Dual 12/15- and 5-Lipoxygenase Deficiency in Macrophages Alters Arachidonic Acid Metabolism and Attenuates Peritonitis and Atherosclerosis in ApoE Knock-out Mice

Lipoxygenase (LO) enzymes catalyze the conversion of arachidonic acid (AA) into biologically active lipid mediators. Two members, 12/15-LO and 5-LO, regulate inflammatory responses and have been studied for their roles in atherogenesis. Both 12/15-LO and 5-LO inhibitors have been suggested as potential therapy to limit the development of atherosclerotic lesions. Here we used a genetic strategy to disrupt both 12/15-LO and 5-LO on an apolipoprotein E (apoE) atherosclerosis-susceptible background to study the impact of dual LO blockade in atherosclerosis and inflammation. Resident peritoneal macrophages are the major cell type that expresses both LO enzymes, and we verified their absence in dual LO-deficient mice. Examination of AA conversion by phorbol myristate acetate-primed and A23187-challenged macrophages from dual LO-deficient mice revealed extensive accumulation of AA with virtually no diversion into the most common cyclooxygenase (COX) products measured (prostaglandin E₂ and thromboxane B₂). Instead the COX-1 by-products 11-hydroxy-eicosatetraenoic acid (HETE) and 15-HETE were elevated. The interrelationship between the two LO pathways in combination with COX-1 inhibition (SC-560) also revealed striking patterns of unique substrate utilization. 5-LO- and dual LO-deficient mice exhibited an attenuated response to zymosan-induced peritoneal inflammation, emphasizing roles for 5-LO in regulating vascular permeability. We observed gender-specific attenuation of atheroma formation at 6 months of age at both the aortic root and throughout the entire aorta in chow-fed female dual LO-deficient mice. We propose that some of the inconsistent data obtained with single LO-deficient mice could be attributable to macrophage-specific patterns of altered AA metabolism.Lipoxygenase (LO)² enzymes are an important source of lipid mediators throughout the plant and animal kingdoms (1, 2). In mammals, these mediators are predominantly formed from arachidonic acid (AA) and act in various physiological and pathological contexts (1–3). Accordingly 5-LO and 12/15-LO are two members of the LO family involved in cardiovascular and inflammatory diseases expressed to variable degrees in several cell types of the myeloid lineage, and their expression is strictly regulated and incompletely understood (2, 4, 5). Despite considerable structural homology between 5-LO and 12/15-LO, both enzymes generate distinct products. The 5-LO metabolite leukotriene (LT) A₄ is precursor to the proinflammatory LTB₄ and cysteinyl LTs, which regulate leukocyte subset-specific chemotaxis (LTB₄) and vascular permeability (cysteinyl LTs), both crucial events during acute peritonitis (1, 6, 7). 12- and 15-HETE, end products synthesized by 12/15-LO, play potential roles in cellular chemotaxis, cancer growth, and inflammation (2, 8). Transcellular interaction products derived from both 12/15-LO and 5-LO, such as lipoxins and maresins, indicate that these enzymes can possess anti-inflammatory activities in innate immunity and the resolution of inflammation (9, 10).In mice, only one cell type is known to express substantial quantities of both 5-LO and 12/15-LO, the peritoneal macrophage (PMΦ) (2, 11, 12). However, differences in subcellular localization, trafficking, and activation (8, 12–16) of these two LOs indicate that they are independently regulated and not functionally coupled. Tissue-resident MΦ (such as PMΦ) represent the first line of defense against invading pathogens and activate the immunological and inflammatory response (17). These phagocytes are capable of elaborating a wide spectrum of bioactive lipid mediators from the LO and cyclooxygenase (COX) pathways. Little is known about the regulation and putative interdependence of these pathways. Some insight was gained using mice lacking 12/15-LO where substrate shunting from the 12/15-LO into the 5-LO pathway was observed (12).The generation of knock-out mice for 12/15-LO (12) and 5-LO (18) has enabled the study of these lipid mediator pathways in models of health and disease. Because 12/15-LO and 5-LO are primarily expressed in distinct hematopoietic cells, their implication in various inflammatory disorders and models of host defense mechanisms have been investigated (2, 3). Atherosclerosis, an inflammatory disease prevalent in societies with high dietary fat intake, is initiated by low density lipoprotein (LDL) retention in the vascular wall (19) and subsequent oxidative modification. This process greatly enhances the LDL atherogenic potential, and intriguingly 12/15-LO can contribute to lipoprotein oxidation (11, 20). Initial studies using 12/15-LO- and 5-LO-deficient mice indicated proatherogenic roles for these enzymes (20, 21). Additionally mice lacking the LTB₄ receptor BLT-1 exhibit protection in early atherogenesis (22), but subsequent data from our laboratory using 5-LO-deficient mouse models have not supported an involvement of 5-LO in atherogenesis (3, 23, 24). Here we studied the consequences of simultaneous 12/15-LO and 5-LO knock-out on peritoneal inflammation and atherosclerosis in apoE-deficient mice and surmised whether some of the capricious results in atherosclerotic lesion studies could be attributable to variable eicosanoid profiles.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Enhanced Virus Clearance by Early Inducible Lymphocytic Choriomeningitis Virus-Neutralizing Antibodies in Immunoglobulin-Transgenic Mice

Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin μ heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host’s capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin μ heavy and the κ light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.Against many cytopathic viruses such as poliovirus, influenza virus, rabies virus, and vesicular stomatitis virus, protective virus-neutralizing antibodies are generated early, within 1 week after infection (3, 31, 36, 44, 49). In contrast, several noncytopathic viruses (e.g., human immunodeficiency virus and hepatitis viruses B and C in humans or lymphocytic choriomeningitis virus [LCMV] in mice) elicit poor and delayed virus-neutralizing antibody responses (1, 7, 20, 24, 27, 35, 45, 48).In the mouse, the natural host of LCMV, the acute LCMV infection is predominantly controlled by cytotoxic T lymphocytes (CTLs) in an obligatory perforin-dependent manner (13, 18, 28, 50). In addition to the CTL response, LCMV-specific antibodies are generated. Early after infection (by day 8), a strong antibody response specific for the internal viral nucleoprotein (NP) is mounted (7, 19, 23, 28). These early LCMV NP-specific antibodies exhibit no virus-neutralizing capacity (7, 10). Results from studies of B-cell-depleted mice and B-cell-deficient mice implied that the early LCMV NP-specific antibodies are not involved in the clearance of LCMV (8, 11, 12, 40). Late after infection (between days 30 and day 60), LCMV-neutralizing antibodies develop (7, 19, 22, 28, 33); these antibodies are directed against the surface glycoprotein (GP) of LCMV (9, 10). LCMV-neutralizing antibodies have an important function in protection against reinfection (4, 6, 38, 41, 47).In some viral infections, subprotective virus-neutralizing antibody titers can enhance disease rather than promote host recovery (i.e., exhibit antibody-dependent enhancement of disease [ADE] [14, 15, 21, 46]). For example, neutralizing antibodies are involved in the resolution of a primary dengue virus infection and in the protection against reinfection. However, if subprotective neutralizing antibody titers are present at the time of reinfection, a severe form of the disease (dengue hemorrhagic fever/dengue shock syndrome [15, 21]), which might be caused by Fc receptor-mediated uptake of virus-antibody complexes leading to an enhanced infection of monocytes (15, 16, 25, 39), can develop. Similarly, an enhancement of disease after intracerebral (i.c.) LCMV infection was observed in mice which had been treated with virus-neutralizing antibodies before the virus challenge (6). ADE in LCMV-infected mice was either due to an enhanced infection of monocytes by Fc receptor-mediated uptake of antibody-virus complexes or due to CTL-mediated immunopathology caused by an imbalanced virus spread and CTL response.To analyze whether LCMV-neutralizing antibodies generated early after infection improve the host’s capacity to clear the virus or enhance immunopathological disease, immunoglobulin (Ig)-transgenic mice expressing LCMV-neutralizing IgM antibodies were generated. After LCMV infection of transgenic mice expressing the Ig heavy chain (H25 transgenic mice), LCMV-neutralizing serum antibodies were mounted within 8 days, which significantly improved the host’s capacity to eliminate LCMV. H25 transgenic mice did not show any signs of ADE after i.c. LCMV infection.Transgenic mice expressing the Ig heavy and light chains (HL25 transgenic mice) exhibited spontaneous LCMV-neutralizing serum antibodies and confirmed the protective role of preexisting LCMV-neutralizing antibodies, even though the neutralizing serum antibodies were of the IgM isotype. Similar to mice which had been treated with LCMV-neutralizing antibodies, HL25 transgenic mice developed an enhanced disease after i.c. LCMV infection, which indicated that ADE was due to an imbalance between virus spread and CTL response. Thus, the early-inducible LCMV-neutralizing antibody response significantly enhanced clearance of the acute infection without any risk of causing ADE.     

A Pipeline for Determining Protein–Protein Interactions and Proximities in the Cellular Milieu

It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from nonspecific interactors, preserving and isolating all unique interactions including those that are weak, transient, or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under nondenaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry. Here, we demonstrate that Stabilized Affinity Capture Mass Spectrometry allows us to stabilize and elucidate local, distant, and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.Insights into many cellular processes require detailed information about interactions between the participating proteins. However, the analysis of such interactions can be challenging because of the often-diverse physicochemical properties and the abundances of the constituent proteins, as well as the sometimes wide range of affinities and complex dynamics of the interactions. One of the key challenges has been acquiring information concerning transient, low affinity interactions in highly complex cellular milieux (3, 4).Methods that allow elucidation of such information include co-localization microscopy (5), fluorescence protein Förster resonance energy transfer (4), immunoelectron microscopy (5), yeast two-hybrid (6), and affinity capture (7, 8). Among these, affinity capture (AC)¹ has the unique potential to detect all specific in vivo interactions simultaneously, including those that interact both directly and indirectly. In recent times, the efficacy of such affinity isolation experiments has been greatly enhanced through the use of sensitive modern mass spectrometric protein identification techniques (9). Nevertheless, AC suffers from several shortcomings. These include the problem of 1) distinguishing specific from nonspecific interactors (10, 11); 2) preserving and isolating all unique interactions including those that are weak and/or transient, as well as those that exchange rapidly (10, 12, 13); and 3) differentiating proximal from more distant interactions (14).We describe here an approach to address these issues, which makes use of chemical stabilization of protein assemblies in the complex cellular milieu prior to AC. Chemical stabilization is an emerging technique for stabilizing and elucidating protein associations both in vitro (15–20) and in vivo (3, 12, 14, 21–29), with mass spectrometric (MS) readout of the AC proteins and their connectivities. Such chemical stabilization methods are indeed well-established and are often used in electron microscopy for preserving complexes and subcellular structures both in the cellular milieu (3) and in purified complexes (30, 31), wherein the most reliable, stable, and established stabilization reagents is glutaraldehyde. Recently, glutaraldehyde has been applied in the “GraFix” protocol in which purified protein complexes are subjected to centrifugation through a density gradient that also contains a gradient of glutaraldehyde (30, 31), allowing for optimal stabilization of authentic complexes and minimization of nonspecific associations and aggregation. GraFix has also been combined with mass spectrometry on purified complexes bound to EM grids to obtain a compositional analysis of the complexes (32), thereby raising the possibility that glutaraldehyde can be successfully utilized in conjunction with AC in complex cellular milieux directly.In this work, we present a robust pipeline for determining specific protein-protein interactions and proximities from cellular milieux. The first steps of the pipeline involve the well-established techniques of flash freezing the cells of interest in liquid nitrogen and cryomilling, which have been known for over a decade (33, 34) to preserve the cellular environment, as well as having shown outstanding performance when used in analysis of macromolecular interactions in yeast (35–39), bacterial (40, 41), trypanosome (42), mouse (43), and human (44–47) systems. The resulting frozen powder, composed of intact micron chunks of cells that have great surface area and outstanding solvent accessibility, is well suited for rapid low temperature chemical stabilization using glutaraldehyde. We selected glutaraldehyde for our procedure based on the fact that it is a very reactive stabilizing reagent, even at lower temperatures, and because it has already been shown to stabilize enzymes in their functional state (48–50). We employed highly efficient, rapid, single stage affinity capture (36, 51) for isolation and bottom-up MS for analysis of the macromolecular assemblies of interest (52–54). For convenience, we have termed this approach Stabilized Affinity-Capture Mass Spectrometry (SAC-MS).     

High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8–9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.Neuropeptides and hormones comprise a diverse class of signaling molecules involved in numerous essential physiological processes, including analgesia, reward, food intake, learning and memory (1). Disorders of the neurosecretory and neuroendocrine systems influence many pathological processes. For example, obesity results from failure of energy homeostasis in association with endocrine alterations (2, 3). Previous work from our lab used crustaceans as model organisms found that multiple neuropeptides were implicated in control of food intake, including RFamides, tachykinin related peptides, RYamides, and pyrokinins (4–6).Crustacean hyperglycemic hormone (CHH)¹ family neuropeptides play a central role in energy homeostasis of crustaceans (7–17). Hyperglycemic response of the CHHs was first reported after injection of crude eyestalk extract in crustaceans. Based on their preprohormone organization, the CHH family can be grouped into two sub-families: subfamily-I containing CHH, and subfamily-II containing molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). The preprohormones of the subfamily-I have a CHH precursor related peptide (CPRP) that is cleaved off during processing; and preprohormones of the subfamily-II lack the CPRP (9). Uncovering their physiological functions will provide new insights into neuroendocrine regulation of energy homeostasis.Characterization of CHH-family neuropeptides is challenging. They are comprised of more than 70 amino acids and often contain multiple post-translational modifications (PTMs) and complex disulfide bridge connections (7). In addition, physiological concentrations of these peptide hormones are typically below picomolar level, and most crustacean species do not have available genome and proteome databases to assist MS-based sequencing.MS-based neuropeptidomics provides a powerful tool for rapid discovery and analysis of a large number of endogenous peptides from the brain and the central nervous system. Our group and others have greatly expanded the peptidomes of many model organisms (3, 18–33). For example, we have discovered more than 200 neuropeptides with several neuropeptide families consisting of as many as 20–40 members in a simple crustacean model system (5, 6, 25–31, 34). However, a majority of these neuropeptides are small peptides with 5–15 amino acid residues long, leaving a gap of identifying larger signaling peptides from organisms without sequenced genome. The observed lack of larger size peptide hormones can be attributed to the lack of effective de novo sequencing strategies for neuropeptides larger than 4 kDa, which are inherently more difficult to fragment using conventional techniques (34–37). Although classical proteomics studies examine larger proteins, these tools are limited to identification based on database searching with one or more peptides matching without complete amino acid sequence coverage (36, 38).Large populations of neuropeptides from 4–10 kDa exist in the nervous systems of both vertebrates and invertebrates (9, 39, 40). Understanding their functional roles requires sufficient molecular knowledge and a unique analytical approach. Therefore, developing effective and reliable methods for de novo sequencing of large neuropeptides at the individual amino acid residue level is an urgent gap to fill in neurobiology. In this study, we present a multifaceted MS strategy aimed at high-definition de novo sequencing and comprehensive characterization of the CHH-family neuropeptides in crustacean central nervous system. The high-definition de novo sequencing was achieved by a combination of three methods: (1) enzymatic digestion and LC-tandem mass spectrometry (MS/MS) bottom-up analysis to generate detailed sequences of proteolytic peptides; (2) off-line LC fractionation and subsequent top-down MS/MS to obtain high-quality fragmentation maps of intact peptides; and (3) on-line LC coupled to top-down MS/MS to allow rapid sequence analysis of low abundance peptides. Combining the three methods overcomes the limitations of each, and thus offers complementary and high-confidence determination of amino acid residues. We report the complete sequence analysis of six CHH-family neuropeptides including the discovery of two novel peptides. With the accurate molecular information, MALDI imaging and ion mobility MS were conducted for the first time to explore their anatomical distribution and biochemical properties.     

From Whole-body Sections Down to Cellular Level,Multiscale Imaging of Phospholipids by MALDI Mass Spectrometry

Significant progress in instrumentation and sample preparation approaches have recently expanded the potential of MALDI imaging mass spectrometry to the analysis of phospholipids and other endogenous metabolites naturally occurring in tissue specimens. Here we explore some of the requirements necessary for the successful analysis and imaging of phospholipids from thin tissue sections of various dimensions by MALDI time-of-flight mass spectrometry. We address methodology issues relative to the imaging of whole-body sections such as those cut from model laboratory animals, sections of intermediate dimensions typically prepared from individual organs, as well as the requirements for imaging areas of interests from these sections at a cellular scale spatial resolution. We also review existing limitations of MALDI imaging MS technology relative to compound identification. Finally, we conclude with a perspective on important issues relative to data exploitation and management that need to be solved to maximize biological understanding of the tissue specimen investigated.Since its introduction in the late 90s (1), MALDI imaging mass spectrometry (MS) technology has witnessed a phenomenal expansion. Initially introduced for the mapping of intact proteins from fresh frozen tissue sections (2), imaging MS is now routinely applied to a wide range of different compounds including peptides, proteins, lipids, metabolites, and xenobiotics (3–7). Numerous compound-specific sample preparation protocols and analytical strategies have been developed. These include tissue sectioning and handling (8–14), automated matrix deposition approaches and data acquisition strategies (15–21), and the emergence of in situ tissue chemistries (22–25). Originally performed on sections cut from fresh frozen tissue specimens, methodologies incorporating an in situ enzymatic digestion step prior to matrix application have been optimized to access the proteome locked in formalin-fixed paraffin-embedded tissue biopsies (25–29). The possibility to use tissues preserved using non-cross-linking approaches has also been demonstrated (30–32). These methodologies are of high importance for the study of numerous diseases because they potentially allow the retrospective analysis for biomarker validation and discovery of the millions of tissue biopsies currently stored worldwide in tissue banks and repositories.In the past decade, instrumentation for imaging MS has also greatly evolved. Whereas the first MS images were collected with time-of-flight instruments (TOF) capable of repetition rates of a few hertz, modern systems are today capable of acquiring data in the kilohertz range and above with improved sensitivity, mass resolving power, and accuracy, significantly reducing acquisition time and improving image quality (33, 34). Beyond time-of-flight analyzers, other MALDI-based instruments have been used such as ion traps (35–37), Qq TOF instruments (38–40), and trap-TOF (16, 41). Ion mobility technology has also been used in conjunction with imaging MS (42–44). More recently, MALDI FT/ICR and Orbitrap mass spectrometers have been demonstrated to be extremely valuable instruments for the performance of imaging MS at very high mass resolving power (45–47). These non-TOF-based systems have proven to be extremely powerful for the imaging of lower molecular weight compounds such as lipids, drugs, and metabolites. Home-built instrumentation and analytical approaches to probe tissues at higher spatial resolution (1–10 μm) have also been described (48–50). In parallel to instrumentation developments, automated data acquisition, image visualization, and processing software packages have now also been developed by most manufacturers.To date, a wide range of biological systems have been studied using imaging MS as a primary methodology. Of strong interest are the organization and identification of the molecular composition of diseased tissues in direct correlation with the underlying histology and how it differs from healthy tissues. Such an approach has been used for the study of cancers (51–54), neurologic disorders (55–57), and other diseases (58, 59). The clinical potential of the imaging MS technology is enormous (7, 60, 61). Results give insights into the onset and progression of diseases, identify novel sets of disease-specific markers, and can provide a molecular confirmation of diagnosis as well as aide in outcome prediction (62–64). Imaging MS has also been extensively used to study the development, functioning, and aging of different organs such as the kidney, prostate, epididymis, and eye lens (65–70). Beyond the study of isolated tissues or organs, whole-body sections from several model animals such as leeches, mice, and rats have been investigated (71–74). For these analyses, specialized instrumentation and protocols are necessary for tissue sectioning and handling (72, 73). Whole-body imaging MS opens the door to the study of the localization and accumulation of administered pharmaceuticals and their known metabolites at the level of entire organisms as well as the monitoring of their efficacy or toxicity as a function of time or dose (72, 73, 75, 76).There is considerable interest in determining the identification and localization of small biomolecules such as lipids in tissues because they are involved in many essential biological functions including cell signaling, energy storage, and membrane structure and function. Defects in lipid metabolism play a role in many diseases such as muscular dystrophy and cardiovascular disease. Phospholipids in tissues have been intensively studied by several groups (37, 40, 77–83). In this respect, for optimal recovery of signal, several variables such as the choice of matrix for both imaging and fragmentation, solvent system, and instrument polarity have been investigated (20, 84). Particularly, the use of lithium cation adducts to facilitate phospholipid identification by tandem MS directly from tissue has also been reported (85). Of significant interest is the recent emergence of two new solvent-free matrix deposition approaches that perform exceptionally well for phospholipid imaging analyses. The first approach, described by Hankin et al. (86), consists in depositing the matrix on the sections through a sublimation process. The described sublimation system consists of sublimation glassware, a heated sand or oil bath (100–200 °C), and a primary vacuum pump (∼5 × 10⁻² torr). Within a few minutes of initiating the sublimation process, an exceptionally homogeneous film of matrix forms on the section. The thickness of the matrix may be controlled by regulating pressure, temperature, and sublimation time. The second approach, described by Puolitaival et al.(87), uses a fine mesh sieve (≤20 μm) to filter finely ground matrix on the tissue sections. Agitation of the sieve results in passage of the matrix through the mesh and the deposition of a fairly homogeneous layer of submicrometer matrix crystals of the surface of the sections. The matrix density on the sections is controlled by direct observation using a standard light microscope. This matrix deposition approach was also found to be ideal to image certain drug compounds (88, 89). Both strategies allow very rapid production of homogeneous matrix coatings on tissue sections with a fairly inexpensive setup. Signal recovery was found to be comparable with those obtained by conventional spray deposition. With the appropriate size sublimation device or sieve, larger sections with dimensions of several centimeters such as those cut from mouse or rat whole bodies can also be rapidly and homogeneously coated.Here we present several examples of MALDI imaging MS of phospholipids from tissue sections using TOF mass spectrometers over a wide range of dimensions from whole-body sections (several centimeters), to individual organs (several millimeters), down to high spatial resolution imaging of selected tissue areas (hundreds of micrometers) at 10-μm lateral resolution and below. For all of these dimension ranges, technological considerations and practical aspects are discussed. In light of the imaging MS results, we also address issues faced for compound identification by tandem MS analysis performed directly on the sections. Finally, we discuss under “Perspective” our vision of the future of the field as well as the technological improvements and analytical tools that need to be improved upon and developed.     

Infection of Glial Cells by the Human Polyomavirus JC Is Mediated by an N-Linked Glycoprotein Containing Terminal α(2-6)-Linked Sialic Acids

The human JC polyomavirus (JCV) is the etiologic agent of the fatal central nervous system (CNS) demyelinating disease progressive multifocal leukoencephalopathy (PML). PML typically occurs in immunosuppressed patients and is the direct result of JCV infection of oligodendrocytes. The initial event in infection of cells by JCV is attachment of the virus to receptors present on the surface of a susceptible cell. Our laboratory has been studying this critical event in the life cycle of JCV, and we have found that JCV binds to a limited number of cell surface receptors on human glial cells that are not shared by the related polyomavirus simian virus 40 (C. K. Liu, A. P. Hope, and W. J. Atwood, J. Neurovirol. 4:49–58, 1998). To further characterize specific JCV receptors on human glial cells, we tested specific neuraminidases, proteases, and phospholipases for the ability to inhibit JCV binding to and infection of glial cells. Several of the enzymes tested were capable of inhibiting virus binding to cells, but only neuraminidase was capable of inhibiting infection. The ability of neuraminidase to inhibit infection correlated with its ability to remove both α(2-3)- and α(2-6)-linked sialic acids from glial cells. A recombinant neuraminidase that specifically removes the α(2-3) linkage of sialic acid had no effect on virus binding or infection. A competition assay between virus and sialic acid-specific lectins that recognize either the α(2-3) or the α(2-6) linkage revealed that JCV preferentially interacts with α(2-6)-linked sialic acids on glial cells. Treatment of glial cells with tunicamycin, but not with benzyl N-acetyl-α-d-galactosaminide, inhibited infection by JCV, indicating that the sialylated JCV receptor is an N-linked glycoprotein. As sialic acid containing glycoproteins play a fundamental role in mediating many virus-cell and cell-cell recognition processes, it will be of interest to determine what role these receptors play in the pathogenesis of PML.Approximately 70% of the human population worldwide is seropositive for JC virus (JCV). Like other polyomaviruses, JCV establishes a lifelong latent or persistent infection in its natural host (40, 49, 50, 68, 72). Reactivation of JCV in the setting of an underlying immunosuppressive illness, such as AIDS, is thought to lead to virus dissemination to the central nervous system (CNS) and subsequent infection of oligodendrocytes (37, 40, 66, 68). Reactivation of latent JCV genomes already present in the CNS has also been postulated to contribute to the development of progressive multifocal leukoencephalopathy (PML) following immunosuppression (19, 48, 55, 70, 75). Approximately 4 to 6% of AIDS patients will develop PML during the course of their illness (10). In the CNS, JCV specifically infects oligodendrocytes and astrocytes. Outside the CNS, JCV genomes have been identified in the urogenital system, in the lymphoid system, and in B lymphocytes (2, 17, 18, 30, 47, 59). In vitro, JCV infects human glial cells and, to a limited extent, human B lymphocytes (3, 4, 39, 41, 42). Recently, JCV infection of tonsillar stromal cells and CD34⁺ B-cell precursors has been described (47). These observations have led to the suggestion that JCV may persist in a lymphoid compartment and that B cells may play a role in trafficking of JCV to the CNS (4, 30, 47).Virus-receptor interactions play a major role in determining virus tropism and tissue-specific pathology associated with virus infection. Viruses that have a very narrow host range and tissue tropism, such as JCV, are often shown to interact with high affinity to a limited number of specific receptors present on susceptible cells (26, 44). In some instances, virus tropism is strictly determined by the presence of specific receptors that mediate binding and entry (7, 16, 27, 35, 46, 53, 56, 67, 73, 74, 76). In other instances, however, successful entry into a cell is necessary but not sufficient for virus growth (5, 8, 45, 57). In these cases, additional permissive factors that interact with viral regulatory elements are required.The receptor binding characteristics of several polyomaviruses have been described. The mouse polyomavirus (PyV) receptor is an N-linked glycoprotein containing terminal α(2-3)-linked sialic acid (12–14, 22, 28). Both the large and small plaque strains of PyV recognize α(2-3)-linked sialic acid. The small-plaque strain also recognizes a branched disialyl structure containing α(2-3)- and α(2-6)-linked sialic acids. Neither strain recognizes straight-chain α(2-6)-linked sialic acid. The ability of the large- and small-plaque strains of PyV to differentially recognize these sialic acid structures has been precisely mapped to a single amino acid in the major virus capsid protein VP1 (21). The large-plaque strains all contain a glycine at amino acid position 92 in VP1, and the small-plaque strains all contain a negatively charged glutamic acid at this position (21). In addition to forming small or large plaques, these strains also differ in the ability to induce tumors in mice (20). This finding suggests that receptor recognition plays an important role in the pathogenesis of PyV.The cell surface receptor for lymphotropic papovavirus (LPV) is an O-linked glycoprotein containing terminal α(2-6)-linked sialic acid (26, 33, 34). Infection with LPV is restricted to a subset of human B-cell lines, and recognition of specific receptors is a major determinant of the tropism of LPV for these cells (26).Unlike the other members of the polyomavirus family, infection of cells by simian virus 40 (SV40) is independent of cell surface sialic acids. Instead, SV40 infection is mediated by major histocompatibility complex (MHC)-encoded class I proteins (5, 11). MHC class I proteins also play a role in mediating the association of SV40 with caveolae, a prerequisite for successful targeting of the SV40 genome to the nucleus of a cell (1, 63). Not surprisingly, SV40 has been shown not to compete with the sialic acid-dependent polyomaviruses for binding to host cells (15, 26, 38, 58).Very little is known about the early steps of JCV binding to and infection of glial cells. Like other members of the polyomavirus family, JCV is known to interact with cell surface sialic acids (51, 52). A role for sialic acids in mediating infection of glial cells has not been described. It is also not known whether the sialic acid is linked to a glycoprotein or a glycolipid. In a previous report, we demonstrated that JCV bound to a limited number of cell surface receptors on SVG cells that were not shared by the related polyomavirus SV40 (38). In this report, we demonstrate that virus binding to and infection of SVG cells is dependent on an N-linked glycoprotein containing terminal α(2-3)- and α(2-6)-linked sialic acids. Competitive binding assays with sialic acid-specific lectins suggest that the virus preferentially interacts with α(2-6)-linked sialic acids. We are currently evaluating the role of this receptor in determining the tropism of JCV for glial cells and B cells.     

Coomassie Blue as a Near-infrared Fluorescent Stain: A Systematic Comparison With Sypro Ruby for In-gel Protein Detection

Quantitative proteome analyses suggest that the well-established stain colloidal Coomassie Blue, when used as an infrared dye, may provide sensitive, post-electrophoretic in-gel protein detection that can rival even Sypro Ruby. Considering the central role of two-dimensional gel electrophoresis in top-down proteomic analyses, a more cost effective alternative such as Coomassie Blue could prove an important tool in ongoing refinements of this important analytical technique. To date, no systematic characterization of Coomassie Blue infrared fluorescence detection relative to detection with SR has been reported. Here, seven commercial Coomassie stain reagents and seven stain formulations described in the literature were systematically compared. The selectivity, threshold sensitivity, inter-protein variability, and linear-dynamic range of Coomassie Blue infrared fluorescence detection were assessed in parallel with Sypro Ruby. Notably, several of the Coomassie stain formulations provided infrared fluorescence detection sensitivity to <1 ng of protein in-gel, slightly exceeding the performance of Sypro Ruby. The linear dynamic range of Coomassie Blue infrared fluorescence detection was found to significantly exceed that of Sypro Ruby. However, in two-dimensional gel analyses, because of a blunted fluorescence response, Sypro Ruby was able to detect a few additional protein spots, amounting to 0.6% of the detected proteome. Thus, although both detection methods have their advantages and disadvantages, differences between the two appear to be small. Coomassie Blue infrared fluorescence detection is thus a viable alternative for gel-based proteomics, offering detection comparable to Sypro Ruby, and more reliable quantitative assessments, but at a fraction of the cost.Gel electrophoresis is an accessible, widely applicable and mature protein resolving technology. As the original top-down approach to proteomic analyses, among its many attributes the high resolution achievable by two dimensional gel-electrophoresis (2DE)¹ ensures that it remains an effective analytical technology despite the appearance of alternatives. However, in-gel detection remains a limiting factor for gel-based analyses; available technology generally permits the detection and quantification of only relatively abundant proteins (35). Many critical components in normal physiology and also disease may be several orders of magnitude less abundant and thus below the detection threshold of in-gel stains, or indeed most techniques. Pre- and post-fractionation technologies have been developed to address this central issue in proteomics but these are not without limitations (1–5). Thus improved detection methods for gel-based proteomics continue to be a high priority, and the literature is rich with different in-gel detection methods and innovative improvements (6–34). This history of iterative refinement presents a wealth of choices when selecting a detection strategy for a gel-based proteomic analysis (35).Perhaps the best known in-gel detection method is the ubiquitous Coomassie Blue (CB) stain; CB has served as a gel stain and protein quantification reagent for over 40 years. Though affordable, robust, easy to use, and compatible with mass spectrometry (MS), CB staining is relatively insensitive. In traditional organic solvent formulations, CB detects ∼ 10 ng of protein in-gel, and some reports suggest poorer sensitivity (27, 29, 36, 37). Sensitivity is hampered by relatively high background staining because of nonspecific retention of dye within the gel matrix (32, 36, 38, 39). The development of colloidal CB (CCB) formulations largely addressed these limitations (12); the concentration of soluble CB was carefully controlled by sequestering the majority of the dye into colloidal particles, mediated by pH, solvent, and the ionic strength of the solution. Minimizing soluble dye concentration and penetration of the gel matrix mitigated background staining, and the introduction of phosphoric acid into the staining reagent enhanced dye-protein interactions (8, 12, 40), contributing to an in-gel staining sensitivity of 5–10 ng protein, with some formulations reportedly yielding sensitivities of 0.1–1 ng (8, 12, 22, 39, 41, 42). Thus CCB achieved higher sensitivity than traditional CB staining, yet maintained all the advantages of the latter, including low cost and compatibility with existing densitometric detection instruments and MS. Although surpassed by newer methods, the practical advantages of CCB ensure that it remains one of the most common gel stains in use.Fluorescent stains have become the routine and sensitive alternative to visible dyes. Among these, the ruthenium-organometallic family of dyes have been widely applied and the most commercially well-known is Sypro Ruby (SR), which is purported to interact noncovalently with primary amines in proteins (15, 18, 19, 43). Chief among the attributes of these dyes is their high sensitivity. In-gel detection limits of < 1 ng for some proteins have been reported for SR (6, 9, 14, 44, 45). Moreover, SR staining has been reported to yield a greater linear dynamic range (LDR), and reduced interprotein variability (IPV) compared with CCB and silver stains (15, 19, 46–49). SR is easy to use, fully MS compatible, and relatively forgiving of variations in initial conditions (6, 15). The chief consequence of these advances remains high cost; SR and related stains are notoriously expensive, and beyond the budget of many laboratories. Furthermore, despite some small cost advantage relative to SR, none of the available alternatives has been consistently and quantitatively demonstrated to substantially improve on the performance of SR under practical conditions (9, 50).Notably, there is evidence to suggest that CCB staining is not fundamentally insensitive, but rather that its sensitivity has been limited by traditional densitometric detection (50, 51). When excited in the near IR at ∼650 nm, protein-bound CB in-gel emits light in the range of 700–800 nm. Until recently, the lack of low-cost, widely available and sufficiently sensitive infrared (IR)-capable imaging instruments prevented mainstream adoption of in-gel CB infrared fluorescence detection (IRFD); advances in imaging technology are now making such instruments far more accessible. Initial reports suggested that IRFD of CB-stained gels provided greater sensitivity than traditional densitometric detection (50, 51). Using CB R250, in-gel IRFD was reported to detect as little as 2 ng of protein in-gel, with a LDR of about an order of magnitude (2 to 20 ng, or 10 to 100 ng in separate gels), beyond which the fluorescent response saturated into the μg range (51). Using the G250 dye variant, it was determined that CB-IRFD of 2D gels detected ∼3 times as many proteins as densitometric imaging, and a comparable number of proteins as seen by SR (50). This study also concluded that CB-IRFD yielded a significantly higher signal to background ratio (S/BG) than SR, providing initial evidence that CB-IRFD may be superior to SR in some aspects of stain performance (50).Despite this initial evidence of the viability of CB-IRF as an in-gel protein detection method, a detailed characterization of this technology has not yet been reported. Here a more thorough, quantitative characterization of CB-IRFD is described, establishing its lowest limit of detection (LLD), IPV, and LDR in comparison to SR. Finally a wealth of modifications and enhancements of CCB formulations have been reported (8, 12, 21, 24, 26, 29, 40, 41, 52–54), and likewise there are many commercially available CCB stain formulations. To date, none of these formulations have been compared quantitatively in terms of their relative performance when detected using IRF. As a general detection method for gel-based proteomics, CB-IRFD was found to provide comparable or even slightly superior performance to SR according to most criteria, including sensitivity and selectivity (50). Furthermore, in terms of LDR, CB-IRFD showed distinct advantages over SR. However, assessing proteomes resolved by 2DE revealed critical distinctions between CB-IRFD and SR in terms of protein quantification versus threshold detection: neither stain could be considered unequivocally superior to the other by all criteria. Nonetheless, IRFD proved the most sensitive method of detecting CB-stained protein in-gel, enabling high sensitivity detection without the need for expensive reagents or even commercial formulations. Overall, CB-IRFD is a viable alternative to SR and other mainstream fluorescent stains, mitigating the high cost of large-scale gel-based proteomic analyses, making high sensitivity gel-based proteomics accessible to all labs. With improvements to CB formulations and/or image acquisition instruments, the performance of this detection technology may be further enhanced.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.Protein–protein interactions (PPIs)¹ play a key role in defining protein functions in biological systems. Aberrant PPIs can have drastic effects on biochemical activities essential to cell homeostasis, growth, and proliferation, and thereby lead to various human diseases (1). Consequently, PPI interfaces have been recognized as a new paradigm for drug development. Therefore, mapping PPIs and their interaction interfaces in living cells is critical not only for a comprehensive understanding of protein function and regulation, but also for describing the molecular mechanisms underlying human pathologies and identifying potential targets for better therapeutics.Several strategies exist for identifying and mapping PPIs, including yeast two-hybrid, protein microarray, and affinity purification mass spectrometry (AP-MS) (2–5). Thanks to new developments in sample preparation strategies, mass spectrometry technologies, and bioinformatics tools, AP-MS has become a powerful and preferred method for studying PPIs at the systems level (6–9). Unlike other approaches, AP-MS experiments allow the capture of protein interactions directly from their natural cellular environment, thus better retaining native protein structures and biologically relevant interactions. In addition, a broader scope of PPI networks can be obtained with greater sensitivity, accuracy, versatility, and speed. Despite the success of this very promising technique, AP-MS experiments can lead to the loss of weak/transient interactions and/or the reorganization of protein interactions during biochemical manipulation under native purification conditions. To circumvent these problems, in vivo chemical cross-linking has been successfully employed to stabilize protein interactions in native cells or tissues prior to cell lysis (10–16). The resulting covalent bonds formed between interacting partners allow affinity purification under stringent and fully denaturing conditions, consequently reducing nonspecific background while preserving stable and weak/transient interactions (12–16). Subsequent mass spectrometric analysis can reveal not only the identities of interacting proteins, but also cross-linked amino acid residues. The latter provides direct molecular evidence describing the physical contacts between and within proteins (17). This information can be used for computational modeling to establish structural topologies of proteins and protein complexes (17–22), as well as for generating experimentally derived protein interaction network topology maps (23, 24). Thus, cross-linking mass spectrometry (XL-MS) strategies represent a powerful and emergent technology that possesses unparalleled capabilities for studying PPIs.Despite their great potential, current XL-MS studies that have aimed to identify cross-linked peptides have been mostly limited to in vitro cross-linking experiments, with few successfully identifying protein interaction interfaces in living cells (24, 25). This is largely because XL-MS studies remain challenging due to the inherent difficulty in the effective MS detection and accurate identification of cross-linked peptides, as well as in unambiguous assignment of cross-linked residues. In general, cross-linked products are heterogeneous and low in abundance relative to non-cross-linked products. In addition, their MS fragmentation is too complex to be interpreted using conventional database searching tools (17, 26). It is noted that almost all of the current in vivo PPI studies utilize formaldehyde cross-linking because of its membrane permeability and fast kinetics (10–16). However, in comparison to the most commonly used amine reactive NHS ester cross-linkers, identification of formaldehyde cross-linked peptides is even more challenging because of its promiscuous nonspecific reactivity and extremely short spacer length (27). Therefore, further developments in reagents and methods are urgently needed to enable simple MS detection and effective identification of in vivo cross-linked products, and thus allow the mapping of authentic protein contact sites as established in cells, especially for protein complexes.Various efforts have been made to address the limitations of XL-MS studies, resulting in new developments in bioinformatics tools for improved data interpretation (28–32) and new designs of cross-linking reagents for enhanced MS analysis of cross-linked peptides (24, 33–39). Among these approaches, the development of new cross-linking reagents holds great promise for mapping PPIs on the systems level. One class of cross-linking reagents containing an enrichment handle have been shown to allow selective isolation of cross-linked products from complex mixtures, boosting their detectability by MS (33–35, 40–42). A second class of cross-linkers containing MS-cleavable bonds have proven to be effective in facilitating the unambiguous identification of cross-linked peptides (36–39, 43, 44), as the resulting cross-linked products can be identified based on their characteristic and simplified fragmentation behavior during MS analysis. Therefore, an ideal cross-linking reagent would possess the combined features of both classes of cross-linkers. To advance the study of in vivo PPIs, we have developed a new XL-MS platform based on a novel membrane-permeable, enrichable, and MS-cleavable cross-linker, Azide-A-DSBSO (azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide), and multistage tandem mass spectrometry (MSⁿ). This new XL-MS strategy has been successfully employed to map in vivo PPIs from mammalian cells at both the proteome scale and the targeted protein complex level.