Circulating Markers of Inflammation Persist in Children and Adults With Giant Aneurysms After Kawasaki Disease.

BACKGROUND: The sequelae of Kawasaki disease (KD) vary widely with the greatest risk for future cardiovascular events among those who develop giant coronary artery aneurysms (CAA). We sought to define the molecular signature associated with different outcomes in pediatric and adult KD patients. METHODS: Molecular profiling was conducted using mass spectrometry-based shotgun proteomics, transcriptomics, and glycomics methods on 8 pediatric KD patients at the acute, subacute, and convalescent time points. Shotgun proteomics was performed on 9 KD adults with giant CAA and matched healthy controls. Plasma calprotectin was measured by ELISA in 28 pediatric KD patients 1 year post-KD, 70 adult KD patients, and 86 healthy adult volunteers. RESULTS: A characteristic molecular profile was seen in pediatric patients during the acute disease, which resolved at the subacute and convalescent periods in patients with no coronary artery sequelae but persisted in 2 patients who developed giant CAA. We, therefore, investigated persistence of inflammation in KD adults with giant CAA by shotgun proteomics that revealed a signature of active inflammation, immune regulation, and cell trafficking. Correlating results obtained using shotgun proteomics in the pediatric and adult KD cohorts identified elevated calprotectin levels in the plasma of patients with CAA. Investigation of expanded pediatric and adult KD cohorts revealed elevated levels of calprotectin in pediatric patients with giant CAA 1 year post-KD and in adult KD patients who developed giant CAA in childhood. CONCLUSIONS: Complex patterns of biomarkers of inflammation and cell trafficking can persist long after the acute phase of KD in patients with giant CAA. Elevated levels of plasma calprotectin months to decades after acute KD and infiltration of cells expressing S100A8 and A9 in vascular tissues suggest ongoing, subclinical inflammation. Calprotectin may serve as a biomarker to inform the management of KD patients following the acute illness.

Plasma N-glycans in colorectal cancer risk.

Aberrant glycosylation has been associated with a number of diseases including cancer. Our aim was to elucidate changes in whole plasma N-glycosylation between colorectal cancer (CRC) cases and controls in one of the largest cohorts of its kind. A set of 633 CRC patients and 478 age and gender matched controls was analysed. Additionally, patients were stratified into four CRC stages. Moreover, N-glycan analysis was carried out in plasma of 40 patients collected prior to the initial diagnosis of CRC. Statistically significant differences were observed in the plasma N-glycome at all stages of CRC, this included a highly significant decrease in relation to the core fucosylated bi-antennary glycans F(6)A2G2 and F(6)A2G2S(6)1 (P < 0.0009). Stage 1 showed a unique biomarker signature compared to stages 2, 3 and 4. There were indications that at risk groups could be identified from the glycome (retrospective AUC = 0.77 and prospective AUC = 0.65). N-glycome biomarkers related to the pathogenic progress of the disease would be a considerable asset in a clinical setting and it could enable novel therapeutics to be developed to target the disease in patients at risk of progression.

High-throughput characterization of the functional impact of IgG Fc glycan aberrancy in juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) encompasses all forms of chronic idiopathic arthritis that arise before age 16. Previous studies have found JIA to be associated with lower Fc galactosylation of circulating IgG, but the overall spectrum of glycan changes and the net impact on IgG function are unknown. Using ultra performance liquid chromatography (UPLC), we compared IgG glycosylation in 54 subjects with recent-onset untreated JIA with 98 healthy pediatric controls, paired to biophysical profiling of affinity for 20 IgG receptors using a high-throughput multiplexed microsphere assay. Patients with JIA exhibited an increase in hypogalactosylated and hyposialylated IgG glycans, but no change in fucosylation or bisection, together with alteration in the spectrum of IgG ligand binding. Supervised machine learning demonstrated a robust capacity to discriminate JIA subjects from controls using either glycosylation or binding data. The binding signature was driven predominantly by enhanced affinity for Fc receptor like protein 5 (FcRL5), a noncanonical Fc receptor expressed on B cells. Affinity for FcRL5 correlated inversely with galactosylation and sialylation, a relationship confirmed through enzymatic manipulation. These results demonstrate the capacity of combined structural and biophysical IgG phenotyping to define the overall functional impact of IgG glycan changes and implicate FcRL5 as a potential cellular sensor of IgG glycosylation.

Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis.

Glycosylation is an important attribute of biopharmaceutical products to monitor from development through production. However, glycosylation analysis has traditionally been a time-consuming process with long sample preparation protocols and manual interpretation of the data. To address the challenges associated with glycan analysis, we developed a streamlined analytical solution that covers the entire process from sample preparation to data analysis. In this communication, we describe the complete analytical solution that begins with a simplified and fast N-linked glycan sample preparation protocol that can be completed in less than 1 hr. The sample preparation includes labelling with RapiFluor-MS tag to improve both fluorescence (FLR) and mass spectral (MS) sensitivities. Following HILIC-UPLC/FLR/MS analyses, the data are processed and a library search based on glucose units has been included to expedite the task of structural assignment. We then applied this total analytical solution to characterize the glycosylation of the NIST Reference Material mAb 8761. For this glycoprotein, we confidently identified 35 N-linked glycans and all three major classes, high mannose, complex, and hybrid, were present. The majority of the glycans were neutral and fucosylated; glycans featuring N-glycolylneuraminic acid and those with two galactoses connected via an α1,3-linkage were also identified.

Comprehensive Profiling of Glycosphingolipid Glycans Using a Novel Broad Specificity Endoglycoceramidase in a High-Throughput Workflow.

The biological function of glycosphingolipids (GSLs) is largely determined by their glycan headgroup moiety. This has placed a renewed emphasis on detailed GSL headgroup structural analysis. Comprehensive profiling of GSL headgroups in biological samples requires the use of endoglycoceramidases with broad substrate specificity and a robust workflow that enables their high-throughput analysis. We present here the first high-throughput glyco-analytical platform for GSL headgroup profiling. The workflow features enzymatic release of GSL glycans with a novel broad-specificity endoglycoceramidase I (EGCase I) from Rhodococcus triatomea, selective glycan capture on hydrazide beads on a robotics platform, 2AB-fluorescent glycan labeling, and analysis by UPLC-HILIC-FLD. R. triatomea EGCase I displayed a wider specificity than known EGCases and was able to efficiently hydrolyze gangliosides, globosides, (n)Lc-type GSLs, and cerebrosides. Our workflow was validated on purified GSL standard lipids and was applied to the characterization of GSLs extracted from several mammalian cell lines and human serum. This study should facilitate the analytical workflow in functional glycomics studies and biomarker discovery.

Co-detection of Target and Total Protein by CyDye Labeling and Fluorescent ECL Plex Immunoblotting in a Standard Proteomics Workflow.

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis have been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with ECL Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27 kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.

A novel role for the fibrinogen Asn-Gly-Arg (NGR) motif in platelet function.

The integrin αIIbß3 on resting platelets can bind to immobilised fibrinogen resulting in platelet spreading and activation but requires activation to bind to soluble fibrinogen. αIIbß3 is known to interact with the general integrin-recognition motif RGD (arginine-glycine-aspartate) as well as the fibrinogen-specific γ-chain dodecapeptide; however, it is not known how fibrinogen binding triggers platelet activation. NGR (asparagine-glycine-arginine) is another integrin-recognition sequence present in fibrinogen and this study aims to determine if it plays a role in the interaction between fibrinogen and αIIbß3. NGR-containing peptides inhibited resting platelet adhesion to fibrinogen with an IC50 of 175 µM but failed to inhibit the adhesion of activated platelets to fibrinogen (IC50> 500 µM). Resting platelet adhesion to mutant fibrinogens lacking the NGR sequences was reduced compared to normal fibrinogen under both static and shear conditions (200 s⁻¹). However, pre-activated platelets were able to fully spread on all types of fibrinogen. Thus, the NGR motif in fibrinogen is the site that is primarily responsible for the interaction with resting αIIbß3 and is responsible for triggering platelet activation.

High-throughput quantitative N-glycan analysis of glycoproteins.

N-linked oligosaccharides are complex non-template-derived structures that are attached to the side chains of asparagine, via the nitrogen atom. Specific changes in the N-glycans of serum glycoproteins have been associated with the pathogenesis of many diseases. The oligosaccharides present on the C(H)2 domain of immunoglobulins are known to modulate the effector functions of the molecule. These glycans provoke various biological effects, necessitating the development of robust high-throughput technology in order to fully characterize the N-glycosylation profile. This chapter describes in detail four methods to release N-glycans from the glycoprotein of interest. Two of these protocols, referred to as the "In-Gel Block" and "1D sodium dodecyl sulfate-polyacrylamide gel electrophoresis" methods, require immobilization of the glycoprotein prior to analysis. An automated method is also described, involving the purification of immunoglobulins directly from fermentation media, and, finally, an "In-solution method" is detailed, which directly releases the N-glycans into solution. HILIC and WAX-HPLC are used to analyze the N-glycan profile. Exoglycosidase enzymes digestion arrays, in combination with computer-assisted data analysis, are used to determine both the sequence and linkage of the N-glycans present.

Antipsychotic treatment of acute paranoid schizophrenia patients with olanzapine results in altered glycosylation of serum glycoproteins.

Atypical antipsychotic drugs, such as olanzapine, have been shown to alleviate the positive, negative and, to a lesser degree, the cognitive symptoms of schizophrenia in many patients. However, the detailed mechanisms of action of these drugs have yet to be elucidated. We have carried out the first investigation aimed at evaluating the effects of olanzapine treatment on the glycosylation of serum proteins in schizophrenia patients. Olanzapine treatment resulted in increased levels of a disialylated biantennary glycan and reduced levels of a number of disialylated bi- and triantennary glycans on whole serum glycoproteins. These changes were not observed on a low-abundance serum protein fraction. α1 acid glycoprotein was identified as a carrier of some of the detected altered oligosaccharides. In addition, glycan analysis of haptoglobin, transferrin, and α1 antitrypsin reported similar findings, although these changes did not reach significance. Exoglycosidase digestion analysis showed that olanzapine treatment increased galactosylation and sialylation of whole serum proteins, suggesting increased activity of specific galactosyltransferases and increased availability of galactose residues for sialylation. Taken together, these findings indicate that olanzapine treatment results in altered glycosylation of serum proteins.

A 2-D gel reference map of the basic human heart proteome.

We have undertaken the identification of basic proteins (pH 6-11) of the human heart using 2-DE. Tissue from the left ventricle of human heart was lysed and proteins were separated in the first dimension on pH 6-11 IPG strips using paper-bridge loading followed by separation on 12% SDS polyacrylamide gels in the second dimension. Proteins were then identified by mass spectrometry and analysed using Proline, a proteomic data analysis platform that was developed in-house. The proteome map contains 176 identified spots with 151 unique proteins and has been made available as part of the UCD-2DPAGE database at http://proteomics-portal.ucd.ie:8082. The associated mass spectrometry data have been submitted to PRIDE (Accession number ♯10098). This reference map, and the other heart reference maps available through the UCD-2DPAGE database, will aid further proteomic studies of heart diseases such as dilated cardiomyopathy and ischaemic heart disease.

Glycomic and glycoproteomic analysis of serum from patients with stomach cancer reveals potential markers arising from host defense response mechanisms.

Despite the reduced incidence of gastric cancer in the developed world, a diagnosis of stomach carcinoma still carries a poor prognosis due to the asymptomatic nature of the disease in the early stages, subsequent advanced stage diagnosis, and a low 5 year survival rate. Endoscopy remains the primary standard for diagnosis of stomach carcinoma and the current marker, carbohydrate antigen 19-9 (CA19-9) lacks the levels of sensitivity and specificity required in order to make it clinically useful for diagnostic monitoring. Therefore, there is a current need for additional markers to improve the diagnostic accuracy for the early stages of stomach cancer. Together, glycomic, proteomic, and glycoproteomic analyses of serum have the potential to identify such probable markers. A discovery study is reported here using preoperative serum from 80 stomach cancer patients, 10 patients bearing benign stomach disease, and 20 matched controls. Glycomic analysis of the total and immunoaffinity depleted serum revealed statistically significant increases in the levels of sialyl Lewis X epitopes (SLe(X)) present on triantennary glycans accompanied by increased levels of core fucosylated agalactosyl biantennary glycans present on IgG (referred to as the IgG G0 glycoform) which are associated with increasing disease pathogenesis. Protein expression analysis using 2D-DiGE returned a number of differentially expressed protein candidates in the depleted serum, many of which were shown to carry triantennary SLe(X) during subsequent glycomic investigations. Biological pathway analysis of the experimental data returned complement activation and acute phase response signaling as the most significantly altered pathways in the stomach cancer patient serum. Upon the basis of these findings, it is suggested that increased expression of IgG G0 and complement activation are a host response to the presence of the stomach tumor while the increased expression of SLe(X) and acute phase response proteins is a result of pro-inflammatory cytokine signaling, including IL-6, during carcinogenesis. The approach presented herein provides an insight into the underlying mechanisms of disease and the resulting changes in the glycome and glycoproteome offer promise as potential markers for diagnosis and prognostic monitoring in stomach cancer.

Two-dimensional reference map for the basic proteome of the human dorsolateral prefrontal cortex (dlPFC) of the prefrontal lobe region of the brain.

We describe a 2-DE proteomic reference map containing 227 basic proteins in the dorsolateral prefrontal cortex region of the human brain. Proteins were separated in the first dimension on pH 6-11 IPG strips using paper-bridge loading and on 12% SDS-PAGE in the second dimension. Proteins were subsequently identified by MS and spectra were analyzed using an in-house proteomics data analysis platform, Proline. The 2-DE reference map is available via the UCD 2-DE Proteome Database (http://proteomics-portal.ucd.ie:8082) and can also be accessed via the WORLD-2DPAGE Portal (http://www.expasy.ch/world-2dpage/). The associated protein identification data have been submitted to the PRIDE database (accession numbers 10018-10033). Separation of proteins in the basic region resolves more membrane associated proteins relevant to the synaptic pathology central to many neurological disorders. The 2-DE reference map will aid with further characterisation of neurological disorders such as bipolar and schizophrenia.

Effect of phosphorylated hsp27 on proliferation of human endothelial and smooth muscle cells.

Recent studies have suggested a protective role of hsp27 against atherosclerosis and transplant graft vasculopathy. Here we have investigated the effects of over-expression of wild-type hsp27 and its phosphorylation mimics on proliferation of human endothelial cells (ECs) and smooth muscle cells (SMCs). ECs and SMCs cultured from human blood vessels or cells lines (human microvascular endothelial cell line and human telomerase reverse transcriptase subunit SMC) were infected with adenovirus containing DNA from wild-type hsp27, hyper-phosphorylated hsp27 mimic (3D hsp27), hypo-phosphorylated hsp27 mimic (3A hsp27) or anti-sense hsp27, and proliferation measured over the next 5 days. Protein extracts from infected cells were subjected to proteomic analysis using 2-D DIGE. Over-expression of 3D hsp27 and anti-sense hsp27 but not 3A hsp27 mimic caused significant inhibition of proliferation of ECs and SMCs. Proteomic analysis focussed on proteins that were significantly down-regulated by the 3D hsp27 mutant. The cell cycling proteins stathmin, cofilin and ubiquitination enzymes fullfilled these criteria. 1-D Western blots of infected human microvascular endothelial cell line and human telomerase reverse transcriptase subunit SMC confirmed down-regulation of stathmin, cofilin and ubiquitination enzymes by 3D hsp27. The phosphorylation status of hsp27 is an important regulator of proliferation of human vascular ECs and SMCs; possibly contributing to cardiovascular protection.

A fluorescent codetection system for immunoblotting and proteomics through ECL-Plex and CyDye labeling.

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis has been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with the recently developed ECL-Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27-kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.

1st Irish proteomics workshop April 17th 2008, University College Dublin, Conway Institute, Dublin, Ireland.

The workshop assembled an excellent collection of speakers from across Ireland and beyond who presented many interesting and diverse topical issues. Various proteomic applications were discussed throughout the day ranging from 2-DE and 2-D DIGE, to GeLC-MS/MS, high density Protein and Antibody Arrays, with a particular focus on the importance of quantitative mass spectrometry in proteomics.

From genome to proteome: back to the future. Report on the 7th Siena meeting, September 3-7, 2006.

This report reviews the 7th Siena Meeting 'From Genome to Proteome: Back to the Future' which took place in Italy from 3-7 September, 2006. There was a significant rise in the number of delegates attending compared with previous Siena meetings. A diversity of speakers and presentations addressed the theme of the meeting in moving proteomics forward to integrate with biology as a whole entity rather than in isolated fractions. In addition, technological advancements in sample preparation and separation as well as identification were discussed.

CyDye immunoblotting for proteomics: co-detection of specific immunoreactive and total protein profiles.

The development of ECL-Plex CyDye-conjugated secondary antibodies allows the advancement of conventional Western blotting, opening up possibilities for highly sensitive and quantitative protein confirmation and identification. We report a novel proteomic method to simultaneously visualise the total protein profile as well as the specific immunodetection of an individual protein species by combining cyanine CyDye pre-labelled proteins and antibody immunoblotting. This technique proposes to revolutionise both 2-D immunoprobing and protein confirmation following MS analysis.