Quality control of allergen products with mass spectrometry part I: Positioning within the EU regulatory framework.

Mass spectrometry (MS) has advanced greatly and many of its applications are ready for utilization within regulatory procedures and could significantly contribute to overcome challenges in standardization of allergen products. It seems sensible to discuss MS within the regulatory framework, before addressing technical questions. While the application to purified proteins is well established from product development to manufacturer's release analytics, its application to complex products such as allergen products is still under development. It needs to be determined where it can complement or replace established methods or where MS offers limited improvement. Despite its technical appeal and versatility, currently MS is mentioned in regulatory guidelines only as one possible measurement method. For example, no specific MS method is given in the European Pharmacopoeia. We discuss applications of MS within the EU regulatory framework. This includes their advantages and disadvantages and their positioning between research, characterization, manufacturer's release analytics and official batch testing. We discuss the qualitative detection of single and multiple allergens as proof of identity, qualitative to semi-quantitative protein profiles for batch to batch consistency testing, and quantification of allergens to state mass units of allergens. MS may also facilitate standardization of allergen products, reference products and reference standards.

Poor transcript-protein correlation in the brain: negatively correlating gene products reveal neuronal polarity as a potential cause.

Transcription, translation, and turnover of transcripts and proteins are essential for cellular function. The contribution of those factors to protein levels is under debate, as transcript levels and cognate protein levels do not necessarily correlate due to regulation of translation and protein turnover. Here we propose neuronal polarity as a third factor that is particularly evident in the CNS, leading to considerable distances between somata and axon terminals. Consequently, transcript levels may negatively correlate with cognate protein levels in CNS regions, i.e., transcript and protein levels behave reciprocally. To test this hypothesis, we performed an integrative inter-omics study and analyzed three interconnected rat auditory brainstem regions (cochlear nuclear complex, CN; superior olivary complex, SOC; inferior colliculus, IC) and the rest of the brain as a reference. We obtained transcript and protein sets in these regions of interest (ROIs) by DNA microarrays and label-free mass spectrometry, and performed principal component and correlation analyses. We found 508 transcript|protein pairs and detected poor to moderate transcript|protein correlation in all ROIs, as evidenced by coefficients of determination from 0.34 to 0.54. We identified 57-80 negatively correlating gene products in the ROIs and intensively analyzed four of them for which the correlation was poorest. Three cognate proteins (Slc6a11, Syngr1, Tppp) were synaptic and hence candidates for a negative correlation because of protein transport into axon terminals. Thus, we systematically analyzed the negatively correlating gene products. Gene ontology analyses revealed overrepresented transport/synapse-related proteins, supporting our hypothesis. We present 30 synapse/transport-related proteins with poor transcript|protein correlation. In conclusion, our analyses support that protein transport in polar cells is a third factor that influences the protein level and, thereby, the transcript|protein correlation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* and *Open Data* because it provided all relevant information to reproduce the study in the manuscript and because it made the data publicly available. The data can be accessed at https://osf.io/ha28n/. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Quality and potency profile of eight recombinant isoallergens, largely mimicking total Bet v 1-specific IgE binding of birch pollen.

BACKGROUND: To date, only limited information on structure, expression levels and IgE binding of Bet v 1 variants, which are simultaneously expressed in birch pollen, is available. OBJECTIVE: To analyse and compare structure and serum IgE/IgG binding of rBet v 1 variants to Bet v 1.0101. METHODS: Recombinant Bet v 1 variants were studied with sera of 20 subjects allergic to birch pollen. Folding, aggregation and solubility of the rBet v 1 variants were analysed to attribute diverging IgE binding to either allergen structure or methodological features. IgE/IgG binding was studied with rBet v 1 in solution or adsorbed to solid phases. Allergen-mediated cross-linking of FcεRI receptors was determined by mediator release of sensitized humanized rat basophil leukaemia cells. RESULTS: All variants, except for rBet v 1.0113, were monomeric and had Bet v 1-type conformation. Serum IgE binding to variants adsorbed to solid phase was reduced to 6.6%-36.5% compared with Bet v 1.0101. In contrast, inhibition of IgE binding to Bet v 1.0101 by rBet v 1 variants ranged from 62% to 83%. Similarly, mediator release ranged from 30.7% to 55.2% for all variants and was only clearly reduced for rBet v 1.0301 (10.4%). The IgE-binding potency of rBet v 1 variants representing their native quantities in birch pollen was only slightly lower compared to extract. IgG binding to variants was between 50.9% and 134.5% compared with rBet v 1.0101 (100%). CONCLUSION AND CLINICAL RELEVANCE: Bet v 1 variants previously classified as hypoallergenic can exhibit similar functional IgE binding as Bet v 1.0101. Eight rBet v 1 variants largely reproduce total Bet v 1-specific IgE binding of birch pollen extracts. Assay format-dependent variation in IgE-binding properties needs to be considered in the development of diagnostic or therapeutic products.

Model for Quality Control of Allergen Products with Mass Spectrometry.

Birch pollen allergy is diagnosed and treated with aqueous extracts from birch pollen, which contain a mixture of allergens and nonallergenic proteins, including large numbers of closely related sequence variants, so-called iso-allergens of the major allergen, Bet v 1. The quality of therapeutic and diagnostic allergen products largely depends on the allergen and iso-allergen composition. Several biochemical methods are currently applied to detect and quantify allergens and to record protein profiles without differentiating between iso-allergens. Mass spectrometry (MS) may entirely replace these technologies, as it allows sequence specific identification and quantification of proteins and protein profiles including sequence variants in one run. However, the protein inference problem still hampers the automatic assignment of peptide sequences to proteins, consequently impeding the quantification of sequence variants. Therefore, the aim of the study was to set up semitargeted analyses of label-free MS data that allow unambiguous identification and quantification of birch pollen allergens and nonallergenic proteins. We combined data independent acquisition with manual assignment of predefined target sequences for quantification of iso-allergens and automatic quantification of other allergens and nonallergenic proteins. The quantitative data for birch pollen allergens and sequence variants of Bet v 1 were further confirmed by multiple reaction monitoring.

Epicocconone staining: a powerful loading control for Western blots.

Western blot analysis is routinely employed for quantifying differences in protein levels between samples. To control equal loading and to arithmetically compensate loading differences, immunodetection of housekeeping proteins is commonly used. Due to potential biases, this approach has been criticized. Here, we evaluate epicocconone-based total protein staining (E-ToPS) as an alternative. We compared it with two other total protein stainings (Coomassie and Sypro Ruby) and with immunodetection of housekeeping proteins (ß-tubulin and glyceraldehyde 3-phosphate dehydrogenase). Evaluation comprised both the natural and the synthetic epicocconone compound. Both compounds produced highly congruent results and showed more sensitive (≤ 1 µg) and less variable staining properties than the other variants. The high sensitivity of E-ToPS, covering minute protein amounts, makes it a powerful loading control, especially for precious samples. Regarding biological and technical variances, E-ToPS outperformed immunostaining against ß-tubulin and glyceraldehyde 3-phosphate dehydrogenase. Furthermore, E-ToPS had no impact on subsequent immunodetection, allowing for an early control of proper loading prior to immunodetection. In contrast to earlier studies, we found logarithmic staining properties for E-ToPS, which should be considered when using it for arithmetic normalization. In conclusion, we demonstrate the superior power of E-ToPS as a loading control for Western blots.

Keratin 23, a novel DPC4/Smad4 target gene which binds 14-3-3ε.

BACKGROUND: Inactivating mutations of SMAD4 are frequent in metastatic colorectal carcinomas. In previous analyses, we were able to show that restoration of Smad4 expression in Smad4-deficient SW480 human colon carcinoma cells was adequate to suppress tumorigenicity and invasive potential, whereas in vitro cell growth was not affected. Using this cellular model system, we searched for new Smad4 targets comparing nuclear subproteomes derived from Smad4 re-expressing and Smad4 negative SW480 cells. METHODS: High resolution two-dimensional (2D) gel electrophoresis was applied to identify novel Smad4 targets in the nuclear subproteome of Smad4 re-expressing SW480 cells. The identified candidate protein Keratin 23 was further characterized by tandem affinity purification. Immunoprecipitation, subfractionation and immunolocalization studies in combination with RNAi were used to validate the Keratin 23-14-3-3ε interaction. RESULTS: We identified keratins 8 and 18, heat shock proteins 60 and 70, plectin 1, as well as 14-3-3ε and γ as novel proteins present in the KRT23-interacting complex. Co-immunoprecipitation and subfractionation analyses as well as immunolocalization studies in our Smad4-SW480 model cells provided further evidence that KRT23 associates with 14-3-3ε and that Smad4 dependent KRT23 up-regulation induces a shift of the 14-3-3ε protein from a nuclear to a cytoplasmic localization. CONCLUSION: Based on our findings we propose a new regulatory circuitry involving Smad4 dependent up-regulation of KRT23 (directly or indirectly) which in turn modulates the interaction between KRT23 and 14-3-3ε leading to a cytoplasmic sequestration of 14-3-3ε. This cytoplasmic KRT23-14-3-3 interaction may alter the functional status of the well described 14-3-3 scaffold protein, known to regulate key cellular processes, such as signal transduction, cell cycle control, and apoptosis and may thus be a previously unappreciated facet of the Smad4 tumor suppressive circuitry.

Publisher Correction: Optimizing the dynamics of protein expression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Optimizing the dynamics of protein expression.

Heterologously expressed genes require adaptation to the host organism to ensure adequate levels of protein synthesis, which is typically approached by replacing codons by the target organism's preferred codons. In view of frequently encountered suboptimal outcomes we introduce the codon-specific elongation model (COSEM) as an alternative concept. COSEM simulates ribosome dynamics during mRNA translation and informs about protein synthesis rates per mRNA in an organism- and context-dependent way. Protein synthesis rates from COSEM are integrated with further relevant covariates such as translation accuracy into a protein expression score that we use for codon optimization. The scoring algorithm further enables fine-tuning of protein expression including deoptimization and is implemented in the software OCTOPOS. The protein expression score produces competitive predictions on proteomic data from prokaryotic, eukaryotic, and human expression systems. In addition, we optimized and tested heterologous expression of manA and ova genes in Salmonella enterica serovar Typhimurium. Superiority over standard methodology was demonstrated by a threefold increase in protein yield compared to wildtype and commercially optimized sequences.

pH and Heat Resistance of the Major Celery Allergen Api g 1.

SCOPE: The major celery allergen Api g 1 is a member of the pathogenesis-related 10 class protein family. This study aims to investigate the impact of heat and pH on the native protein conformation required for Immunoglobulin E (IgE) recognition. METHODS AND RESULTS: Spectroscopic methods, MS and IgE-binding analyses are used to study the effects of pH and thermal treatment on Api g 1.0101. Heat processing results in a loss of the native protein fold via denaturation, oligomerization, and precipitation along with a subsequent reduction of IgE recognition. The induced effects and timescales are strongly pH dependent. While Api g 1 refolds partially into an IgE-binding conformation at physiological pH, acidic pH treatment leads to the formation of structurally heat-resistant, IgE-reactive oligomers. Thermal processing in the presence of a celery matrix or at pH conditions close to the isoelectric point (pI = 4.63) of Api g 1.0101 results in almost instant precipitation. CONCLUSION: This study demonstrates that Api g 1.0101 is not intrinsically susceptible to heat treatment in vitro. However, the pH and the celery matrix strongly influence the stability of Api g 1.0101 and might be the main reasons for the observed temperature lability of this important food allergen.

How proteomics reveals potential biomarkers in brain diseases.

The brain is complex, and so are the proteomics studies of brain tissue and its diseases, including Alzheimer's Disease, Parkinson's Disease and schizophrenia. In this review, general considerations and strategies of proteomics technologies, the advantages and challenges as well as the special needs for brain tissue are described and summarized. In addition, the results of the first studies are presented including a quality evaluation of the candidate proteins for these diseases. A paragraph is dedicated to the efforts of standardization in this field.

Developmental changes of the protein repertoire in the rat auditory brainstem: a comparative proteomics approach in the superior olivary complex and the inferior colliculus with DIGE and iTRAQ.

Protein profiles of developing neural circuits undergo manifold changes. The aim of this proteomic analysis was to quantify postnatal changes in two auditory brainstem areas in a comparative approach. Protein samples from the inferior colliculus (IC) and the superior olivary complex (SOC) were obtained from neonatal (P4) and young adult (P60) rats. The cytosolic fractions of both areas were examined by 2-D DIGE, and the plasma membrane-enriched fraction of the IC was analyzed via iTRAQ. iTRAQ showed a regulation in 34% of the quantified proteins. DIGE revealed 12% regulated spots in both the SOC and IC and, thus, numeric congruency. Although regulation in KEGG pathways displayed a similar pattern in both areas, only 13 of 71 regulated DIGE proteins were regulated in common, implying major area-specific differences. 89% of regulated glycolysis/gluconeogenesis and citrate cycle proteins were up-regulated in the SOC or IC, suggesting a higher energy demand in adulthood. Seventeen cytoskeleton proteins were regulated, consistent with complex morphological reorganization between P4 and P60. Fourteen were uniquely regulated in the SOC, providing further evidence for area-specific differences. Altogether, we provide the first elaborate catalog of proteins involved in auditory brainstem development, several of them possibly of particular developmental relevance.

Chloride cotransporters, chloride homeostasis, and synaptic inhibition in the developing auditory system.

The role of glycine and GABA as inhibitory neurotransmitters in the adult vertebrate nervous system has been well characterized in a variety of model systems, including the auditory, which is particularly well suited for analyzing inhibitory neurotransmission. However, a full understanding of glycinergic and GABAergic transmission requires profound knowledge of how the precise organization of such synapses emerges. Likewise, the role of glycinergic and GABAergic signaling during development, including the dynamic changes in regulation of cytosolic chloride via chloride cotransporters, needs to be thoroughly understood. Recent literature has elucidated the developmental expression of many of the molecular components that comprise the inhibitory synaptic phenotype. An equally important focus of research has revealed the critical role of glycinergic and GABAergic signaling in sculpting different developmental aspects in the auditory system. This review examines the current literature detailing the expression patterns and function (chapter 1), as well as the regulation and pharmacology of chloride cotransporters (chapter 2). Of particular importance is the ontogeny of glycinergic and GABAergic transmission (chapter 3). The review also surveys the recent work on the signaling role of these two major inhibitory neurotransmitters in the developing auditory system (chapter 4) and concludes with an overview of areas for further research (chapter 5).

Proteome analysis of a plasma membrane-enriched fraction at the placental feto-maternal barrier.

PURPOSE: We want to identify proteins that are part of or associated with the plasma membrane of the human feto-maternal barrier, which is crucially important for nutrient, gas, and waste exchange between the mother and the fetus. All transfer processes occur through one specialized endothelial cell layer, the multinuclear syncytiotrophoblast (STB). Specifically, the apical plasma membrane of the STB interacts with the maternal blood and is the site of initial transport processes across the placenta. EXPERIMENTAL DESIGN: We used a proteomic approach that employed the enrichment of apical STB membranes isolated from healthy placentae by ultracentrifugation and saccharose gradient centrifugation steps in combination with 1-D SDS-PAGE and ESI-MS analysis. RESULTS: We identified 296 different proteins, 175 of which were integral and peripheral membrane proteins, partially containing 1-12 transmembrane domains or lipid anchors. One hundred and sixty-one proteins (54%) were allocated to the plasma membrane. CONCLUSIONS AND CLINICAL RELEVANCE: A high number of transporters, receptors, and proteins involved in signal transduction processes and vesicular trafficking were identified for the first time at the feto-maternal barrier. Our results are valuable sources for further studies of the cell physiology of the healthy placenta at the time of birth or the pathophysiology of several pregnancy disorders.

Analysis of the pancreatic tumor progression by a quantitative proteomic approach and immunhistochemical validation.

To increase the knowledge about the development of pancreatic ductal adenocarcinoma, (PDAC) detailed analysis of the tumor progression is required. To identify proteins differentially expressed in the pancreatic intraepithelial neoplasia (PanIN), the precursor lesions of PDAC, we conducted a quantitative proteome study on microdissected PanIN cells. Proteins from 1000 microdissected cells were subjected to a procedure combining fluorescence dye saturation labeling with high resolution two-dimensional gel electrophoresis (2-DE). Differentially regulated protein spots were identified using protein lysates from PDAC tissues as a reference proteome followed by nanoLC-ESI-MS/MS. Thirty-seven single lesions of different PanIN grade (PanIN 1A/B, PanIN 2, PanIN 3) from nine patients were analyzed. Their protein expression was compared with each other, with PDAC cells and with normal ductal cells. The differential expression of differentially regulated protein spots was validated by means of immunohistochemistry using tissue microarrays. Of 2500 protein spots, 86 were found to be significantly regulated (p < 0.05, ratio > 1.6) during PanIN progression. Thirty-one nonredundant proteins were identified by mass spectrometry. Immunohistochemistry revealed that the differential expression of the selected candidate proteins major vault protein (MVP), anterior gradient 2 (AGR 2) and 14-3-3 sigma, annexin A4, and S100A10 could be successfully validated in PanIN lesions. The highly sensitive and robust proteome analysis revealed differentially regulated proteins involved in pancreatic tumor progression. The analysis of normal preneoplastic and neoplastic pancreatic tissue establishes a basis for identification of candidate biomarkers in PanIN progression that can be detected in pancreatic juice and in serum or are candidates for in vivo imaging approaches.

'Proteomic basics--sample preparation and separation': the 1st European Summer School in Kloster Neustift, 12-18 August, 2007 Brixen/Bressanone, South Tyrol, Italy.

Proteomics is rapidly developing into a routine approach for protein analysis in many laboratories. The series of European-wide Summer Schools 'Proteomics Basics' (http://www.proteomic-basics.eu/) aims at teaching of comprehensive knowledge in proteomics research and applied technologies for master and graduate students and postdocs currently moving into the field of proteomic research. In the next 3 years the series will cover the theoretical basis of the fundamental topics in the various areas of proteomic analysis, i.e. sample preparation and handling, mass spectrometry, post-translational modifications and quantitation given by leading experts in the field. This summer school series embodies a unique advantage in comparison with conventional scientific meetings and university curricula: internationally renowned experts will give a detailed perspective view of the fundamentals of their particular proteome research area, something which is usually not encountered at conferences and congresses. Here, we give a report on the first European Summer School 'Sample Preparation and Handling' within the series 'Proteomic Basics' that was held at the monastery in Neustift close to Bressanone/Brixen, Italy from August 12 to 18, 2007.

The glomerular proteome in a model of chronic kidney disease.

Adequate kidney function is crucial in sustaining vertebrate homeostasis. Certain diseases can diminish renal function and lead to end-stage renal disease. Diabetes mellitus and hypertension are the main causes of glomerulosclerosis and albuminuria in adults. The molecular mechanisms that trigger these maladaptive changes are still unsatisfyingly described. We previously introduced 2-D DIGE in combination with focused tissue isolation methods to analyze protein expression in glomeruli. Glomeruli, the crucial compartments in albuminuric renal diseases, were extracted using magnetic particles from subtotally nephrectomized FVB mice (n = 6); this 5/6 nephrectomy in FVB mice is a model of chronic kidney disease. Analysis of protein expression levels from glomerular protein lysates was performed using 2-D DIGE and compared with glomerular protein lysates from mice that underwent sham surgery. The comparison of about 2100 detectable spots between both groups revealed 48 protein spots that showed significant differential expression. Of those, 33 proteins could be identified using nanoLC-ESI MS. The metalloproteinase meprin 1 alpha, the beta galactoside-binding-lectin galectin-1 and dimethylarginine dimethylaminohydrolase 1, a key enzyme in NO metabolism, were found to be differentially regulated, thus implying a role in the pathogenesis and pathophysiology of progressive kidney disease. In conclusion, 2-D DIGE protein analysis of smallest sample sizes from specific organ compartments provides focused protein expression results, which help in gaining an understanding of the molecular mechanisms of chronic kidney disease.

Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling.

Loss of renal function is often associated with the injury of kidney glomeruli. It is therefore necessary to understand the mechanisms leading to progressive glomerular diseases; this may be addressed using proteomics. Until now, however, analysis of the glomeruli proteome using 2-DE has been technically hampered by low protein yields from scarce samples. To circumvent this problem, we developed a procedure which allows the human and mouse glomeruli proteome to be analysed. In this study, two different approaches were used to isolate mouse and human glomerular protein from kidney cortex. Mouse glomeruli were extracted by embolisation magnetic beads into the glomerular capillaries. Laser capture microdissection (LCM) was utilised to harvest glomeruli from human biopsy material. Human and murine samples were analysed using a fluorescence saturation labelling technique. Using 3 microg mouse glomerular protein a total of 2900 spots were resolved for differential proteome analysis. Moreover, it was also demonstrated for the first time that only ten glomeruli (0.5 microg) picked by LCM from a slide of a human kidney biopsy material were sufficient to visualise 900 spots. This novel strategy paves the way for future experiments aimed at investigating functional proteomics of glomerular diseases in humans and in mice.

2-D differential membrane proteome analysis of scarce protein samples.

Proteome studies with small sample amounts are difficult to perform, especially when membrane proteins are the focus of interest. In our study a new method for the analysis of scarce membrane protein samples combining large gel 2-D-CTAB/SDS-PAGE with fluorescence dye saturation labelling (satDIGE) was developed, allowing a highly sensitive differential analysis of different cell states. After Triton X-114 phase partitioning, enriched membrane protein samples of T cells were labelled at cysteine residues using fluorescence dyes and separated by large gel 2D-CTAB/SDS-PAGE. For a differential analysis 3 mug protein was found to be sufficient to detect proteins in a widespread well-separated diagonal spot pattern.