[Show abstract][Hide abstract] ABSTRACT: Although DNA encodes the molecular instructions that underlie control of cell function, it is the proteins that are primarily responsible for implementing those instructions. Therefore, quantitative analyses of the proteome would be expected to yield insights into important candidates for the detection and treatment of disease. We present an iTRAQ (Isobaric Tagging for Relative and Absolute Quantification)-based proteomic analysis of 10 ovarian cancer cell lines and 2 normal ovarian surface epithelial cell lines. We profiled the abundance of 2659 cellular proteins, of which 1273 were common to all 12 cell lines. Of the 1273, 75 proteins exhibited elevated expression, and 164 proteins had diminished expression in the cancerous cells compared to the normal cell lines. The iTRAQ expression profiles allowed us to segregate cell lines based upon sensitivity and resistance to carboplatin. Importantly, we observed no substantial correlation between protein abundance and RNA expression or epigenetic DNA methylation data. Furthermore, we could not discriminate between sensitivity and resistance to carboplatin on the basis of RNA expression and DNA methylation data alone. This study illustrates the importance of proteomics-based discovery for defining the basis for the carboplatin response in ovarian cancer and highlights candidate proteins, particularly involved in cellular redox regulation, homologous recombination and DNA damage repair, that otherwise could not have been predicted from whole genome and expression data sources alone.
[Show abstract][Hide abstract] ABSTRACT: Oncogenic RAS (H-RAS(V12)) induces premature senescence in primary cells by triggering production of reactive oxygen species (ROS), but the molecular role of ROS in senescence remains elusive. We investigated whether inhibition of protein tyrosine phosphatases by ROS contributed to H-RAS(V12)-induced senescence. We identified protein tyrosine phosphatase 1B (PTP1B) as a major target of H-RAS(V12)-induced ROS. Inactivation of PTP1B was necessary and sufficient to induce premature senescence in H-RAS(V12)-expressing IMR90 fibroblasts. We identified phospho-Tyr 393 of argonaute 2 (AGO2) as a direct substrate of PTP1B. Phosphorylation of AGO2 at Tyr 393 inhibited loading with microRNAs (miRNAs) and thus miRNA-mediated gene silencing, which counteracted the function of H-RAS(V12)-induced oncogenic miRNAs. Overall, our data illustrate that premature senescence in H-RAS(V12)-transformed primary cells is a consequence of oxidative inactivation of PTP1B and inhibition of miRNA-mediated gene silencing.
[Show abstract][Hide abstract] ABSTRACT: Chromatographed peptide signals form the basis of further data processing that eventually results in functional information derived from data-dependent bottom-up proteomics assays. We seek to rank LC/MS parent ions by the quality of their extracted ion chromatograms (ExICs.) Ranked extracted ion chromatograms (RExICs) act as an intuitive physical/chemical preselection filter to improve the quality of MS/MS fragment scans submitted for database search. We identify more than 4,900 proteins when considering detector shifts of less than 7ppm. High quality parent ions for which the database search yields no hits become candidates for subsequent unrestricted analysis for post-translational modifications. Following this rational approach, we prioritize identification of more than 5,000 spectrum matches from modified peptides and confirmed the presence of acetylaldehyde modified His/Lys. We present a logical workflow that scores data-dependent selected ion chromatograms and leverage information about semi-analytical LC/LC dimension prior to mass spectrometry. Our method can be successfully used to identify unexpected modifications in peptides with excellent chromatography characteristics, independent of fragmentation pattern and activation methods. We illustrate analysis of ion chromatograms detected in two different modes by RF linear ion trap and electrostatic field orbitrap. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: I. Overview We have developed a streamlined, optimized sample workflow for 2D MudPIT analysis of complex peptide samples labeled with iTRAQ. The workflow employs a triple-phase vented MudPIT trap column and up to 16 discrete salt steps delivered by autosampler. Pooled iTRAQ labeled samples can be dried immediately after reaction, and then loaded directly onto the trap column with no additional cleanup. Each salt step is eluted with a 100 minute gradient, so the entire analysis is completed in ~36hr of instrument time. We have used the protocol extensively for proteins isolated from cell culture, tissue, and serum and plasma samples. Between 4,000 and 5,000 proteins can be identified and quantified in a single analysis (1% FDR) when using 50-80ug of total initial load and 8-plex iTRAQ. Close to 1,000 proteins have also been identified from whole-cell lysates with initial loads of < 5ug. This is similar to the best we had achieved using OffGel fractionation of iTRAQ labeled peptides, but has proven faster, more convenient and much more reproducible. Quantitative peptide shotgun proteomics requires fast and robust protocols that cover cell lysis, enzymatic digestion, labeling (e.g. iTRAQ) and 2D fractionation prior to MS analysis. As large-scale proteomic screens require replicates to obtain statistically significant results, these protocols must also be inherently reproducible. iTRAQ labeling adds additional considerations to the sample workflow, in particular the final cleanup steps immediately prior to chromatography where excess non-volatile reagent and buffer must be almost completely removed for successful capture by strong cation exchange (SCX) resins. In the past, we have used OffGel fractionation for the first dimension of peptide separation, but this requires an additional step of C18 cleanup (stage-tip), and then 36hrs to achieve focus with the high-resolution (24 fraction) protocol . This introduces a significant sample processing overhead to Core laboratories, where analysis time-per-sample is often a severe constraint. Individual OffGel fractions then require another C18 ziptip or stage-tip cleanup to remove glycerol and buffer ampholytes before autosampler injection, with the potential for sample loss. To streamline this process, we began optimization of an in-line vented MudPIT protocol optimized for iTRAQ labeled samples that achieves good chromatographic resolution, high protein counts, and minimal sample handling. Triphasic trap column is loaded directly after iTRAQ reaction (after drying) with no additional sample cleanup
NERLSCD 2012, Mountain View Grand Hotel Whitefield, NH; 10/2012
[Show abstract][Hide abstract] ABSTRACT: Tumour suppressor genes encode a broad class of molecules whose mutational attenuation contributes to malignant progression. In the canonical situation, the tumour suppressor is completely inactivated through a two-hit process involving a point mutation in one allele and chromosomal deletion of the other. Here, to identify tumour suppressor genes in lymphoma, we screen a short hairpin RNA library targeting genes deleted in human lymphomas. We functionally identify those genes whose suppression promotes tumorigenesis in a mouse lymphoma model. Of the nine tumour suppressors we identified, eight correspond to genes occurring in three physically linked 'clusters', suggesting that the common occurrence of large chromosomal deletions in human tumours reflects selective pressure to attenuate multiple genes. Among the new tumour suppressors are adenosylmethionine decarboxylase 1 (AMD1) and eukaryotic translation initiation factor 5A (eIF5A), two genes associated with hypusine, a unique amino acid produced as a product of polyamine metabolism through a highly conserved pathway. Through a secondary screen surveying the impact of all polyamine enzymes on tumorigenesis, we establish the polyamine-hypusine axis as a new tumour suppressor network regulating apoptosis. Unexpectedly, heterozygous deletions encompassing AMD1 and eIF5A often occur together in human lymphomas and co-suppression of both genes promotes lymphomagenesis in mice. Thus, some tumour suppressor functions can be disabled through a two-step process targeting different genes acting in the same pathway.
[Show abstract][Hide abstract] ABSTRACT: Although originally considered toxic, hydrogen sulfide (H(2)S) has been implicated in mediating various biological processes. Nevertheless, its cellular targets and mode of action are not well understood. Protein tyrosine phosphatases (PTPs), which regulate numerous signal transduction pathways, use an essential cysteine residue at the active site, which is characterized by a low pK(a) and is susceptible to reversible oxidation. Here, we report that PTP1B was reversibly inactivated by H(2)S, in vitro and in cells, through sulfhydration of the active-site cysteine residue. Unlike oxidized PTP1B, the sulfhydrated enzyme was preferentially reduced in vitro by thioredoxin, compared to glutathione or dithiothreitol. Sulfhydration of PTP1B in cells required the presence of cystathionine γ-lyase (CSE), a critical enzyme in H(2)S production, and resulted in inhibition of phosphatase activity. Suppression of CSE decreased H(2)S production and decreased the phosphorylation of tyrosine-619 in PERK [protein kinase-like endoplasmic reticulum (ER) kinase], thus reducing its activation in response to ER stress. PERK, which phosphorylates the eukaryotic translational initiation factor 2, leading to attenuation of protein translation, was a direct substrate of PTP1B. In addition, CSE knockdown led to activation of the nonreceptor tyrosine kinase SRC, previously shown to be mediated by PTP1B. These effects of suppressing H(2)S production on the response to ER stress were abrogated by a small-molecule inhibitor of PTP1B. Together, these data define a signaling function for H(2)S in inhibiting PTP1B activity and thereby promoting PERK activity during the response to ER stress.
[Show abstract][Hide abstract] ABSTRACT: The challenge of understanding the widespread biological roles of animal microRNAs (miRNAs) has prompted the development of genetic and functional genomics technologies for miRNA loss-of-function studies. However, tools for exploring the functions of entire miRNA families are still limited. We developed a method that enables antagonism of miRNA function using seed-targeting 8-mer locked nucleic acid (LNA) oligonucleotides, termed tiny LNAs. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant upregulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues and in breast tumors in mice, coinciding with long-term miRNA silencing. Transcriptional and proteomic profiling suggested that tiny LNAs have negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny LNA complementary sites. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families in vivo.
[Show abstract][Hide abstract] ABSTRACT: Combining RNAi in cultured cells and analysis of mutant animals, we probed the roles of known Piwi-interacting RNA (piRNA) pathway components in the initiation and effector phases of transposon silencing. Squash associated physically with Piwi, and reductions in its expression led to modest transposon derepression without effects on piRNAs, consistent with an effector role. Alterations in Zucchini or Armitage reduced both Piwi protein and piRNAs, indicating functions in the formation of a stable Piwi RISC (RNA-induced silencing complex). Notably, loss of Zucchini or mutations within its catalytic domain led to accumulation of unprocessed precursor transcripts from flamenco, consistent with a role for this putative nuclease in piRNA biogenesis.
Genes & development 10/2010; 24(22):2499-504. · 12.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The qualitative and quantitative characterization of protein abundance profiles over a series of time points or a set of environmental conditions is becoming increasingly important. Using isobaric mass tagging experiments, mass spectrometry-based quantitative proteomics deliver accurate peptide abundance profiles for relative quantitation. Associated data analysis workflows need to provide tailored statistical treatment that (i) takes the correlation structure of the normalized peptide abundance profiles into account and (ii) allows inference of protein-level similarity. We introduce a suitable distance measure for relative abundance profiles, derive a statistical test for equality and propose a protein-level representation of peptide-level measurements. This yields a workflow that delivers a similarity ranking of protein abundance profiles with respect to a defined reference. All procedures have in common that they operate based on the true correlation structure that underlies the measurements. This optimizes power and delivers more intuitive and efficient results than existing methods that do not take these circumstances into account.
We use protein profile similarity screening to identify candidate proteins whose abundances are post-transcriptionally controlled by the Anaphase Promoting Complex/Cyclosome (APC/C), a specific E3 ubiquitin ligase that is a master regulator of the cell cycle. Results are compared with an established protein correlation profiling method. The proposed procedure yields a 50.9-fold enrichment of co-regulated protein candidates and a 2.5-fold improvement over the previous method.
A MATLAB toolbox is available from http://hci.iwr.uni-heidelberg.de/mip/proteomics.
[Show abstract][Hide abstract] ABSTRACT: In this work, we explore the potential of the metalloendopeptidase Lys-N for MALDI-MS/MS proteomics applications. Initially we digested a HEK293 cellular lysate with Lys-N and, for comparison, in parallel with the protease Lys-C. The resulting peptides were separated by strong cation exchange to enrich and isolate peptides containing a single N-terminal lysine. MALDI-MS/MS analysis of these peptides yielded CID spectra with clear and often complete sequence ladders of b-ions. To test the applicability for de novo sequencing we next separated an ostrich muscle tissue protein lysate by one-dimensional SDS-PAGE. A protein band at 42 kDa was in-gel digested with Lys-N. Relatively straightforward sequencing resulted in the de novo identification of the two ostrich proteins creatine kinase and actin. We therefore conclude that this method that combines Lys-N, strong cation exchange enrichment, and MALDI-MS/MS analysis provides a valuable alternative proteomics strategy.
[Show abstract][Hide abstract] ABSTRACT: Triply and doubly charged iTRAQ ( isobaric tagging for relative and absolute quantitation) labeled peptide cations from a tryptic peptide mixture of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications associated with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissociation (ETD) of triply charged or activated ETD (ETD and supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addition of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissociation (CID) of the product ion at m/ z 162 yielded the reporter ion at m/ z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quantitative information similar to that directly produced from CID. A 103 Da neutral loss species observed in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents.
Journal of Proteome Research 09/2008; 7(9):3643-8. · 5.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ligand binding to cell surface receptors initiates a cascade of signaling events regulated by dynamic phosphorylation events on a multitude of pathway proteins. Quantitative features, including intensity, timing, and duration of phosphorylation of particular residues, may play a role in determining cellular response, but experimental data required for analysis of these features have not previously been available. To understand the dynamic operation of signaling cascades, we have developed a method enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins in a time-resolved manner, downstream of epidermal growth factor receptor (EGFR) activation. Tryptic peptides from four different EGFR stimulation time points were labeled with four isoforms of the iTRAQ reagent to enable downstream quantification. After mixing of the labeled samples, tyrosine-phosphorylated peptides were immunoprecipitated with an anti-phosphotyrosine antibody and further enriched by IMAC before LC/MS/MS analysis. Database searching and manual confirmation of peptide phosphorylation site assignments led to the identification of 78 tyrosine phosphorylation sites on 58 proteins from a single analysis. Replicate analyses of a separate biological sample provided both validation of this first data set and identification of 26 additional tyrosine phosphorylation sites and 18 additional proteins. iTRAQ fragment ion ratios provided time course phosphorylation profiles for each site. The data set of quantitative temporal phosphorylation profiles was further characterized by self-organizing maps, which resulted in identification of several cohorts of tyrosine residues exhibiting self-similar temporal phosphorylation profiles, operationally defining dynamic modules in the EGFR signaling network consistent with particular cellular processes. The presence of novel proteins and associated tyrosine phosphorylation sites within these modules indicates additional components of this network and potentially localizes the topological action of these proteins. Additional analysis and modeling of the data generated in this study are likely to yield more sophisticated models of receptor tyrosine kinase-initiated signal transduction, trafficking, and regulation.
[Show abstract][Hide abstract] ABSTRACT: We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.