Seattle Proteome Center (SPC)
NHLBI Proteomics Center at the Institute for Systems Biology

PILOT PROJECTS

Pilots are exploratory projects of limited duration. The objective of pilots is to rapidly establish proof-of-principle for an idea, technique or concept and thus serve as catalysts of new proteome research directions pursued by the Center. At any given time, the Center will maintain 2-4 pilot projects. If successful, pilots may be developed into full proteome research projects. New pilot projects will be selected by the Executive Committee from submissions from Center researchers or from the greater Seattle research community.

Pilot Project A: Isolation of DNA binding protein regulatory complexes associated with transcribing human Beta-globin locus in vivo - Mark Groudine (FHCRC), P.I.
The objective of this pilot project is to develop an approach based on quantitative proteomics for the systematic analysis of the proteins binding to gene regulatory sequences. A major problem with current approaches for probing gene regulatory complexes is that they are analyzed for known or proposed binding partners (e.g., DNA binding sequences) that are limited by scientific bias and limited quantities of purified complexes. The proposed strategy is expected to provide an unbiased view of the proteins interacting with gene regulatory sequences and to be able to distinguish proteins binding to specific DNA sequences from those binding non-specifically

Pilot project B: Analysis of protein-protein and protein-lipid interactions on the cytosolic surface of mammalian cell plasma membranes - John Glomset (UW), P.I.
The objective of this pilot is to develop a new approach to the systematic analysis of the proteins associated with lipid membranes in eukaryotic cells. The proteins of mammalian cell membranes include not only proteins that span the membrane lipid bilayer (transmembrane proteins), but also proteins that associate with only one side of the bilayer (peripheral membrane proteins). Some bind primarily to transmembrane proteins, some associate with membranes through covalently bound lipid anchors, some contain amphipathic regions that insert between the fatty acyl chains of membrane phosphoglycerides, and some bind primarily to the polar head groups of the phosphoglycerides. Beyond this, the protein and lipid compositions of different cell membranes can vary, and some membrane proteins and lipids can change in response to stimuli. This raises the possibility that the surfaces of membranes may act as "super scaffolds" that bind different groups of cytosolic proteins under different circumstances and promote specific interactions within these groups. This remarkable potential for complexity poses a major challenge for those who wish to use proteomics approaches to try to understand the molecular basis of protein-protein and protein-lipid interactions on membrane surfaces. This pilot project will explore the use of quantitative proteomics for the analysis of this functionally important class of proteins. This project will be carried out in the second year, and the detailed research plan will therefore be developed in July-September 2003 time frames.

Pilot Project C: Generation of a macrophage annotated peptide database - Alan Aderem (ISB), Elaine Raines (UW), David Goodlett (ISB), P.I.s
One of the central goals of the Center is the development of proteomics technologies to systematically study macrophage biology. The ability to rapidly and accurately generate quantitative protein profiles of differentially perturbed macrophages and different macrophage populations is a necessary prerequisite for this goal. We believe that the required sample throughput cannot be achieved as long as each peptide has to be sequenced anew in every profiling experiment. The need to repeatedly sequence the same peptides is a generic bottleneck in proteomics. In this pilot, we will explore a new strategy that attempts to obviate the need for repetitive sequencing of the same peptide. We have termed this the Annotated Peptide Database (APD) project. The essence of the APD project is the ability to generate quantitative profiles of peptides that will be identified by matching to a bar-coded peptide library generated from the cell type under investigation, in this case, the macrophage. If successful, such a database will provide a necessary tool for the high throughput, quantitative proteomic experiments to probe human clinical specimens and the well-characterized inflammatory response of the mouse in phase two of the program.

Pilot Project D - Michael Gelb (UW), P.I.
This new pilot will be initiated late in Year 1 or Year 2 of the program and will complement Technology Module D, quantitative analysis of protein complexes. This project will be headed by Dr. Mike Gelb, Department of Chemistry, University of Washington. He and his group will develop a new class of protein crosslinkers. The discriminating feature of these crosslinking reagents will be that they contain an isotope tag and an affinity tag so that the crosslinked peptides can be selectively isolated from complex peptide mixtures and quantified by isotope dilution theory. These reagents will be used to determine the protein interfaces in protein complexes and to determine changes in the stoichiometry of complexes.

Pilot Project E - Elaine Raines, Peter Gough (UW), P.I.s
Mass-spectrometry has been used to identify protein-protein interactions, but is limited by the inability to generate highly-purified protein complexes. The recent development of the tandem-affinity purification (TAP) tag methodology, in which “bait” proteins are expressed as a fusion protein containing a TAP eptiope tag that can be used for high-efficiency protein purification, has allowed the rapid analysis of cytoplasmic protein-protein interactions in eukaryotic cells. However, conditions to utilize this approach for the isolation of cell-surface protein complexes have not been established. In order to identify novel substrates for ADAM17, we will express a catalytically-inactive ADAM17 mutant as a fusion protein with a TAP epitope tag and utilize this system to establish a protocol for isolation of cell surface protein complexes.

Pilot Project F - Steve Hauschka, Charis Himeda (UW), P.I.s
This project seeks to identify transcription factors binding to the muscle creatine kinase (MCK) enhancer, and their associated protein complexes. Recently, the Trex-binding factor has been identified as Six4 using a DNA affinity isolation strategy and a recently described quantitative proteomics technique developed by the SPC. In that study, Six4 was identified from a background of nearly 1,000 unique proteins/protein groups (5), demonstrating the power and utility of quantitative mass spectrometry for the identification of site-specific DNA-binding factors in partially purified samples. This project seeks to utilize a similar approach to identify factors associated with the complete 206-bp MCK enhancer. Initial studies will concentrate on the selective purification of enhancer-binding complexes using magnetic beads coupled to DNA containing either a wild-type enhancer, or a control enhancer sequence mutated at each of the seven control elements. Both wild-type and control templates will contain a recognition site for the restriction enzyme PstI. Nuclear extracts from differentiated skeletal myocytes will be incubated with the immobilized templates to allow protein binding. After washing, the templates with associated factors will be cleaved using PstI. Recovery of known enhancer-binding factors will be assessed by gel mobility shift assays. Enhancer-binding factors and their associated protein complexes will then be identified using a quantitative proteomics strategy based on Isotope Coded Affinity Tag (ICAT) reagents and tandem mass spectrometry.

Pilot Project G: Chromatin Components at Imprinting Control Regions - Tony Krumm (UW), P.I.
The goal of this pilot project is to identify the protein components at impriniting control regions and chromatin boundaries/insulators. Our large-scale genomic survey using the ChIP-CHIP approach has recently identified a number of genomic regions that are required for the maintenance of transcriptionally active domains. These elements, initially identified through their association with the nuclear factor CTCF, encompass regions larger than 300 bp and harbor multiple functions that are mediated through cooperatively bound protein complexes. We will employ a DNA affinity purification protocol in conjunction with ICAT mass spectrometry to identify proteins that are recruited by the CCCTC-binding factor. Protein complexes will be isolated using immobilized templates. Templates with mutations that abolish CTCF binding will be used as a control for the non-specific binding of nuclear proteins.. Bound protein will be identified by quantitative mass spectrometry. The significance of the identified proteins in vivo will be assessed by chromatin immunoprecipitation experiments. This strategy will permit the identification of protein components that bind to the target region in cooperation with CTCF.

Pilot Project H: LIBRA – a software tool to identify ubiquitin-like modifiers (ULMs)- Brian Raught and Patrick Pedrioli, P.I.s
Modification by the ubiquitin-like modifiers (ULMs) represents a novel, poorly understood signaling mechanism. To understand how ULM modification affects the function of a particular protein conjugate, the modified target protein lysine residue(s) must be identified. Recent advances in mass spectrometry and accompanying software tools have allowed for the rapid identification of proteins in complex biological samples. However, standard peptide identification software cannot identify many types of ULM-modified (SUMO, URM1, FAT10, HUB1) peptides. We have thus developed a software tool, LIBRA/SUMmON, which uses user-specifiable search parameters for the identification of ULM target peptides. We propose to further develop this software platform for use with other types of post-translational modifications, to use this tool to scan available large scale MS/MS data for novel ULM modified peptides, and to make these tools available to the scientific community.

Pilot Project I: Cell Surface Scanning - Bernd Wollscheid, P.I.
This project will further develop and apply the cell surface glycocapturing technology towards the identification of cell surface proteins and their respective glycosites in various human and mouse cell lines, as well as in primary cells. As a alternate enrichment strategy, we will explore in the initial phase of this project novel magnetic hydrazide microbeads for covalent attachment to cell surface glycoproteins (in collaboration with Mitenyi Biotech). In an in vivo approach we will explore in addition a metabolic labeling strategy with a modified sugar that gets incorporated into cell surface glycoproteins, similarly allowing for their selective isolation and characterization (synthesis of the modified sugar in M.Gelb’s lab at UW). The final goal of this project is to combine the mature cell surface glycoprotein labeling strategy with quantitative proteomics methods. To do so, amino-reactive isobaric tags (iTRAQ) reagents (Applied Biosystems) and SILAC labeling reagents will be employed to simultaneously identify and provide relative quantitation of the protein samples in a proof-of-principle experiment. Time permitting, we would like to elucidate the role of newly identified key cell surface molecules in follow-up experiments as potential differentiation or bio-markers (GFP-tagging, TAP-tagging, generation of antibodies). In order to catalog, annotate and dissiminate the generated data we will develop the SISYPHUS GlycoBase, a relational database which will be available to the community as a repository for identified cell surface proteins and their glycosites.

Pilot Project J: Identify the acetylation status of proteins during erythroid differentiation - Marjorie Brand, Jeff Ranish, P.I.s
The objective of this pilot project is to develop a method based on quantitative proteomics to study post-translational modifications on proteins during erythroid differentiation. Post-translational modifications play crucial roles in regulating protein function, by modifying the activity state, localization, turnover and/or interactions with DNA and other proteins. The problem with current approaches is that they rely on the mass spectrometer to identify specific post-translational modifications in the context of an important background of non-modified proteins. Here we propose to circumvent these limitations by isolating peptides carrying a specific post-translational modification (e.g. acetylation) prior to their identification and quantification by mass spectrometry. The quantitative aspect of this project is crucial to reveal variations of post-translational modifications during erythroid differentiation. The proposed strategy will allow us to:

Finally, this method has the potential to be generalized to other types of post-translational modifications (i.e. methylation, ubiquitination, phosphorylation) as well as additional types of cells.

Pilot Project K: Quantitative proteomics by soluble polymer-based isotopic labeling – Weiguo Andy Tao, P.I.
A new strategy and reagents for selective isolation, labeling and identification of proteins from complex mixture and in living cells, termed soluble polymer-based isotopic labeling (SoPIL), has been developed. This is a continuation of work started by Andy Tao when he was postdoc at the Center. The central feature of the technology is that it utilizes a soluble nanopolymer as the support and platform for quantitative proteomics. The application of soluble polymer-based proteomics takes advantages of the homogeneity of solution-phase reaction, convenience of solid-phase capture and release process, and characteristics of cell-permeable nanoparticles. Soluble polymer was functionalized to selectively tag Cys-containing peptides followed by analyses by microcapillary liquid chromatography and tandem mass spectrometry for identification and relative quantities.

The method was applied in a proof-of-principle study to detect difference in protein abundance in standard protein mixtures and in two snake venoms. We observed for the first time extensive cleavage products by proteases in snake venoms. Quantitative analysis by SoPIL reagents allowed us to measure difference of protease activities in two snake venoms.

Pilot Project L: Identification of physiological substrates of MMP12;N-terminal peptide enrichment - Thomas Vaisar, P.I.
Obesity is the most common metabolic disease in industrialized society and is a major risk factor for diabetes, atherosclerosis, and the metabolic syndrome. The cellular hallmark of obesity is adipocyte proliferation and expansion. However, recent studies have demonstrated that macrophages accumulate in adipose tissue of obese mice and humans, raising the possibility that these inflammatory cells of the innate system play a previously unsuspected role in body weight regulation. Matrix metalloproteinases (MMPs) are implicated in cytokine release, matrix remodeling, angiogenesis, and the release of inflammatory mediators. Levels of MMP-12, a macrophage specific metalloelastase, increase dramatically in adipose tissue when mice are fed a high fat diet. To determine whether MMP-12 plays a role in obesity, we monitored the weight of wild-type and MMP-12-/- mice fed a high fat diet. Mice deficient in MMP-12 gained markedly more weight than wild-type mice, indicating that MMP-12 plays a previously unsuspected role in diet-induced obesity. These observations raise the exciting possibility that proteolytic activity of MMP-12 plays a critical role in body weight regulation. However, very little is known about the substrates for MMPs in vivo.

We propose three specific aims. First, on a model of known MMP-12 substrates, we will develop methodology for selective enrichment of N-terminal peptides (including the new termini produced by MMP-12 cleavage) by acylation of proteins' N-termini followed by depletion of internal peptides produced by further proteolytic degradation (e.g. trypsin digestion), and their identification by mass spectrometry. Using isotopic labeling of the acyl group we will establish a method for relative quantitation of the enriched N-terminal peptides. Second, we will confirm efficiency of this methodology for selective enrichment and relative quantitation of specific N-terminal peptides from complex mixture. After digestion of model proteins with MMP-12 we will inactivate MMP-12 and combine the digest with various amounts of plasma. Third, using the optimized approach developed in model system studies, we will use the wild-type macrophage cells (which lack detectable MMP-12) and the MMP-12 expressing cells to search for novel cellular substrates of the proteinase. Candidate proteolytic substrates will be confirmed using traditional biochemical approaches.

Pilot Project M: Quantitative MS analysis of crosslinked chromatin to annotate the human epigenome – Tony Krumm, P.I.
The goal of this project is to apply quantitative ICAT mass spectrometry on cross-linked, affinity-purified chromatin to identify proteins that co-localize in vivo with specific nuclear factors.

In the first quarter of this pilot project, we attempted to establish an experimental protocol for efficient crosslinking and stringent purification of crosslinked chromatin complexes on a small scale. To achieve this, the quality, specificity and efficiency of the antibody is the single most important factor. We have tested four different commercially available antibody preparations (anti-TAFIIp250 or anti-TFIIB) in chromatin immunoprecipitations. The efficiency of immunoprecipitation was assessed by a PCR analysis to measure the degree of recovery of active vs. inactive promoter sequences. We require an at least tenfold enrichment of active promoters. Only one antibody fulfilled these requirements. In the next quarter of this pilot project, the scale of the chromatin IP will be increased to obtain immunopurified crosslinked complexes for mass spec analysis.

Pilot Project N: Mechanisms of Regulation of L-type Calcium Channels - William Catterall and Michelle Emrick, P.I.s
We plan to examine the effects of adrenergic stimulation/inhibition and IGF signaling on L-type calcium channels in vivo. Firstprotein purification procedures will be adapted/improved to more automated methods using a recently acquired AKTA-purifier (FPLC). Then New Zealand White Rabbits will be treated with/without isoproterenol, propranolol, or insulin-like growth factor 1, tissue will be harvested, and L-type calcium channel (CaV1.1) purified from skeletal muscle tissue using new methods. Next mass spectrometric (MS) methods will be determined and optimized to quantitatively determine differences in posttranslational modification (PTM) on CaV1.1 under the four drug treatment conditions. Once PTM sites are identified, their functional significance will be analyzed by mutagenesis and whole-cell voltage clamp recordings in mammalian cells. To identify the modifying enzymes involved, predictive software will be used to identify putative enzymes and in vitro assays will be performed. Once enzymes are identified, pharmacological inhibitors and activators will be used in cell culture to confirm enzyme involvement in regulating channel activity. The various signaling pathways involved under the four drug treatment conditions will be compared and a signaling network for L-type calcium channel regulation will be proposed.

Pilot Project O: Mapping the dynamic composition of transcriptional regulatory complexes by mass spectrometry - Jeff Ranish, P.I.
In this pilot project, we propose to develop a quantitative mass spectrometric approach to comprehensively characterize the dynamic composition of transcription complexes associated with specific regulatory elements in yeast. This involves the development of an efficient approach for isolating specific transcriptional regulatory complexes from formaldehyde cross-linked cells, and comprehensively analyzing their composition by mass spectrometry.

Pilot Project P: Isolation and Characterization of RNA regulatory machines in yeast - John Aitchison and Aimee Dudley, P.I.s
We propose to an integrated system for 1) following the association of specific mRNA transcripts with the post-transcriptional regulatory machinery and 2) assaying in an unbiased way, the complete set of RNA transcripts associated with posttranscriptional factors. While the pilot project will focus on the transcripts and factors identified in a previous study, the general methods developed will be widely applicable to the analysis of a wide variety of protein-RNA interactions.