Alex D. Colella

Comparison of Stain-Free gels with traditional immunoblot loading control methodology.

Loading controls are necessary for semiquantitative Western blotting to compensate for loading errors. Loading control methods include the reprobing of membranes with an antibody against a constitutively expressed protein or staining the membrane with a total protein stain. We compared the loading control performance of recently released Stain-Free (SF) gels with Sypro Ruby (SR) and reprobing using β-actin. SF gels demonstrated superior performance in that they were faster, required fewer steps and consumables, and allowed the quality of electrophoresis and Western transfer to be assessed before committing to costly and time-consuming Western blots.

Drosophila orthologue of WWOX, the chromosomal fragile site FRA16D tumour suppressor gene, functions in aerobic metabolism and regulates reactive oxygen species

Common chromosomal fragile sites FRA3B and FRA16D are frequent sites of DNA instability in cancer, but their contribution to cancer cell biology is not yet understood. Genes that span these sites (FHIT and WWOX, respectively) are often perturbed (either increased or decreased) in cancer cells and both are able to suppress tumour growth. While WWOX has some tumour suppressor characteristics, its normal role and functional contribution to cancer has not been fully determined. We find that a significant proportion of Drosophila Wwox interactors identified by proteomics and microarray analyses have roles in aerobic metabolism. Functional relationships between Wwox and either CG6439/isocitrate dehydrogenase (Idh) or Cu–Zn superoxide dismutase (Sod) were confirmed by genetic interactions. In addition, altered levels of Wwox resulted in altered levels of endogenous reactive oxygen species. Wwox (like FHIT) contributes to pathways involving aerobic metabolism and oxidative stress, providing an explanation for the ‘non-classical tumour suppressor’ behaviour of WWOX. Fragile sites, and the genes that span them, are therefore part of a protective response mechanism to oxidative stress and likely contributors to the differences seen in aerobic glycolysis (Warburg effect) in cancer cells.

An immunodominant La/SSB autoantibody proteome derives from public clonotypes

The La/SSB autoantigen is a major target of long‐term humoral autoimmunity in primary Sjögrens Syndrome (SS) and systemic lupus erythematosus. A majority of patients with linked anti‐Ro60/Ro52/La responses target an NH2‐terminal epitope designated LaA that is expressed on Ro/La ribonucleoprotein complexes and the surface membrane of apoptotic cells. In this study, we used high‐resolution Orbitrap mass spectrometry to determine the clonality, isotype and V‐region sequences of LaA‐specific autoantibodies in seven patients with primary SS. Anti‐LaA immunoglobulin (Ig)Gs purified from polyclonal sera by epitope‐specific affinity chromatography were analysed by combined database and de‐novo mass spectrometric sequencing. Autoantibody responses comprised two heavily mutated IgG1 kappa‐restricted monoclonal species that were shared (public) across unrelated patients; one clonotype was specified by an IGHV3‐30 heavy chain paired with IGKV3‐15 light chain and the second by an IGHV3‐43/IGKV3‐20 pairing. Shared amino acid replacement mutations were also seen within heavy and light chain complementarity‐determining regions, consistent with a common breach of B cell tolerance followed by antigen‐driven clonal selection. The discovery of public clonotypic autoantibodies directed against an immunodominant epitope on La, taken together with recent findings for the linked Ro52 and Ro60 autoantigens, supports a model of systemic autoimmunity in which humoral responses against protein–RNA complexes are mediated by public sets of autoreactive B cell clonotypes.

Secreted autoantibody repertoires in Sjögren's syndrome and systemic lupus erythematosus: A proteomic approach.

The structures of epitopes bound by autoantibodies against RNA-protein complexes have been well-defined over several decades, but little is known of the clonality, immunoglobulin (Ig) variable (V) gene usage and mutational status of the autoantibodies themselves at the level of the secreted (serum) proteome. A novel proteomic workflow is presented based on affinity purification of specific Igs from serum, high-resolution two-dimensional gel electrophoresis, and de novo and database-driven sequencing of V-region proteins by mass spectrometry. Analysis of anti-Ro52/Ro60/La proteomes in primary Sjögrens syndrome (SS) and anti-Sm and anti-ribosomal P proteomes in systemic lupus erythematosus (SLE) has revealed that these antibody responses are dominated by restricted sets of public (shared) clonotypes, consistent with common pathways of production across unrelated individuals. The discovery of shared sets of specific V-region peptides can be exploited for diagnostic biomarkers in targeted mass spectrometry platforms and for tracking and removal of pathogenic clones.

Lupus anti-ribosomal P autoantibody proteomes express convergent biclonal signatures

Lupus‐specific anti‐ribosomal P (anti‐Rib‐P) autoantibodies have been implicated in the pathogenesis of neurological complications in systemic lupus erythematosus (SLE). The aim of the present study was to determine variable (V)‐region signatures of secreted autoantibody proteomes specific for the Rib‐P heterocomplex and investigate the molecular basis of the reported cross‐reactivity with Sm autoantigen. Anti‐Rib‐P immunoglobulins (IgGs) were purified from six anti‐Rib‐P‐positive sera by elution from enzyme‐linked immunosorbent assay (ELISA) plates coated with either native Rib‐P proteins or an 11‐amino acid peptide (11‐C peptide) representing the conserved COOH‐terminal P epitope. Rib‐P‐ and 11‐C peptide‐specific IgGs were analysed for heavy (H) and light (L) chain clonality and V‐region expression using an electrophoretic and de‐novo and database‐driven mass spectrometric sequencing workflow. Purified anti‐Rib‐P and anti‐SmD IgGs were tested for cross‐reactivity on ELISA and their proteome data sets analysed for shared clonotypes. Anti‐Rib‐P autoantibody proteomes were IgG1 kappa‐restricted and comprised two public clonotypes defined by unique H/L chain pairings. The major clonotypic population was specific for the common COOH‐terminal epitope, while the second shared the same pairing signature as a recently reported anti‐SmD clonotype, accounting for two‐way immunoassay cross‐reactivity between these lupus autoantibodies. Sequence convergence of anti‐Rib‐P proteomes suggests common molecular pathways of autoantibody production and identifies stereotyped clonal populations that are thought to play a pathogenic role in neuropsychiatric lupus. Shared clonotypic structures for anti‐Rib‐P and anti‐Sm responses suggest a common B cell clonal origin for subsets of these lupus‐specific autoantibodies.

Extended boiling of peanut progressively reduces IgE allergenicity while retaining T cell reactivity.

Current peanut oral immunotherapy is hampered by frequent adverse events. It has been shown that boiling can reduce peanut allergenicity. Hypoallergenic peanut products have the potential to reduce treatment‐related reactions during desensitization.

Purification of α-synuclein containing inclusions from human post mortem brain tissue.

UNLABELLED Comparison with existing methods. BACKGROUND Neurodegenerative disorders affect a large proportion of the elderly population. A group of disorders, known as the α-synucleinopathies, are characterised by the presence of α-synuclein-containing protein inclusions, such as Lewy Bodies (LBs) found in neurons from Parkinsons Disease (PD) and Dementia with Lewy Bodies (DLB), and Glial Cytoplasmic Inclusions (GCIs) found in oligodendrocytes from Multiple System Atrophy (MSA). The analysis of the protein composition of inclusions has been hindered by limitations of methods for isolating the inclusions from the surrounding tissue. METHOD Four modifications were made to the published method for GCI purification by Gai et al. (1999) which were: collecting the entire inclusion-containing part of the Percoll gradient; lysis of nuclei prior to DNAse digestion; limited tryptic digestion to release inclusions from the cytoskeletal meshwork; and increased antibody and magnetic bead concentrations/volumes to capture the larger amounts of inclusions. RESULTS The optimised method gave a 28-fold increase in yield compared to the published method of Gai et al. (1999). A 2D-DIGE comparison revealed a 3.8-fold increase in α-synuclein enrichment and a corresponding 5.2-fold reduction in tubulin contamination. This method was also successfully adapted to the purification of LBs from DLB tissue. A 2D-DIGE comparison of purified GCIs (n=2) revealed that GCIs consist of 11.7% α-synuclein, 1.9% α-β-crystallin and 2.3% 14-3-3 proteins compared to 8.5%, 2.0% and 1.5% in LBs, respectively. CONCLUSIONS This study has generated an improved method for the purification of α-synuclein-containing inclusions with a yield sufficient for multiple forms of analysis.

IgV peptide mapping of native Ro60 autoantibody proteomes in primary Sjögren's syndrome reveals molecular markers of Ro/La diversification.

We have used high-resolution mass spectrometry to sequence precipitating anti-Ro60 proteomes from sera of patients with primary Sjögrens syndrome and compare immunoglobulin variable-region (IgV) peptide signatures in Ro/La autoantibody subsets. Anti-Ro60 were purified by elution from native Ro60-coated ELISA plates and subjected to combined de novo amino acid sequencing and database matching. Monospecific anti-Ro60 Igs comprised dominant public and minor private sets of IgG1 kappa and lambda restricted heavy and light chains. Specific IgV amino acid substitutions stratified anti-Ro60 from anti-Ro60/La responses, providing a molecular fingerprint of Ro60/La determinant spreading and suggesting that different forms of Ro60 antigen drive these responses. Sequencing of linked anti-Ro52 proteomes from individual patients and comparison with their anti-Ro60 partners revealed sharing of a dominant IGHV3-23/IGKV3-20 paired clonotype but with divergent IgV mutational signatures. In summary, anti-Ro60 IgV peptide mapping provides insights into Ro/La autoantibody diversification and reveals serum-based molecular markers of humoral Ro60 autoimmunity.

Molecular Profiling and Clonal Tracking of Secreted Rheumatoid Factors in Primary Sjögren's Syndrome

Rheumatoid factors (RFs) are associated with systemic disease in primary Sjögrens syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane‐bound IgM‐RF in primary SS and identify unique heavy‐chain peptide signatures for RF clonotype tracking.

Proteomic analysis of influenza haemagglutinin-specific antibodies following vaccination reveals convergent immunoglobulin variable region signatures

Analysis of the anti-haemagglutinin serum antibody proteome from six H1N1pdm09 influenza A vaccinated subjects demonstrated restricted IgG1 heavy chain species encoded by IGHV5-51 and IGHV3-7 gene families in 2 subjects and either IGHV5-51 or IGHV3-7 in 4 individuals. All subjects exhibited a dominant IGKV3-20 light chain, however 5 subjects also exhibited IGKV3-11 and IGKV4-1 families. Sequences were closely aligned with the matched germline sequence, with few shared mutations. This study illustrates the feasibility of using a proteomic approach to determine the expressed V region signatures of serum antibodies induced by vaccination.