Joseph A. Loo

Studying noncovalent protein complexes by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry has been used to study protein interactions driven by noncovalent forces. The gentleness of the electrospray ionization process allows intact protein complexes to be directly detected by mass spectrometry. Evidence from the growing body of literature suggests that the ESI-MS observations for these weakly bound systems reflect, to some extent, the nature of the interaction found in the condensed phase. Stoichiometry of the complex can be easily obtained from the resulting mass spectrum because the molecular weight of the complex is directly measured. For the study of protein interactions, ESI-MS is complementary to other biophysical methods, such as NMR and analytical ultracentrifugation. However, mass spectrometry offers advantages in speed and sensitivity. The experimental variables that play a role in the outcome of ESI-MS studies of noncovalently bound complexes are reviewed. Several applications of ESI-MS are discussed, including protein interactions with metal ions and nucleic acids and subunit protein structures (quaternary structure).

Electrospray ionization mass spectrometry: a technology for studying noncovalent macromolecular complexes

Abstract Electrospray ionization mass spectrometry (ESI-MS) has demonstrated utility for the detection and study of weakly bound, noncovalent complexes, including protein interactions with inhibitors, cofactors, metal ions, carbohydrates, other peptides and proteins, enzyme–substrate pairings, and nucleic acid complexes. From the measurement of molecular mass of the intact complex and the individual binding partners, the binding stoichiometry can be derived. In many examples, the relative and absolute binding affinities can be deduced by the MS-based method. A review of the experimental principles of the method for studying noncovalent complexes, with emphasis on proteins, and the early studies that aided in the development of ESI-MS for this application are presented. Examples of protein complexes, such as the calcium-bound calmodulin-melittin complex, streptavidin homotetramer, and the enolase protein dimer are used to illustrate important features of the technique. A discussion on current and future applications of ESI-MS, such as the determination of the topology of macromolecular complexes, is provided.

Salivary Proteomics for Oral Cancer Biomarker Discovery

Purpose: This study aims to explore the presence of informative protein biomarkers in the human saliva proteome and to evaluate their potential for detection of oral squamous cell carcinoma (OSCC). Experimental Design: Whole saliva samples were collected from patients (n = 64) with OSCC and matched healthy subjects (n = 64). The proteins in pooled whole saliva samples of patients with OSCC (n = 16) and matched healthy subjects (n = 16) were profiled using shotgun proteomics based on C4 reversed-phase liquid chromatography for prefractionation, capillary reversed-phase liquid chromatography with quadruple time-of-flight mass spectrometry, and Mascot sequence database searching. Immunoassays were used for validation of the candidate biomarkers on a new group of OSCC (n = 48) and matched healthy subjects (n = 48). Receiver operating characteristic analysis was exploited to evaluate the diagnostic value of discovered candidate biomarkers for OSCC. Results: Subtractive proteomics revealed several salivary proteins at differential levels between the OSCC patients and matched control subjects. Five candidate biomarkers were successfully validated using immunoassays on an independent set of OSCC patients and matched healthy subjects. The combination of these candidate biomarkers yielded a receiver operating characteristic value of 93%, sensitivity of 90%, and specificity of 83% in detecting OSCC. Conclusion: Patient-based saliva proteomics is a promising approach to searching for OSCC biomarkers. The discovery of these new targets may lead to a simple clinical tool for the noninvasive diagnosis of oral cancer. Long-term longitudinal studies with large populations of individuals with oral cancer and those who are at high risk of developing oral cancer are needed to validate these potential biomarkers.

Use of Proteomics to Demonstrate a Hierarchical Oxidative Stress Response to Diesel Exhaust Particle Chemicals in a Macrophage Cell Line

Epidemiological studies demonstrate an association between short term exposure to ambient particulate matter (PM) and cardiorespiratory morbidity and mortality. Although the biological mechanisms of these adverse effects are unknown, emerging data suggest a key role for oxidative stress. Ambient PM and diesel exhaust particles (DEP) contain redox cycling organic chemicals that induce pro-oxidative and pro-inflammatory effects in the lung. These responses are suppressed by N-acetylcysteine (NAC), which directly complexes to electrophilic DEP chemicals and exert additional antioxidant effects at the cellular level. A proteomics approach was used to study DEP-induced responses in the macrophage cell line, RAW 264.7. We demonstrate that in the dose range 10–100 μg/ml, organic DEP extracts induce a progressive decline in the cellular GSH/GSSG ratio, in parallel with a linear increase in newly expressed proteins on the two-dimensional gel. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electrospray ionization-liquid chromatography/mass spectrometry/mass spectrometry analysis, 32 newly induced/NAC-suppressed proteins were identified. These include antioxidant enzymes (e.g. heme oxygenase-1 and catalase), pro-inflammatory components (e.g. p38MAPK and Rel A), and products of intermediary metabolism that are regulated by oxidative stress. Heme oxygenase-1 was induced at low extract dose and with minimal decline in the GSH/GSSG ratio, whereas MAP kinase activation required a higher chemical dose and incremental levels of oxidative stress. Moreover, at extract doses >50 μg/ml, there is a steep decline in cellular viability. These data suggest that DEP induce a hierarchical oxidative stress response in which some of these proteins may serve as markers for oxidative stress during PM exposures.

The Proteomes of Human Parotid and Submandibular/Sublingual Gland Salivas Collected as the Ductal Secretions

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

Target identification using drug affinity responsive target stability (DARTS)

Identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive target stability (DARTS), which takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. DARTS is universally applicable because it requires no modification of the drug and is independent of the mechanism of drug action. We demonstrate use of DARTS to identify known small-molecule–protein interactions and to reveal the eukaryotic translation initiation machinery as a molecular target for the longevity-enhancing plant natural product resveratrol. We envisage that DARTS will also be useful in global mapping of protein–metabolite interaction networks and in label-free screening of unlimited varieties of compounds for development as molecular imaging agents.

Peptide and protein analysis by electrospray ionization-mass spectrometry and capillary electrophoresis-mass spectrometry

The extension of mass spectrometry to high molecular weight biopolymers based upon electrospray ionization and the on-line combination with capillary electrophoresis is described. Electrospray ionization produces gas-phase intact multiply charged molecular ions of biomolecules from highly charged liquid droplets by a high electric field. For high molecular weight substances electrospray ionization results in a characteristic bell-shaped distribution of multiply charged ions, with each adjacent major peak in the spectrum differing by one charge. Multiply charged molecular ions of proteins with molecular weights greater than 130,000 have been observed with a quadrupole mass spectrometer of limited mass-to-charge range (m/z 1700). Molecular weights can be readily determined for large proteins with accuracies in the range of +/- 0.01 to 0.05%; at least an order of magnitude further improvement appears feasible with improved techniques and instrumentation. The electrospray ionization method is sensitive, presently requiring samples in the 100 fmol to 10 pmol range for proteins. Initial results combining rapid separations by capillary zone electrophoresis with on-line mass spectrometric detection via the electrospray ionization source are demonstrated for myoglobin and other proteins and polypeptides. The potential for extension of these methods to molecular weights on the order of 10(6) is discussed.

Cardiolipin defines the interactome of the major ADP/ATP carrier protein of the mitochondrial inner membrane

Defined mutations in the mitochondrial ADP/ATP carrier (AAC) are associated with certain types of progressive external ophthalmoplegia. AAC is required for oxidative phosphorylation (OXPHOS), and dysregulation of AAC has been implicated in apoptosis. Little is known about the AAC interactome, aside from a known requirement for the phospholipid cardiolipin (CL) and that it is thought to function as a homodimer. Using a newly developed dual affinity tag, we demonstrate that yeast AAC2 physically participates in several protein complexes of distinct size and composition. The respiratory supercomplex and several smaller AAC2-containing complexes, including other members of the mitochondrial carrier family, are identified here. In the absence of CL, most of the defined interactions are destabilized or undetectable. The absence of CL and/or AAC2 results in distinct yet additive alterations in respiratory supercomplex structure and respiratory function. Thus, a single lipid can significantly alter the functional interactome of an individual protein.

Carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins

Defensins are peptides that protect the host against microorganisms. Here we show that the θ-defensin retrocyclin 2 (RC2) inhibited influenza virus infection by blocking membrane fusion mediated by the viral hemagglutinin. RC2 was effective even after hemagglutinin attained a fusogenic conformation or had induced membrane hemifusion. RC2, a multivalent lectin, prevented hemagglutinin-mediated fusion by erecting a network of crosslinked and immobilized surface glycoproteins. RC2 also inhibited fusion mediated by Sindbis virus and baculovirus. Human β-defensin 3 and mannan-binding lectin also blocked viral fusion by creating a protective barricade of immobilized surface proteins. This general mechanism might explain the broad-spectrum antiviral activity of many multivalent lectins of the innate immune system.

Heme is involved in microRNA processing

MicroRNAs (miRNAs) regulate the expression of a large number of protein-coding genes. Their primary transcripts (pri-miRNAs) have to undergo multiple processing steps to reach the functional form. Little is known about how the processing of miRNAs is modulated. Here we show that the RNA-binding protein DiGeorge critical region-8 (DGCR8), which is essential for the first processing step, is a heme-binding protein. The association with heme promotes dimerization of DGCR8. The heme-bound DGCR8 dimer seems to trimerize upon binding pri-miRNAs and is active in triggering pri-miRNA cleavage, whereas the heme-free monomer is much less active. A heme-binding region of DGCR8 inhibits the pri-miRNA–processing activity of the monomer. This putative autoinhibition is overcome by heme. Our finding that heme is involved in pri-miRNA processing suggests that the gene-regulation network of miRNAs and signal-transduction pathways involving heme might be connected.