Caroline A. Evans

iTRAQ underestimation in simple and complex mixtures: "the good, the bad and the ugly".

The increasing popularity of iTRAQ for quantitative proteomics applications makes it necessary to evaluate its relevance, accuracy, and precision for biological interpretation. Here, we have assessed (a) the accuracy and precision of iTRAQ quantification in a controlled experimental setup, using low- and high-complexity protein mixtures; and (b) the potential pitfalls that hamper the applicability and attainable dynamic range of iTRAQ: isotopic contamination, background interference, and signal-to-noise ratio. Our data suggest greater dynamic crosstalk between interfering factors affecting underestimations, and that these interferences were largely scenario-specific, dependent on sample complexity. The good is the potential for iTRAQ to provide accurate quantification spanning 2 orders of magnitude. This potential is however limited by two factors. (1) The bad: the existence of isotopic impurities that can be corrected for; provided accurate isotopic factors are at ones disposal. (2) The ugly: we demonstrate here the interference of mixed MS/MS contribution occurring during precursor selection, an issue that is currently very difficult to minimize. In light of our results, we propose a list of advice for iTRAQ data analysis that could routinely ameliorate quantitative interpretation of proteomic data sets.

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/PDGFRβ, FIP1/PDGFRα, 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/PDGFRα. 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.

PEDRo: A database for storing, searching and disseminating experimental proteomics data

BackgroundProteomics is rapidly evolving into a high-throughput technology, in which substantial and systematic studies are conducted on samples from a wide range of physiological, developmental, or pathological conditions. Reference maps from 2D gels are widely circulated. However, there is, as yet, no formally accepted standard representation to support the sharing of proteomics data, and little systematic dissemination of comprehensive proteomic data sets.ResultsThis paper describes the design, implementation and use of a P roteome E xperimental D ata R epo sitory (PEDRo), which makes comprehensive proteomics data sets available for browsing, searching and downloading. It is also serves to extend the debate on the level of detail at which proteomics data should be captured, the sorts of facilities that should be provided by proteome data management systems, and the techniques by which such facilities can be made available.ConclusionsThe PEDRo database provides access to a collection of comprehensive descriptions of experimental data sets in proteomics. Not only are these data sets interesting in and of themselves, they also provide a useful early validation of the PEDRo data model, which has served as a starting point for the ongoing standardisation activity through the Proteome Standards Initiative of the Human Proteome Organisation.

iTRAQ identification of candidate serum biomarkers associated with metastatic progression of human prostate cancer.

A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer ‘BPH’, (ii) localised cancer with no evidence of progression, ‘non-progressing’ (iii) localised cancer with evidence of biochemical progression, ‘progressing’, and (iv) bone metastasis at presentation ‘metastatic’. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and ‘panels’ of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation.

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

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.

Proteomic Analysis of Chronic Lymphocytic Leukemia Subtypes with Mutated or Unmutated Ig VH Genes

Chronic lymphocytic leukemia (CLL) is a common hematopoietic malignant disease with variable outcome. CLL has been divided into distinct groups based on whether somatic hypermutation has occurred in the variable region of the immunoglobulin heavy-chain locus or alternatively if the cells express higher levels of the CD38 protein. We have analyzed the proteome of 12 cases of CLL (six mutated (M-CLL) and six unmutated (UM-CLL) immunoglobulin heavy-chain loci; seven CD38-negative and five CD38-positive) using two-dimensional electrophoresis and mass spectrometry. Statistical evaluation using principal component analysis indicated significant differences in patterns of protein expression between the cases with and without somatic mutation. Specific proteins indicated by principal component analysis as varying between the prognostic groups were characterized using mass spectrometry. The levels of F-actin-capping protein β subunit, 14-3-3 β protein, and laminin-binding protein precursor were significantly increased in M-CLL relative to UM-CLL. In addition, primary sequence data from tandem mass spectrometry showed that nucleophosmin was present as several protein spots in M-CLL but was not detected in UM-CLL samples, suggesting that several post-translationally modified forms of nucleophosmin vary between these two sample groups. No specific differences were found between CD38-positive and -negative patient samples using the same approach. The results presented show that proteomic analysis can complement other approaches in identifying proteins that may have potential value in the biological and diagnostic distinction between important clinical subtypes of CLL.

Proteomic analyses of intermediate filaments reveals cytokeratin8 is highly acetylated : implications for colorectal epithelial homeostasis

Butyrate is a critical cancer‐preventive element in the colon milieu whose mechanism of action is unclear, but appears to be mediated through inhibition of histone deacetylases (HDACs) and consequent alterations in global protein acetylation. Cytokeratins (CKs) have roles in cytoskeletal function as components of the intermediate filaments (IFs) and this involves CKs in the regulation of tissue homeostasis of high‐turnover epithelia such as the colon. We used a 2‐D gel/MS analysis to characterise the proteome of IFs, and a novel monitoring‐initiated detection and sequencing (MIDAS) approach to identify acetylation sites on principal proteins. We report that CKs are highly acetylated in a colon cancer cell line, with five acetylation sites characterised on CK8 and a further one on CK18. Acetylation of CK8 is responsive to butyrate. HDAC5 is the deacetylase associated with IFs. These data indicate a novel action of butyrate as a cancer preventive agent. Acetylation of CK8 may be associated with IFs stabilisation and thereby provide a candidate mechanism for the appropriate retention or loss of epithelial cells from the flat mucosa.

Changes in the Proteome Associated with the Action of Bcr-Abl Tyrosine Kinase Are Not Related to Transcriptional Regulation

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease, the hallmark of which is the Bcr-Abl protein tyrosine kinase (PTK). Without intervention the disease progresses from a benign chronic phase to a rapidly fatal blast crisis. To identify the molecular mechanisms underlying disease progression we used two-dimensional gel electrophoresis on a model we have previously described using the expression of a conditional mutant of Bcr-Abl PTK in a multipotent stem cell line, FDCP-Mix. Long term exposure of FDCP-Mix cells to Bcr-Abl mimics disease progression in CML. Four major differences were observed as a consequence of long term exposure to the Bcr-Abl PTK compared with cells exposed short term. The proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry-generated peptide mass fingerprint data and liquid chromatography-tandem mass spectrometry-generated sequence information. Leukotriene A4 hydrolase, an enzyme known to be deregulated in CML, was found to be up-regulated. Annexin VI, vacuolar ATP synthase catalytic subunit A, and mortalin were found to be down-regulated. Poly(A) PCR cDNA analysis showed there was no correlation between the protein expression changes and mRNA levels. Western blot analysis also indicated no change in the levels of mortalin or leukotriene A4 hydrolase, indicating that post-translational events may modify protein content of the specific spots. Leukotriene B4 levels (product of leukotriene A4 hydrolase) were, however, reduced in cells exposed long term to Bcr-Abl activity. This study demonstrates the potential of proteomic analysis to define novel effects of oncogenes.

Effects of heavy metals on Cyanothece sp. CCY 0110 growth, extracellular polymeric substances (EPS) production, ultrastructure and protein profiles

UNLABELLED The effects of several heavy metals on the growth/survival, EPS production, ultrastructure and protein profiles of the highly efficient extracellular polymeric substances (EPS)-producer cyanobacterium Cyanothece sp. CCY 0110 were evaluated. Our results clearly show that each heavy metal affects the cells in a particular manner, triggering distinctive responses. Concerning chronic exposure, cells were more affected by Cu(2+) followed by Pb(2+), Cd(2+), and Li(+). The presence of metal leads to remarkable ultrastructural changes, mainly at the thylakoid level. The comparison of the proteomes (iTRAQ) allowed to follow the stress responses and to distinguish specific effects related to the time of exposure and/or the concentration of an essential (Cu(2+)) and a non-essential (Cd(2+)) metal. The majority of the proteins identified and with fold changes were associated with photosynthesis, CO2 fixation and carbohydrate metabolism, translation, and nitrogen and amino acid metabolism. Moreover, our results indicate that during chronic exposure to sub-lethal concentrations of Cu(2+), the cells tune down their metabolic rate to invest energy in the activation of detoxification mechanisms, which eventually result in a remarkable recovery. In contrast, the toxic effects of Cd(2+) are cumulative. Unexpectedly, the amount of released polysaccharides (RPS) was not enhanced by the presence of heavy metals. BIOLOGICAL SIGNIFICANCE This work shows the holistic effects of different heavy metals on the cells of the highly efficient EPS-producer the cyanobacterium Cyanothece sp. CCY 0110. The growth/survival, EPS production, ultrastructure, protein profiles and stress response were evaluated. The knowledge generated by this study will contribute to the implementation of heavy-metal removal systems based on cyanobacteria EPS or their isolated polymers.

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.

BackgroundA 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 designThere 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.DiscussionThis 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 RegistrationTrial Registration Number: ISRCTN90852168