Christine C. Wu

A method for the comprehensive proteomic analysis of membrane proteins.

We describe a method that allows for the concurrent proteomic analysis of both membrane and soluble proteins from complex membrane-containing samples. When coupled with multidimensional protein identification technology (MudPIT), this method results in (i) the identification of soluble and membrane proteins, (ii) the identification of post-translational modification sites on soluble and membrane proteins, and (iii) the characterization of membrane protein topology and relative localization of soluble proteins. Overlapping peptides produced from digestion with the robust nonspecific protease proteinase K facilitates the identification of covalent modifications (phosphorylation and methylation). High-pH treatment disrupts sealed membrane compartments without solubilizing or denaturing the lipid bilayer to allow mapping of the soluble domains of integral membrane proteins. Furthermore, coupling protease protection strategies to this method permits characterization of the relative sidedness of the hydrophilic domains of membrane proteins.

The application of mass spectrometry to membrane proteomics

Membrane proteins perform some of the most important functions in the cell, including the regulation of cell signaling through surface receptors, cell–cell interactions, and the intracellular compartmentalization of organelles. Recent developments in proteomic strategies have focused on the inclusion of membrane proteins in high-throughput analyses. While slow and steady progress continues to be made in gel-based technologies, significant advances have been reported in non-gel shotgun methods using liquid chromatography coupled to mass spectrometry (LC/MS). These latter strategies facilitate the identification of large numbers of membrane proteins and modifications, and have the potential to provide insights into protein topology and orientation in membranes.

Direct Qualitative Analysis of Triacylglycerols by Electrospray Mass Spectrometry Using a Linear Ion Trap

Triacylglycerols (TAGs) isolated from a biological sample provide a challenge for mass spectrometric analysis because of the complexity of naturally occurring TAGs, which may contain different fatty acyl substituents resulting in a large number of molecular species having the identical elemental composition. We have investigated the use of mass spectrometry to obtain unambiguous information as to the individual TAG molecular species present in a complex mixture of triacylglycerols using a linear ion trap mass spectrometer. Ammonium adducts of TAGs, [M+NH4]+, were generated by electrospray ionization, which permitted the molecular weight of each TAG molecular species to be determined. The mechanisms involved in the decomposition of the [M+NH4]+ and subsequent fragment ions were investigated using deuterium labeling, MS/MS, and MS3 experiments. Collision induced decomposition of [M+NH4]+ ions resulted in the neutral loss of NH3 and an acyl side-chain (as a carboxylic acid) to generate a diacyl product ion. MS/MS data were used to identify each acyl group present for a given [M+NH4]+ ion, and this information could be combined with molecular weight data to identify possible TAG molecular species present in a biological extract. Subsequent MS3 experiments on the resultant diacyl product ions, which gave rise to acylium (RCO+) and related ions, enabled unambiguous TAG molecular assignments. These strategies of MS, MS/MS, and MS3 experiments were applied to identify components within a complex mixture of neutral lipids extracted from RAW 264. 7 cells.

Sequential Cyk-4 binding to ECT2 and FIP3 regulates cleavage furrow ingression and abscission during cytokinesis

Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytoskeleton and membrane compartments. Recently, FIP3 has been implicated in targeting of recycling endosomes to the mid‐body of dividing cells and is found required for abscission. Here, we demonstrate that the centralspindlin component Cyk‐4 is a FIP3‐binding protein. Furthermore, we show that FIP3 binds to Cyk‐4 at late telophase and that centralspindlin may be required for FIP3 recruitment to the mid‐body. We have mapped the FIP3‐binding region on Cyk‐4 and show that it overlaps with the ECT2‐binding domain. Finally, we demonstrate that FIP3 and ECT2 form mutually exclusive complexes with Cyk‐4 and that dissociation of ECT2 from the mid‐body at late telophase may be required for the recruitment of FIP3 and recycling endosomes to the cleavage furrow. Thus, we propose that centralspindlin complex not only regulates acto‐myosin ring contraction but also endocytic vesicle transport to the cleavage furrow and it does so through sequential interactions with ECT2 and FIP3.

GMx33: A Novel Family of trans-Golgi Proteins Identified by Proteomics

The known functions of the Golgi complex include the sorting, packaging, post‐translational modification, and transport of secretory proteins, membrane proteins, and lipids. Other functions still remain elusive to cell biologists. With the goal of identifying novel Golgi proteins, a proteomics project was undertaken to map the major proteins of the organelle using two‐dimensional gels, to identify the unknowns using tandem mass spectrometry, and to screen for Golgi residents using GFP‐fusion constructs. Multiple unknowns were identified, and the initial characterization of one of these proteins is reported here. GMx33α is a member of a conserved family of cytosolic Golgi‐associated proteins with no known homology to any known functional domain or protein. Biochemical analyses show that GMx33α differentially partitions into all phases of multiple detergent extractions, and two‐dimensional immunoblots reveal that there are multiple differentially modified forms of GMx33α associated with the Golgi, several of which are phosphorylated. Evidence suggests that these post‐translational modifications regulate its association with the Golgi. GMx33α was not found on Golgi budded vesicles, and immuno‐electron microscopy co‐localizes GMx33α to the trans‐face on the same three cisternae as TGN38 in normal rat kidney cells. This work represents the preliminary characterization of a novel family of trans‐Golgi‐associated proteins.

Proteomic Analysis of Two Functional States of the Golgi Complex in Mammary Epithelial Cells

Organellar compartments involved in secretion are expanded during the transition from late pregnancy (basal secretory state) to lactation (maximal secretory state) to accommodate for the increased secretory function required for copious milk production in mammary epithelial cells. The Golgi complex is a major organelle of the secretory pathway and functions to sort, package, distribute, and post‐translationally modify newly synthesized proteins and membrane lipids. These complex functions of the Golgi are reflected in the protein complement of the organelle. Therefore, using proteomics, the protein complements of Golgi fractions isolated at two functional states (basal and maximal) were compared to identify some of the molecular changes that occur during this transition. This global analysis has revealed that only a subset of the total proteins is up‐regulated from steady state during the transition. Identification of these proteins by tandem mass spectrometry has revealed several classes of proteins involved in the regulation of membrane fusion and secretion. This first installment of the functional proteomic analysis of the Golgi complex begins to define the molecular basis for the transition from basal to maximal secretion.

Human ARF4 expression rescues sec7 mutant yeast cells.

Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics.

Quantitative proteomic analysis of mammalian organisms using metabolically labeled tissues

Metabolic labeling of mammalian organisms with stable isotopes can be used to provide tissue-specific internal standards for use in quantitative proteomic analyses. This method provides an alternative and complementary strategy to covalent modification approaches using isotope-coded mass tags. This chapter will focus on the generation of the isotope-labeled tissues, the analysis of the sample using Multidimensional Protein Identification Technology, and the computational analysis of the mass spectrometric data acquired.

Computational Analysis of Quantitative Proteomics Data Using Stable Isotope Labeling

Over the last few years, new proteomics methods have been developed for making quantitative comparisons using stable isotope labeling. Although these methods have paved the way for quantitative proteomics, the analysis of these data is often the rate-limiting step. In fact, many analyzes are still carried out manually, which adds a level of subjectivity to the data that will vary between laboratories and even analysts. In this chapter, we have attempted to summarize several of the key steps necessary for an individual to automate the analysis of quantitative proteomics data. The approach is straightfoward to implement for an individual with moderate programming experience and used to process proteomics data in an objective manner.