Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays

Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody''s linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on- and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.Antibodies are used in proteomics both as imaging reagents for the analysis of tissue specificity (1) and subcellular localization (2) and as capturing agents for targeted proteomics (3), in particular for the enrichment of peptides for immunoaffinity methods such as Stable Isotope Standards and Capture by Anti-peptide Antibodies (4). In fact, the Human Proteome Project (5) has announced that one of the three pillars of the project will be antibody-based, with one of the aims being to generate antibodies to at least one representative protein from all protein-coding genes. Knowledge about the binding site (epitope) of an antibody toward a target protein is thus important for gaining basic insights into antibody specificity and sensitivity and facilitating the identification and design of antigens to be used for reagents in proteomics, as well as for the generation of therapeutic antibodies and vaccines (1, 6). With over 20 monoclonal-antibody-based drugs now on the market and over 100 in clinical trials, the field of antibody therapeutics has become a central component of the pharmaceutical industry (7). One of the key parameters for antibodies includes the nature of the binding recognition toward the target, involving either linear epitopes formed by consecutive amino acid residues or conformational epitopes consisting of amino acids brought together by the fold of the target protein (8).A large number of methods have therefore been developed to determine the epitopes of antibodies, including mass spectrometry (9), solid phase libraries (10, 11), and different display systems (12–14) such as bacterial display (15) and phage display (16). The most common method for epitope mapping involves the use of soluble and immobilized (tethered) peptide libraries, often in an array format, exemplified by the “Geysen Pepscan” method (11) in which overlapping “tiled” peptides are synthesized and used for binding analysis. The tiled peptide approach can also be combined with alanine scans (17) in which alanine substitutions are introduced into the synthetic peptides and the direct contribution of each amino acid can be investigated. Maier et al. (18) described a high-throughput epitope-mapping screen of a recombinant peptide library consisting of a total of 2304 overlapping peptides of the vitamin D receptor, and recently Buus et al. (19) used in situ synthesis on microarrays to design and generate 70,000 peptides for epitope mapping of antibodies using a range of peptides with sizes from 4-mer to 20-mer.So far it has not been possible to investigate on- and off-target binding in a proteome-wide manner, but the emergence of new methods for in situ synthesis of peptides on ultra-dense arrays has made this achievable. Here, we describe the design and use of peptide arrays generated with parallel in situ photolithic synthesis (20) of a total of 2.1 million overlapping peptides covering all human proteins with overlapping peptides. Miniaturization of the peptide arrays (21) has led to improved density of the synthesized peptides and consequently has improved the resolution and coverage of the epitope mapping. This has allowed us to study the specificity and cross-reactivity of both monoclonal and polyclonal antibodies across the whole “epitome” with the use of both proteome-wide arrays and focused-content peptide arrays covering selected antigen sequences to precisely map the contribution of each amino acid of the target protein for binding recognition of the corresponding antibodies. The results show the usefulness of proteome-wide epitope mapping, showing a path forward for high-throughput analysis of antibody interactions.     

When It Hurts (and Helps) to Try: The Role of Effort in Language Learning

Compared to children, adults are bad at learning language. This is counterintuitive; adults outperform children on most measures of cognition, especially those that involve effort (which continue to mature into early adulthood). The present study asks whether these mature effortful abilities interfere with language learning in adults and further, whether interference occurs equally for aspects of language that adults are good (word-segmentation) versus bad (grammar) at learning. Learners were exposed to an artificial language comprised of statistically defined words that belong to phonologically defined categories (grammar). Exposure occurred under passive or effortful conditions. Passive learners were told to listen while effortful learners were instructed to try to 1) learn the words, 2) learn the categories, or 3) learn the category-order. Effortful learners showed an advantage for learning words while passive learners showed an advantage for learning the categories. Effort can therefore hurt the learning of categories.     

Evolutionary Origins of C-Terminal (GPP)n 3-Hydroxyproline Formation in Vertebrate Tendon Collagen

Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPP)_n) in addition to the fully occupied A1 site at Pro986. The C-terminal (GPP)_n motif has five consecutive GPP triplets in α1(I), four in α2(I) and three in α1(II), all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin) and type II collagen (cartilage and notochord) were examined by mass spectrometry. The (GPP)_n domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human), up to five 3-hydroxyproline residues per (GPP)_n motif were found in α1(I) and four in α2(I), with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPP)_n site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.     

An Assessment of the Measurement Equivalence of English and French Versions of the Center for Epidemiologic Studies Depression (CES-D) Scale in Systemic Sclerosis

Objectives

Center for Epidemiologic Studies Depression (CES-D) Scale scores in English- and French-speaking Canadian systemic sclerosis (SSc) patients are commonly pooled in analyses, but no studies have evaluated the metric equivalence of the English and French CES-D. The study objective was to examine the metric equivalence of the CES-D in English- and French-speaking SSc patients.

Methods

The CES-D was completed by 1007 English-speaking and 248 French-speaking patients from the Canadian Scleroderma Research Group Registry. Confirmatory factor analysis (CFA) was used to assess the factor structure in both samples. The Multiple-Indicator Multiple-Cause (MIMIC) model was utilized to assess differential item functioning (DIF).

Results

A two-factor model (Positive and Negative affect) showed excellent fit in both samples. Statistically significant, but small-magnitude, DIF was found for 3 of 20 CES-D items, including items 3 (Blues), 10 (Fearful), and 11 (Sleep). Prior to accounting for DIF, French-speaking patients had 0.08 of a standard deviation (SD) lower latent scores for the Positive factor (95% confidence interval [CI]−0.25 to 0.08) and 0.09 SD higher scores (95% CI−0.07 to 0.24) for the Negative factor than English-speaking patients. After DIF correction, there was no change on the Positive factor and a non-significant increase of 0.04 SD on the Negative factor for French-speaking patients (difference = 0.13 SD, 95% CI−0.03 to 0.28).

Conclusions

The English and French versions of the CES-D, despite minor DIF on several items, are substantively equivalent and can be used in studies that combine data from English- and French-speaking Canadian SSc patients.     

Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.     

Late mitotic failure in mice lacking Sak, a polo-like kinase

Polo-like kinases in yeast, flies, and mammals regulate key events in mitosis. Such events include spindle formation at G2/M, the anaphase-promoting complex (APC) at the exit from mitosis, the cleavage structure at cytokinesis, and DNA damage checkpoints in G2/M. Polo-like kinases are distinguished by two C-terminal polo box (pb) motifs, which localize the enzymes to mitotic structures. We previously identified Sak, a novel polo-like kinase found in Drosophila and mammals. Here, we demonstrate that the Sak kinase has a functional pb domain that localizes the enzyme to the nucleolus during G2, to the centrosomes in G2/M, and to the cleavage furrow during cytokinesis. To study the role of Sak in embryo development, we generated a Sak null allele, the first polo-like kinase to be mutated in mice. Sak(-/-) embryos arrested after gastrulation at E7.5, with a marked increase in mitotic and apoptotic cells. Sak(-/-) embryos displayed cells in late anaphase or telophase that continued to express cyclin B1 and phosphorylated histone H3. Our results suggest that Sak is required for the APC-dependent destruction of cyclin B1 and for exit from mitosis in the postgastrulation embryo.     

Transcriptional CDK inhibitors,CYC065 and THZ1 promote Bim-dependent apoptosis in primary and recurrent GBM through cell cycle arrest and Mcl-1 downregulation

Activation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation or overexpression of CDKs can support tumourigenesis including glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. The incurability of GBM highlights the need to discover novel and more effective treatment options. Since CDKs 2, 7 and 9 were found to be overexpressed in GBM, we tested the therapeutic efficacy of two CDK inhibitors (CKIs) (CYC065 and THZ1) in a heterogeneous panel of GBM patient-derived cell lines (PDCLs) cultured as gliomaspheres, as preclinically relevant models. CYC065 and THZ1 treatments suppressed invasion and induced viability loss in the majority of gliomaspheres, irrespective of the mutational background of the GBM cases, but spared primary cortical neurons. Viability loss arose from G2/M cell cycle arrest following treatment and subsequent induction of apoptotic cell death. Treatment efficacies and treatment durations required to induce cell death were associated with proliferation velocities, and apoptosis induction correlated with complete abolishment of Mcl-1 expression, a cell cycle-regulated antiapoptotic Bcl-2 family member. GBM models generally appeared highly dependent on Mcl-1 expression for cell survival, as demonstrated by pharmacological Mcl-1 inhibition or depletion of Mcl-1 expression. Further analyses identified CKI-induced Mcl-1 loss as a prerequisite to establish conditions at which the BH3-only protein Bim can efficiently induce apoptosis, with cellular Bim amounts strongly correlating with treatment efficacy. CKIs reduced proliferation and promoted apoptosis also in chick embryo xenograft models of primary and recurrent GBM. Collectively, these studies highlight the potential of these novel CKIs to suppress growth and induce cell death of patient-derived GBM cultures in vitro and in vivo, warranting further clinical investigation.Subject terms: Cancer models, Apoptosis, RNAi     

Are Scores on English and French Versions of the PHQ-9 Comparable? An Assessment of Differential Item Functioning

Background

Medical research increasingly utilizes patient-reported outcome measures administered and scored in different languages. In order to pool or compare outcomes from different language versions, instruments should be measurement equivalent across linguistic groups. The objective of this study was to examine the cross-language measurement equivalence of the Patient Health Questionnaire-9 (PHQ-9) between English- and French-speaking Canadian patients with systemic sclerosis (SSc).

Methods

The sample consisted of 739 English- and 221 French-speaking SSc patients. Multiple-Indicator Multiple-Cause (MIMIC) modeling was used to identify items displaying possible differential item functioning (DIF).

Results

A one-factor model for the PHQ-9 fit the data well in both English- and French-speaking samples. Statistically significant DIF was found for 3 of 9 items on the PHQ-9. However, the overall estimate in depression latent scores between English- and French-speaking respondents was not influenced substantively by DIF.

Conclusions

Although there were several PHQ-9 items with evidence of minor DIF, there was no evidence that these differences influenced overall scores meaningfully. The PHQ-9 can reasonably be used without adjustment in Canadian English- and French-speaking samples. Analyses assessing measurement equivalence should be routinely conducted prior to pooling data from English and French versions of patient-reported outcome measures.