Unusual 1-3 peptidoglycan cross-links in Acetobacteraceae are made by L,D-transpeptidases with a catalytic domain distantly related to YkuD domains.

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position four of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software that can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a bioinformatics approach, we identified a G. oxydans mutant from a transposon library with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling.

Clostridioides difficile canonical L,D-transpeptidases catalyze a novel type of peptidoglycan cross-links and are not required for beta-lactam resistance.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerization of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 cross-links, catalyzed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the three canonical Ldts encoded by C. difficile (LdtCd1, LdtCd2, and LdtCd3) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyze the formation of novel types of peptidoglycan cross-links using meso-diaminopimelic acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross-links in C. difficile is catalyzed by as yet unidentified noncanonical Ldt enzymes.

Colorectal keratins: Integrating nutrition, metabolism and colorectal health.

The colon mucosa is lined with crypts of circa 300 cells, forming a continuous barrier whose roles include absorption of water, recovery of metabolic energy sources (notably short chain fatty acids), secretion of a protective mucus barrier, and physiological signalling. There is high turnover and replenishment of cells in the mucosa, disruption of this may lead to bowel pathologies including cancer and inflammatory bowel disease. Keratins have been implicated in the processes of cell death, epithelial integrity, response to inflammation and as a result are often described as guardians of the colonic epithelium. Keratin proteins carry extensive post-translational modifications, the cofactors for kinases, acetyl transferases and other modification-regulating enzymes are themselves products of metabolism. A cluster of studies has begun to reveal a bidirectional relationship between keratin form and function and metabolism. In this paper we hypothesise a mechanistic interaction between keratins and metabolism is governed through regulation of post-translational modifications and may contribute significantly to the normal functioning of the colon, placing keratins at the centre of a nutrition-metabolism-health triangle.

LFQRatio: A Normalization Method to Decipher Quantitative Proteome Changes in Microbial Coculture Systems.

The value of synthetic microbial communities in biotechnology is gaining traction due to their ability to undertake more complex metabolic tasks than monocultures. However, a thorough understanding of strain interactions, productivity, and stability is often required to optimize growth and scale up cultivation. Quantitative proteomics can provide valuable insights into how microbial strains adapt to changing conditions in biomanufacturing. However, current workflows and methodologies are not suitable for simple artificial coculture systems where strain ratios are dynamic. Here, we established a workflow for coculture proteomics using an exemplar system containing two members, Azotobacter vinelandii and Synechococcus elongatus. Factors affecting the quantitative accuracy of coculture proteomics were investigated, including peptide physicochemical characteristics such as molecular weight, isoelectric point, hydrophobicity, and dynamic range as well as factors relating to protein identification such as varying proteome size and shared peptides between species. Different quantification methods based on spectral counts and intensity were evaluated at the protein and cell level. We propose a new normalization method, named "LFQRatio", to reflect the relative contributions of two distinct cell types emerging from cell ratio changes during cocultivation. LFQRatio can be applied to real coculture proteomics experiments, providing accurate insights into quantitative proteome changes in each strain.

Metastasising Fibroblasts Show an HDAC6-Dependent Increase in Migration Speed and Loss of Directionality Linked to Major Changes in the Vimentin Interactome.

Metastasising cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumours in the clinic. We aimed to clarify how vimentin regulates the motility of metastasising fibroblasts. STED super-resolution microscopy, live-cell imaging and quantitative proteomics revealed that oncogene-expressing and metastasising fibroblasts show a less-elongated cell shape, reduced cell spreading, increased cell migration speed, reduced directionality, and stronger coupling between these migration parameters compared to normal control cells. In total, we identified and compared 555 proteins in the vimentin interactome. In metastasising cells, the levels of keratin 18 and Rab5C were increased, while those of actin and collagen were decreased. Inhibition of HDAC6 reversed the shape, spreading and migration phenotypes of metastasising cells back to normal. Inhibition of HDAC6 also decreased the levels of talin 1, tropomyosin, Rab GDI ß, collagen and emilin 1 in the vimentin interactome, and partially reversed the nanoscale vimentin organisation in oncogene-expressing cells. These findings describe the changes in the vimentin interactome and nanoscale distribution that accompany the defective cell shape, spreading and migration of metastasising cells. These results support the hypothesis that oncogenes can act through HDAC6 to regulate the vimentin binding of the cytoskeletal and cell-extracellular matrix adhesion components that contribute to the defective motility of metastasising cells.

Application of the broadband collision-induced dissociation (bbCID) mass spectrometry approach for protein glycosylation and phosphorylation analysis.

RATIONALE: Analysis of post-translationally modified peptides by mass spectrometry (MS) remains incomplete, in part due to incomplete sampling of all peptides which is inherent to traditional data-dependent acquisition (DDA). An alternative MS approach, data-independent acquisition (DIA), enables comprehensive recording of all detectable precursor and product ions, independent of precursor intensity. The use of broadband collision-induced dissociation (bbCID), a DIA method, was evaluated for the identification of protein glycosylation and phosphorylation. METHODS: bbCID was applied to identify glycopeptides and phosphopeptides generated from standard proteins using a high-resolution Bruker maXis 3G mass spectrometer. In bbCID, precursor and product ion spectra were obtained by alternating low and high collision energy. Precursor ions were assigned manually based on the detection of diagnostic ions specific to either glycosylation or phosphorylation. The composition of the glycan modification was resolved in the positive ion mode, while the level of phosphorylation was investigated in the negative ion mode. RESULTS: The results demonstrate for the first time that the use of a bbCID approach is suitable for the identification of glycopeptides and phosphopeptides based on the detection of specific diagnostic and associated precursor ions. The novel use of bbCID in negative ion mode allowed the discrimination of singly and multiply phosphorylated peptides based on the detection of phosphate diagnostic ions. The results also demonstrate the ability of this approach to allow the identification of glycan composition in N- and O-linked glycopeptides, in positive ion mode. CONCLUSIONS: We contend that bbCID is a valuable addition to the existing toolkit for PTM discovery. Moreover, this technique could be employed to direct targeted proteomics methods, particularly where there is no a priori information on glycosylation or phosphorylation status. This technique is immediately relevant to the characterisation of individual proteins or biological samples of low complexity, as demonstrated for the analysis of the glycosylation status of a therapeutic protein.

Quantitative proteomic analysis reveals maturation as a mechanism underlying glucocorticoid resistance in B lineage ALL and re-sensitization by JNK inhibition.

Glucocorticoid (GC) resistance is a continuing clinical problem in childhood acute lymphoblastic leukaemia (ALL) but the underlying mechanisms remain unclear. A proteomic approach was used to compare profiles of the B-lineage ALL GC-sensitive cell line, PreB 697, and its GC-resistant sub-line, R3F9, pre- and post-dexamethasone exposure. PAX5, a transcription factor critical to B-cell development was differentially regulated in the PreB 697 compared to the R3F9 cell line in response to GC. PAX5 basal protein expression was less in R3F9 compared to its GC-sensitive parent and confirmed to be lower in other GC-resistant sub-lines of Pre B 697 and was associated with a decreased expression of the PAX5 transcriptional target, CD19. Gene set enrichment analysis showed that increasing GC-resistance was associated with differentiation from preB-II to an immature B-lymphocyte stage. GC-resistant sub-lines were shown to have higher levels of phosphorylated JNK compared to the parent line and JNK inhibition caused re-sensitization to GC. Exploiting this maturation may be key to overcoming GC resistance and targeting signalling pathways linked to the maturation state, such as JNK, may be a novel approach.

Quantitative proteomics analysis of BMS-214662 effects on CD34 positive cells from chronic myeloid leukaemia patients.

Chronic myeloid leukaemia (CML) arises in a haemopoietic stem cell and is driven by the Bcr-Abl oncoprotein. Abl kinase inhibitors (protein tyrosine kinase inhibitors) represent standard treatment for CML and induce remission in the majority of patients with early disease, however these drugs do not target leukaemic stem cells (LSCs) effectively, thus preventing cure. Previously, we identified the farnesyl transferase inhibitor BMS-214662 as a selective inducer of apoptosis in LSCs of CML patients relative to normal controls; however, the mechanism underlying LSC-specific apoptosis remains unclear. To identify pathways involved in the favourable effects of BMS-214662 in CML, we employed a proteomic approach (based on iTRAQ) to analyse changes in protein expression in response to drug treatment in the nuclear and cytoplasmic fractions of CD34(+) CML cells. The study identified 88 proteins as altered after drug treatment, which included proteins known to be involved in nucleic acid metabolism, oncogenesis, developmental processes and intracellular protein trafficking. We found that expression of Ebp1, a negative regulator of proliferation, was upregulated in the nucleus of BMS-214662-treated cells. Furthermore, proteins showing altered levels in the cytosol, such as histones, were predominantly derived from the nucleus and BMS-214662 affected expression levels of nuclear pore complex proteins. Validation of key facets of these observations suggests that drug-induced alterations in protein localisation, potentially via loss of nuclear membrane integrity, contributes to the LSC specificity of BMS-214662, possibly via Ran proteins as targets.

Exploring Predictive Biomarkers of Relapse in Ulcerative Colitis: A Proteomics Approach.

INTRODUCTION AND AIMS: Risk stratification of subjects with a history of inflammatory bowel disease (IBD) into those likely to relapse and those who will remain quiescent continues to be a significant challenge. The aim of this study was to investigate whether certain proteomic signature profiles or biomarkers during remission are associated with future disease relapse in patients with ulcerative colitis (UC). METHODS: Endoscopic rectal samples from patients with UC in clinical, endoscopic, and histological remission at index endoscopy were collected, as well as samplers from normal control individuals. The patients were stratified to early relapsers (ERs) if they developed clinical signs of UC flare within 6 months of index endoscopy or nonrelapsers (NRs) if there was no relapse after 36 months of follow-up. The pooled rectal samples from ERs, NRs, and control individuals were subjected to nano-liquid chromatography and tandem mass spectrometry as per standard iTRAQ (isobaric tags for relative and absolute quantitation) workflow methodology. Selected proteomics-yielded candidates were subjected to orthogonal validation via immunoblotting, in a biomarker discovery exercise. RESULTS: Sixty-one patients were included, of whom 8 had clinical relapse within 6 months from the index endoscopy, and 43 patients had no clinical symptoms of relapse within the 36-month follow-up period. Ten patients who had clinical signs of relapse between 6 and 36 months were excluded. Seventeen control individuals were also included. Soluble proteomics analyses between ERs, NRs, and control individuals revealed a series of upregulated and downregulated proteins. Following orthogonal validation, upregulated TRX (P = .001) and IGHA1 (P = .001) were observed in ERs relative to NRs. CONCLUSIONS: Several novel candidate tissue biomarkers have been identified in this study, which could discriminate patients with UC at risk of early relapse from those in long-term sustained remission. Our findings may pave the way for pre-emptive UC disease monitoring and therapeutic decision making.

This study aimed to investigate if certain proteins (biomarkers) could predict whether patients with Ulcerative Colitis (UC) would have a disease relapse. Rectal samples were collected from UC patients who were in remission and from healthy individuals. The patients were categorised into two groups: those who had a flare-up within 6 months (early relapsers) and those who did not have a relapse after 36 months (non-relapsers). Using proteomics methodology, it was found that certain proteins were more common in the early relapsers compared to the non-relapsers and healthy individuals. Two proteins, TRX and IGHA1, were significantly higher in the early relapsers. These proteins could potentially be used as markers to identify UC patients who are at risk of having an early relapse. This could help monitoring UC patients more effectively and making better treatment decisions.

Keratins in colorectal epithelial function and disease.

Keratins are the largest subgroup of intermediate filament proteins, which are an important constituent of the cellular cytoskeleton. The principally expressed keratins (K) of the intestinal epithelium are K8, K18 and K19. The specific keratin profile of a particular epithelium provides it with strength and integrity. In the colon, keratins have been shown to regulate electrolyte transport, likely by targeting ion transporters to their correct location in the colonocytes. Keratins are highly dynamic and are subject to post-translational modifications including phosphorylation, acetylation and glycosylation. These affect the filament dynamics and hence solubility of keratins and may contribute to protection against degradation. Keratin null mice (K8(-/-) ) develop colitis, and abnormal keratin mutations have been shown to be associated with inflammatory bowel disease (IBD). Abnormal expression of K7 and K20 has been noted in colitis-associated dysplasia and cancers. In sporadic colorectal cancers (CRCs) may be useful in predicting tumour prognosis; a low K20 expression is noted in CRCs with high microsatellite instability; and keratins have been noted as dysregulated in peri-adenomatous fields. Caspase-cleaved fragment of K18 (M30) in the serum of patients with CRC has been used as a marker of cancer load and to assess response to therapy. These data suggest an emerging importance of keratins in maintaining normal function of the gastrointestinal epithelium as well as being a marker of various colorectal diseases. This review will primarily focus on the biology of these proteins, physiological functions and alterations in IBD and CRCs.

An integrated workflow for extraction and solubilization of intermediate filaments from colorectal biopsies for proteomic analysis.

We report a technique for isolation and solubilization of intermediate filament (IF) proteins from colonic biopsies compatible with both gel electrophoresis and liquid chromatography "shotgun" proteomics using mass spectrometry (MS). This is important because changes in the IF proteome, particularly in keratin expression and modification, are noted in colonic mucosa of patients with colorectal cancer. Though keratins have traditionally been dissolved in high concentration of urea, the latter solvent precludes efficient proteolytic digestion by trypsin prior to gel-free LC-MS/MS approaches. The extraction of cytoskeletal proteins was initially evaluated using MCF-7 cancer cell lines using a published, differential detergent solubilization protocol. IF proteins were extracted from colonic biopsies using a combination of homogenization and sonication. Since comparable efficiency of solubilization was noted on the extracted IF from cell lines between urea and guanidine hydrochloride (GuHCl) in triethylammonium bicarbonate buffer, isolated proteins from endoscopic biopsies were solubilized in GuHCl. Using immunoblotting techniques, we successfully demonstrated isolation of keratins and preservation of posttranslational modifications (phosphorylation, acetylation). Dissolved proteins were tryptically digested and peptides analyzed by MS, showing the functionality of the workflow in shotgun proteomic applications, specifically compatibility of the workflow for isobaric tagging relative and absolute quantification based quantitation approaches.

Comparative Proteomics Reveals Evidence of Enhanced EPA Trafficking in a Mutant Strain of Nannochloropsis oculata.

The marine microalga Nannochloropsis oculata is a bioproducer of eicosapentaenoic acid (EPA), a fatty acid. EPA is incorporated into monogalactosyldiacylglycerol within N. oculata thylakoid membranes, and there is a biotechnological need to remodel EPA synthesis to maximize production and simplify downstream processing. In this study, random mutagenesis and chemical inhibitor-based selection method were devised to increase EPA production and accessibility for improved extraction. Ethyl methanesulfonate was used as the mutagen with selective pressure achieved by using two enzyme inhibitors of lipid metabolism: cerulenin and galvestine-1. Fatty acid methyl ester analysis of a selected fast-growing mutant strain had a higher percentage of EPA (37.5% of total fatty acids) than the wild-type strain (22.2% total fatty acids), with the highest EPA quantity recorded at 68.5 mg/g dry cell weight, while wild-type cells had 48.6 mg/g dry cell weight. Label-free quantitative proteomics for differential protein expression analysis revealed that the wild-type and mutant strains might have alternative channeling pathways for EPA synthesis. The mutant strain showed potentially improved photosynthetic efficiency, thus synthesizing a higher quantity of membrane lipids and EPA. The EPA synthesis pathways could also have deviated in the mutant, where fatty acid desaturase type 2 (13.7-fold upregulated) and lipid droplet surface protein (LDSP) (34.8-fold upregulated) were expressed significantly higher than in the wild-type strain. This study increases the understanding of EPA trafficking in N. oculata, leading to further strategies that can be implemented to enhance EPA synthesis in marine microalgae.

Relative quantification in proteomics: new approaches for biochemistry.

Recent developments in mass spectrometry and protein arrays provide opportunities to derive systematically proteomic information from small samples of cellular material. Relative quantification among samples can be achieved with either gel-based or gel-free approaches. Furthermore, the adaptation of specific techniques facilitates absolute quantification. Here, relative quantification in two-dimensional gel electrophoresis is contrasted with that in non-gel-based approaches such as isobaric tagging of peptides, pre-labelling of living cells with isotopomeric forms of essential amino acids and protein array platforms. In addition, novel flow-cytometry-based approaches are considered. These technologies can all be used to determine accurately the levels of proteins or biomarkers in a wide range of samples.

Assessment of downstream effectors of BCR/ABL protein tyrosine kinase using combined proteomic approaches.

Leukaemic transformation is frequently associated with the aberrant activity of a protein tyrosine kinase (PTK). As such it is of clinical relevance to be able to map the effects of these leukaemogenic PTKs on haemopoietic cells at the level of phosphorylation modulation. In this paradigm study we have employed a range of proteomic approaches to analyse the effects of one such PTK, BCR/ABL. We have employed phosphoproteome enrichment techniques allied to peptide and protein quantification to identify proteins and pathways involved in cellular transformation. Amongst the proteins shown to be regulated at the post-translational level were cofilin, an actin-severing protein thus linked to altered motility and Cbl an E3 ubiquitin ligase integrally linked to the control of tyrosine kinase signalling (regulated by 5 and 6 PTKs respectively). The major class of proteins identified however were molecular chaperones. We also showed that HSP90 phosphorylation is altered by BCR/ABL action and that HSP90 plays a crucial role in oncogene stability. Further investigation with another six leukaemogenic PTKs demonstrates that this HSP90 role in oncogene stability appears to be a common phenomenon in a range of leukaemias. This opens up the potential opportunity to treat different leukaemias with HSP90 inhibitors.

Eight-plex iTRAQ analysis of variant metastatic human prostate cancer cells identifies candidate biomarkers of progression: An exploratory study.

BACKGROUND: Due to the heterogeneity in the biological behavior of prostate cancer, biomarkers that can reliably distinguish indolent from aggressive disease are urgently needed to inform treatment choices. METHODS: We employed 8-plex isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to profile the proteomes of two distinct panels of isogenic prostate cancer cells with varying growth and metastatic potentials, in order to identify novel biomarkers associated with progression. The LNCaP, LNCaP-Pro5, and LNCaP-LN3 panel of cells represent a model of androgen-responsive prostate cancer, while the PC-3, PC-3M, and PC-3M-LN4 panel represent a model of androgen-insensitive disease. RESULTS: Of the 245 unique proteins identified and quantified (>or=95% confidence; >or=2 peptides/protein), 17 showed significant differential expression (>or=+/-1.5), in at least one of the variant LNCaP cells relative to parental cells. Similarly, comparisons within the PC-3 panel identified 45 proteins to show significant differential expression in at least one of the variant PC-3 cells compared with parental cells. Differential expression of selected candidates was verified by Western blotting or immunocytochemistry, and corresponding mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Immunostaining of prostate tissue microarrays for ERp5, one of the candidates identified, showed a significant higher immunoexpression in pre-malignant lesions compared with non-malignant epithelium (P < 0.0001, Mann-Whitney U-test), and in high Gleason grade (4-5) versus low grade (2-3) cancers (P < 0.05). CONCLUSIONS: Our study provides proof of principle for the application of an 8-plex iTRAQ approach to uncover clinically relevant candidate biomarkers for prostate cancer progression.

Eight-channel iTRAQ enables comparison of the activity of six leukemogenic tyrosine kinases.

There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL, TEL/PDGFRbeta, FIP1/PDGFRalpha, D816V KIT, NPM/ALK, and FLT3ITD) revealed few common effects on the transcriptome. It is apparent, however, that proteome changes are not directly governed by transcriptome changes. Therefore, we assessed and used a new generation of iTRAQ tagging, enabling eight-channel relative quantification discovery proteomics, to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRalpha. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin, although this did not correspond to changes in motility. However, correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes, illustrating the utility of such a combined approach.

Phosphopeptide enrichment for phosphoproteomic analysis - A tutorial and review of novel materials.

Significant technical advancements in phosphopeptide enrichment have enabled the identification of thousands of p-peptides (mono and multiply phosphorylated) in a single experiment. However, it is still not possible to enrich all p-peptide species in a single step. A range of new techniques and materials has been developed, with the potential to provide a step-change in phosphopeptide enrichment. The first half of this review contains a tutorial for new potential phosphoproteomic researchers; discussing the key steps of a typical phosphoproteomic experiment used to investigate canonical phosphorylation sites (serine, threonine and tyrosine). The latter half then show-cases the latest developments in p-peptide enrichment including: i) Strategies to mitigate non-specific binding in immobilized metal ion affinity chromatography and metal oxide affinity chromatography protocols; ii) Techniques to separate multiply phosphorylated peptides from monophosphorylated peptides (including canonical from non-canonical phosphorylated peptides), or to simultaneously co-enrich other post-translational modifications; iii) New hybrid materials and methods directed towards enhanced selectivity and efficiency of metal-based enrichment; iv) Novel materials that hold promise for enhanced phosphotyrosine enrichment. A combination of well-understood techniques and materials is much more effective than any technique in isolation; but the field of phosphoproteomics currently requires benchmarking of novel materials against current methodologies to fully evaluate their utility in peptide based proteoform analysis.

A study protocol to investigate the relationship between dietary fibre intake and fermentation, colon cell turnover, global protein acetylation and early carcinogenesis: the FACT study.

BACKGROUND: A number of studies, notably EPIC, have shown a descrease in colorectal cancer risk associated with increased fibre consumption. Whilst the underlying mechanisms are likely to be multifactorial, production of the short-chain fatty-acid butyrate fro butyratye is frequently cited as a major potential contributor to the effect. Butyrate inhibits histone deacetylases, which work on a wide range of proteins over and above histones. We therefore hypothesized that alterations in the acetylated proteome may be associated with a cancer risk phenotype in the colorectal mucosa, and that such alterations are candidate biomarkers for effectiveness of fibre interventions in cancer prevention. METHODS AN DESIGN: There are two principal arms to this study: (i) a cross-sectional study (FACT OBS) of 90 subjects recruited from gastroenterology clinics and; (ii) an intervention trial in 40 subjects with an 8 week high fibre intervention. In both studies the principal goal is to investigate a link between fibre intake, SCFA production and global protein acetylation. The primary measure is level of faecal butyrate, which it is hoped will be elevated by moving subjects to a high fibre diet. Fibre intakes will be estimated in the cross-sectional group using the EPIC Food Frequency Questionnaire. Subsidiary measures of the effect of butyrate on colon mucosal function and pre-cancerous phenotype will include measures of apoptosis, apoptotic regulators cell cycle and cell division. DISCUSSION: This study will provide a new level of mechanistic data on alterations in the functional proteome in response to the colon microenvironment which may underwrite the observed cancer preventive effect of fibre. The study may yield novel candidate biomarkers of fibre fermentation and colon mucosal function. TRIAL REGISTRATION NUMBER: ISRCTN90852168.

Reducing Complexity? Cysteine Reduction and S-Alkylation in Proteomic Workflows: Practical Considerations.

Reduction and alkylation are common processing steps in sample preparation for qualitative and quantitative proteomic analyses. In principle, these steps mitigate the limitations resulting from the presence of disulfide bridges. There has been recurring debate in the proteomics community around their use, with concern over negative impacts that result from overalkylation (off-target, non-thiol sites) or incomplete reduction and/or S-alkylation of cysteine. This chapter integrates findings from a number of studies on different reduction and alkylation strategies, to guide users in experimental design for their optimal use in proteomic workflows.

Application of Proteomics to Inflammatory Bowel Disease Research: Current Status and Future Perspectives.

Inflammatory bowel disease (IBD) is a chronic relapsing/remitting inflammatory illness of the gastrointestinal tract of unknown aetiology. Despite recent advances in decoding the pathophysiology of IBD, many questions regarding disease pathogenesis remain. Genome-wide association studies (GWAS) and knockout mouse models have significantly advanced our understanding of genetic susceptibility loci and inflammatory pathways involved in IBD pathogenesis. Despite their important contribution to a better delineation of the disease process in IBD, these genetic findings have had little clinical impact to date. This is because the presence of a given gene mutation does not automatically correspond to changes in its expression or final metabolic or structural effect(s). Furthermore, the existence of these gene susceptibility loci in the normal population suggests other driving prerequisites for the disease manifestation. Proteins can be considered the main functional units as almost all intracellular physiological functions as well as intercellular interactions are dependent on them. Proteomics provides methods for the large-scale study of the proteins encoded by the genome of an organism or a cell, to directly investigate the proteins and pathways involved. Understanding the proteome composition and alterations yields insights into IBD pathogenesis as well as identifying potential biomarkers of disease activity, mucosal healing, and cancer progression. This review describes the state of the art in the field with respect to the study of IBD and the potential for translation from biomarker discovery to clinical application.