH, and revealed allelic heterogeneity for CPS1 and PRODH. Combined GWAS and exome analyses of metabolites detected by high-resolution 1H-NMR is a robust approach to uncover metabolite quantitative trait loci (mQTL), and the likely causative variants in these loci. It is anticipated that insight in the genetics of intermediate phenotypes will provide additional insight into the genetics of complex traits.

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.

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.

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.

Anti-citrullinated protein antibodies acquire a pro-inflammatory Fc glycosylation phenotype prior to the onset of rheumatoid arthritis.

OBJECTIVE: To determine whether anticitrullinated protein antibodies (ACPA) exhibit specific changes in Fc glycosylation prior to the onset of arthritis. METHODS: Serum samples of patients with ACPA-positive arthralgia (n=183) were collected at baseline and at various time points of follow-up. 105 patients developed arthritis after a median of 12 months (IQR 6-24) and were classified as having either rheumatoid arthritis (RA, n=48) or undifferentiated arthritis (UA, n=57) based on the 1987 American College of Rheumatology (ACR) criteria. ACPA and total serum IgG were isolated by affinity purification and cleaved by trypsin. ACPA-IgG1 Fc-glycopeptides were subsequently analysed by nano-liquid chromatography mass spectrometry and compared to those of total IgG1. RESULTS: At baseline, ACPA-IgG1 and total IgG1 from arthralgia patients displayed similar Fc glycosylation patterns. By contrast, at the onset of arthritis, ACPA exhibited a decrease in galactose residues in RA patients, but not in UA patients. This decrease occurred around 3 months prior to diagnosis and was paralleled by an increase in systemic inflammation (erythrocyte sedimentation rate). Galactosylation of total IgG1 was also decreased in RA, but this did not precede the onset of arthritis. Interestingly, we additionally noted a higher degree of ACPA-IgG1 Fc core fucosylation at baseline as compared with total IgG1, which further increased prior to diagnosis. CONCLUSIONS: ACPA display significant changes in Fc galactosylation and fucosylation prior to the onset of RA. These changes towards a more pro-inflammatory phenotype could be involved in driving the disease process.

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.

Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels.

The potential of various quantitative lateral flow (LF) based assays utilizing up-converting phosphor (UCP) reporters for the diagnosis of schistosomiasis is reviewed including recent developments. Active infections are demonstrated by screening for the presence of regurgitated worm antigens (genus specific polysaccharides), whereas anti-Schistosoma antibodies may indicate ongoing as well as past infections. The circulating anodic antigen (CAA) in serum or urine (and potentially also saliva) is identified as the marker that may allow detection of single-worm infections. Quantitation of antigen levels is a reliable method to study effects of drug administration, worm burden and anti-fecundity mechanisms. Moreover, the ratio of CAA and circulating cathodic antigen (CCA) is postulated to facilitate identification of either Schistosoma mansoni or Schistosoma haematobium infections. The UCP-LF assays allow simultaneous detection of multiple targets on a single strip, a valuable feature for antibody detection assays. Although antibody detection in endemic regions is not a useful tool to diagnose active infections, it gains potential when the ratio of different classes of antibody specific for the parasite/disease can be determined. The UCP-LF antibody assay format allows this type of multiplexing, including testing a linear array of up to 20 different targets. Multiple test spots would allow detection of specific antibodies, e.g. against different Schistosoma species or other pathogens as soil-transmitted helminths. Concluding, the different UCP-LF based assays for diagnosis of schistosomiasis provide a collection of tests with relatively low complexity and high sensitivity, covering the full range of diagnostics needed in control programmes for mapping, screening and monitoring.