An Export-Specific Reporter Designed for Gram-Positive Bacteria: Application to Lactococcus lactis

The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of the Staphylococcus aureus secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called Δ_SPNuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in Lactococcus lactis, thus demonstrating the suitability of Δ_SPNuc to report protein export. The shuttle vector pFUN was designed to construct Δ_SPNuc translational fusions whose expression signals are provided by inserted DNA. The capacity of Δ_SPNuc to reveal and identify exported proteins was tested by generating an L. lactis genomic library in pFUN and by screening for Nuc activity directly in L. lactis. All Δ_SPNuc fusions displaying a strong Nuc⁺ phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in L. lactis. Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding. Our results with L. lactis show that Δ_SPNuc is well suited to report both protein export and membrane protein topology.Most exported proteins are targeted for transport by a primary export signal comprising a hydrophobic domain. The signal can be present at the protein N terminus and cleaved during transport (i.e., signal peptide), but it can also remain embedded in the membrane (i.e., transmembrane segment) (63). Exported proteins are estimated to represent about 20% of total cellular proteins in gram-negative bacteria (39, 44), and contribute to various essential processes like nutrient uptake, macromolecular transport and assembly, envelope biogenesis and integrity, motility, cell division, energy generation, scavenging and detoxification, signal transduction, stress resistance, cell communication, and virulence in the case of pathogens.Several years ago, the elegant strategy of translational fusion to an export-specific reporter protein was designed to specifically isolate genes encoding exported proteins. This kind of reporter is translocation competent but unable to direct its own export (it corresponds to a signal peptideless form of an exported protein), and its activity requires an extracytoplasmic location. Among a library of proteins N-terminally fused to such a reporter, only fusions having the proper signal are exported and active. This strategy was first described for Escherichia coli using alkaline phosphatase (PhoA) as a reporter (16, 36); since then it has been applied to many gram-negative bacteria, particularly pathogens (for reviews, see references 24 and 35 and references therein).Export-specific reporters have a potentially important use in gram-positive bacteria, not only for protein identification and structural analyses, but also for technological applications. Most studies directly adopted the gram-negative reporters available, PhoA and the E. coli TEM β-lactamase (BlaM) (5). The Bacillus licheniformis α-amylase, AmyL, has also been used (17). Surprisingly, relatively few fusion studies allowed identification and characterization of the exported proteins (32, 42). In many cases, only the export signal was characterized (17, 18, 43, 51, 54, 55), possibly because only very short polypeptides (60 amino acids) were fused to the reporter.The rather limited results obtained by using reporter fusions may reveal that the reporters used are not fully adapted for use in gram-positive bacteria. (i) Fusions to gram-negative reporters PhoA and BlaM seem to display little activity and/or to be less stable in gram-positive bacteria, probably because of improper folding (42, 54). Both PhoA (active as a dimer) and BlaM folding require disulfide bond formation, which is catalyzed by DsbA in various gram-negative bacteria (3, 22); it is not yet clear whether such a process exists in gram-positive bacteria (19). Furthermore, altered codon usage and GC content may decrease expression of reporter genes. (ii) Selection of BlaM fusions has been routinely performed in E. coli, possibly due to difficulties of direct ampicillin resistance selection in gram-positive bacteria (43, 51, 54). Such preselection may create a bias due to species specificity of export signals, which, for signal peptides, are significantly longer in gram-positive bacteria (65). (iii) AmyL, a reporter of gram-positive origin, may be the best suited for use in gram-positive bacteria. However, the plate detection test results in loss of cell viability (18a), and thus its use requires replica plating (17, 18).The above-mentioned considerations led us to design a protein export reporter which would be suitable for use in a broad host range of gram-positive bacteria. The reporter we chose is based on the Staphylococcus aureus secreted nuclease (Nuc), a small, stable, monomeric, extensively studied enzyme (EC 3.1.31.1 [9]), having a mature form devoid of cysteine residues (50). Nuc is efficiently secreted by various gram-positive bacteria as an active 168-amino-acid polypeptide which may undergo subsequent proteolytic cleavage of the N-terminal 19- to 21-amino-acid propeptide to give rise to another active form, called NucA (27, 30, 31, 38, 58). The enzymatic activity test for Nuc is sensitive and nontoxic to colonies (28, 29, 50). Several features of Nuc thus make it a potentially optimal candidate for reporting protein export in gram-positive bacteria.In this study, we show that a truncated form of Nuc lacking its export signal (called Δ_SPNuc) is an export-specific reporter. A shuttle vector, pFUN (for fusion to nuclease), was designed to specifically identify genes encoding exported proteins as translational fusions to Δ_SPNuc. pFUN was developed and used to study protein export in Lactococcus lactis, a gram-positive microaerophilic industrial microorganism used in dairy fermentations (37). Despite the technological importance of surface and extracellular proteins in this organism, export of relatively few proteins (excluding plasmid- or transposon-encoded proteins) has been reported to date (4, 6, 12, 13, 15, 26, 40, 60–62). In this work, we characterize 16 previously unknown exported L. lactis proteins. Our results confirm that Δ_SPNuc is a sensitive and specific export reporter for L. lactis and potentially for other gram-positive bacteria.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[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]     

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]     

Electrochemical Measurement of Electron Transfer Kinetics by Shewanella oneidensis MR-1

Shewanella oneidensis strain MR-1 can respire using carbon electrodes and metal oxyhydroxides as electron acceptors, requiring mechanisms for transferring electrons from the cell interior to surfaces located beyond the cell. Although purified outer membrane cytochromes will reduce both electrodes and metals, S. oneidensis also secretes flavins, which accelerate electron transfer to metals and electrodes. We developed techniques for detecting direct electron transfer by intact cells, using turnover and single turnover voltammetry. Metabolically active cells attached to graphite electrodes produced thin (submonolayer) films that demonstrated both catalytic and reversible electron transfer in the presence and absence of flavins. In the absence of soluble flavins, electron transfer occurred in a broad potential window centered at ∼0 V (versus standard hydrogen electrode), and was altered in single (ΔomcA, ΔmtrC) and double deletion (ΔomcA/ΔmtrC) mutants of outer membrane cytochromes. The addition of soluble flavins at physiological concentrations significantly accelerated electron transfer and allowed catalytic electron transfer to occur at lower applied potentials (−0.2 V). Scan rate analysis indicated that rate constants for direct electron transfer were slower than those reported for pure cytochromes (∼1 s⁻¹). These observations indicated that anodic current in the higher (>0 V) window is due to activation of a direct transfer mechanism, whereas electron transfer at lower potentials is enabled by flavins. The electrochemical dissection of these activities in living cells into two systems with characteristic midpoint potentials and kinetic behaviors explains prior observations and demonstrates the complementary nature of S. oneidensis electron transfer strategies.Respiratory electron flow typically occurs at the inner membrane, where oxidation and reduction can be easily linked to intracellular electron carriers and used to generate a membrane potential. However, when the electron acceptor or donor is insoluble, bacteria must possess a mechanism for transferring electrons beyond their inner membrane (1). This is especially true for Proteobcteria, which have an outer membrane that further insulates cytoplasmic and inner membrane processes from insoluble substrates. Metal oxides (such as Fe(III) and Mn(IV) oxyhydroxides) are well recognized naturally occurring electron acceptors that demand such an electron transfer strategy (2–4).Shewanella oneidensis MR-1, a metabolically versatile member of the gammaproteobacteria (5), is capable of reducing insoluble metals, and this phenotype has been linked to a collection of interacting multiheme cytochromes spanning the inner membrane, periplasmic space, and outer membrane (6–12). There is also evidence that some of these cytochromes decorate the surface of pili-like structures extending from the cell surface (13, 14). Regardless of the ultimate location of the cytochromes, in all models of electron transfer, electrons must hop from these proteins to a solid surface or be transferred to a soluble mediator that can diffuse to a final destination (15, 16). Although chelation of a metal oxide is a third option (17, 18), the fact that Shewanella is able to transfer electrons to solid graphite electrodes (19–23) underscores the need for a direct or diffusion-based electron transfer mechanism to link cellular proteins and surfaces.Recent work has shown that Shewanella species secrete soluble flavins (FMN and riboflavin) that facilitate electron transfer to both metals and electrodes (23, 24). For example, removal of accumulated soluble flavins decreases the rate of electron transfer by Shewanella biofilms to electrodes over 80%. Consistent with this observation, kinetic measurements with pure MtrC and OmcA (25) showed that direct reduction of solid metal oxides by these cytochromes was too slow to explain physiological rates of electron transfer, whereas turnover rates of these enzymes with soluble flavins were orders of magnitude larger. These studies suggest that the kinetics of electron transfer from cytochromes on the outer surface of Shewanella to electrodes will be significantly altered in the absence of diffusible mediators (9–11, 26–34).Voltammetry has proven a useful technique for the analysis of electron transfer rates and pathways using purified proteins (35–39) and has recently been extended to the study of intact bacteria (23, 40–42). In slow scan rate cyclic voltammetry, the rate of electron transfer from respiring Shewanella biofilms to electrodes rises sharply at the E°′ of riboflavin and FMN (−0.2 V versus SHE)² (23). Such measurements relating thermodynamic driving force to turnover kinetics would be difficult with whole cell:Fe(III) oxide incubations, which do not allow fine control over the electron acceptor redox potential or real time recording of electron transfer rates. In addition, voltammetry provides tools to observe electron movement under single-turnover conditions (in the absence of electron donor), allowing reversible oxidation and reduction of proteins accessible to the electrode and study of kinetic behavior (43, 44).In this work, techniques of turnover (sustained electron transfer from cells to electrode in the presence of electron donor) and single turnover (reversible oxidation and reduction in the absence of electron donor) voltammetry were harnessed to investigate the role of outer membrane proteins in electron transfer from Shewanella to electrodes. In all of these studies, intact metabolically active cells were used, along with electrode surfaces known to act as acceptors for Shewanella. The results in the absence of soluble mediators provide evidence that electron transfer between MtrC and OmcA and surfaces requires a higher potential compared with when flavins are present to shuttle electrons to the surface. Mutant analysis also demonstrates that cells possessing different outer membrane cytochromes have differing abilities for direct and mediator-enabled electron transfer.     

Stomatin-like Protein-1 Interacts with Stomatin and Is Targeted to Late Endosomes

The human stomatin-like protein-1 (SLP-1) is a membrane protein with a characteristic bipartite structure containing a stomatin domain and a sterol carrier protein-2 (SCP-2) domain. This structure suggests a role for SLP-1 in sterol/lipid transfer and transport. Because SLP-1 has not been investigated, we first studied the molecular and cell biological characteristics of the expressed protein. We show here that SLP-1 localizes to the late endosomal compartment, like stomatin. Unlike stomatin, SLP-1 does not localize to the plasma membrane. Overexpression of SLP-1 leads to the redistribution of stomatin from the plasma membrane to late endosomes suggesting a complex formation between these proteins. We found that the targeting of SLP-1 to late endosomes is caused by a GYXXΦ (Φ being a bulky, hydrophobic amino acid) sorting signal at the N terminus. Mutation of this signal results in plasma membrane localization. SLP-1 and stomatin co-localize in the late endosomal compartment, they co-immunoprecipitate, thus showing a direct interaction, and they associate with detergent-resistant membranes. In accordance with the proposed lipid transfer function, we show that, under conditions of blocked cholesterol efflux from late endosomes, SLP-1 induces the formation of enlarged, cholesterol-filled, weakly LAMP-2-positive, acidic vesicles in the perinuclear region. This massive cholesterol accumulation clearly depends on the SCP-2 domain of SLP-1, suggesting a role for this domain in cholesterol transfer to late endosomes.Human stomatin-like protein-1 (SLP-1),³ also known as STOML-1, STORP (1), slipin-1 (2), or hUNC-24 (3), is the human orthologue of Caenorhabditis elegans UNC-24 and a member of the stomatin protein family that comprises 5 human members: stomatin (4–6), SLP-1 (1, 7), SLP-2 (8), SLP-3 (9, 10), and podocin (11). SLP-1 is predominantly expressed in the brain, heart, and skeletal muscle (7, 8) and can be identified in most other tissues (1). Its structure contains a hydrophilic N terminus, a 30-residue hydrophobic domain that is thought to anchor the protein to the cytoplasmic side of the membrane, followed by a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (12) that is also known as prohibitin (PHB) domain (13), and a C-terminal sterol carrier protein-2 (SCP-2)/nonspecific lipid transfer protein domain (14, 15). This unique structure that was first revealed in C. elegans UNC-24 (16) suggests that SLP-1 may be involved in lipid transfer and transport (17).The founder of the family, stomatin, is a major protein of the red blood cell membrane (band 7.2) and is ubiquitously expressed (18). It is missing in red cells of patients with overhydrated hereditary stomatocytosis, a pathological condition characterized by increased permeability of the red cells for monovalent ions and stomatocytic morphology (19, 20). However, the lack of stomatin is not due to a mutation in its gene but rather to a transport defect (21, 22). Stomatin is a monotopic, oligomeric, palmitoylated, cholesterol-binding membrane protein (18) that is associated with lipid rafts (23, 24) or raft-like detergent-resistant membranes (DRMs) (25), serving as a respective marker (26–28). Other stomatin family members like podocin (29, 30) and SLP-3 (9) are also enriched in DRMs. Many SPFH/PHB proteins share this property suggesting that the SPFH/PHB domain plays an important role in lipid raft/DRM targeting (13, 31). Several interactions of stomatin with membrane proteins have been revealed, notably with the acid sensing ion channels (32) and the glucose transporter GLUT1 (33, 34). Interestingly, stomatin functions as a switch of GLUT1 specificity from glucose to dehydroascorbate in the human red blood cell thus increasing vitamin C recycling and compensating the human inability to synthesize vitamin C (35).The C. elegans genome contains 10 members of the stomatin family. Defects in three of these genes (mec-2, unc-1, and unc-24) cause distinct neuropathologic phenotypes, namely uncoordinated movement and defect in mechanosensation, respectively (36, 37). These are explained by dysfunction of the respective stomatin-like proteins in complex with degenerin/epithelial sodium channels that also affects the sensitivity to volatile anesthetics (38, 39). Importantly, MEC-2 and human podocin bind cholesterol and form large supercomplexes with various ion channels thus modulating channel activity (40). The biological functions of the SLP-1 orthologue UNC-24 and stomatin orthologue UNC-1 are associated, because the unc-24 gene controls the distribution or stability of the UNC-1 protein (41). In addition, UNC-24 co-localizes and interacts with MEC-2 and is essential for touch sensitivity (36). Based on these observations, we hypothesize that human stomatin and SLP-1 similarly interact and modify the distribution of each other. These proteins may have important functions in regulating the activity of ion channels in the human brain and muscle tissues. Despite its putative role in cellular lipid distribution, SLP-1 has not been studied to date.In this work, we characterized human SLP-1 as a late endosomal protein and identified an N-terminal GYXXΦ motif as the targeting signal. We found that SLP-1 interacts with stomatin in vitro and in vivo and associates with DRMs. Regarding the proposed lipid transfer function, we showed that SLP-1 induces the formation of large, cholesterol-rich vesicles or vacuoles when cholesterol trafficking from the late endosomes is blocked suggesting a net cholesterol transfer to the late endosomes and/or lysosomes. This effect was clearly attributed to the SCP-2/nonspecific lipid transfer protein domain of SLP-1, in line with the original hypothesis.     

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]     

Plasma Protein Pentosidine and Carboxymethyllysine, Biomarkers for Age-related Macular Degeneration

Age-related macular degeneration (AMD) causes severe vision loss in the elderly; early identification of AMD risk could help slow or prevent disease progression. Toward the discovery of AMD biomarkers, we quantified plasma protein N^ε-carboxymethyllysine (CML) and pentosidine from 58 AMD and 32 control donors. CML and pentosidine are advanced glycation end products that are abundant in Bruch membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaris. We measured CML and pentosidine by LC-MS/MS and LC-fluorometry, respectively, and found higher mean levels of CML (∼54%) and pentosidine (∼64%) in AMD (p < 0.0001) relative to normal controls. Plasma protein fructosyl-lysine, a marker of early glycation, was found by amino acid analysis to be in equal amounts in control and non-diabetic AMD donors, supporting an association between AMD and increased levels of CML and pentosidine independent of other diseases like diabetes. Carboxyethylpyrrole (CEP), an oxidative modification from docosahexaenoate-containing lipids and also abundant in AMD Bruch membrane, was elevated ∼86% in the AMD cohort, but autoantibody titers to CEP, CML, and pentosidine were not significantly increased. Compellingly higher mean levels of CML and pentosidine were present in AMD plasma protein over a broad age range. Receiver operating curves indicate that CML, CEP adducts, and pentosidine alone discriminated between AMD and control subjects with 78, 79, and 88% accuracy, respectively, whereas CML in combination with pentosidine provided ∼89% accuracy, and CEP plus pentosidine provided ∼92% accuracy. Pentosidine levels appeared slightly altered in AMD patients with hypertension and cardiovascular disease, indicating further studies are warranted. Overall this study supports the potential utility of plasma protein CML and pentosidine as biomarkers for assessing AMD risk and susceptibility, particularly in combination with CEP adducts and with concurrent analyses of fructosyl-lysine to detect confounding factors.Age-related macular degeneration (AMD)¹ is a progressive, multifactorial disease and a major cause of severe vision loss in the elderly (1). Deposition of debris (drusen) in the macular region of Bruch membrane, the extracellular matrix separating the choriocapillaris from the retinal pigment epithelium (RPE), is an early, hallmark risk factor of AMD. The disease can progress to advanced dry AMD (geographic atrophy), which is characterized by regional degeneration of photoreceptor and RPE cells, or to advanced wet AMD (choroidal neovascularization (CNV)), which is characterized by abnormal blood vessels growing from the choriocapillaris through Bruch membrane beneath the retina. CNV accounts for over 80% of debilitating vision loss in AMD; however, only 10–15% of AMD cases progress to CNV.There is growing consensus that AMD is an age-related inflammatory disease involving dysregulation of the complement system; however, triggers of the inflammatory response have yet to be well defined. Oxidative stress appears to be involved as smoking significantly increases the risk of AMD (2), antioxidant vitamins can selectively slow AMD progression (3), and a host of oxidative protein and DNA modifications have been detected at elevated levels in AMD Bruch membrane, drusen, retina, RPE, and plasma (4–11). Oxidative protein modifications like carboxyethylpyrrole (CEP) and N^ε-carboxymethyllysine (CML), both elevated in AMD Bruch membrane, stimulate neovascularization in vivo (12, 13), suggesting possible roles in CNV. Other studies have shown that mice immunized with CEP protein modifications develop an AMD-like phenotype (14). Accordingly oxidative modifications may be catalysts or triggers of AMD pathology (6).AMD has long been hypothesized to be a systemic disease (15) based in part on the presence of retinal drusen in patients with membranoproliferative glomerulonephritis type II (16) and systemic complement activation in AMD (17). Support for this hypothesis also comes from mounting evidence that advanced glycation end products (AGEs) may play a role in AMD (4, 5, 7, 18, 19). AGEs are a heterogeneous group of mostly oxidative modifications resulting from the Maillard nonenzymatic glycation reaction that have been associated with age-related diseases and diabetic complications (20, 21). In 1998, CML was the first AGE to be found in AMD Bruch membrane and drusen (4). Other AGEs have since been detected in AMD ocular tissues (5, 7, 18) and in Bruch membrane, drusen, RPE, and choroidal extracellular matrix from healthy eyes (6, 22). CML, a nonfluorescent AGE, and pentosidine, a fluorescent cross-linking AGE, increase with age in Bruch membrane (18, 23). Receptors for AGEs (RAGE and AGE-R1) appear elevated on RPE and photoreceptor cells in early and advanced dry AMD (7) especially in RPE overlying drusen-like deposits on Bruch membrane (19). AGE-R3, also known as galectin-3, is elevated in AMD Bruch membrane (24).Although AMD susceptibility genes now account for over 50% of AMD cases (25), many individuals with AMD risk genotypes may never develop advanced disease with severe vision loss. Nevertheless the prevalence of advanced AMD is increasing (26). Toward the discovery of better methods to detect those at risk for advanced AMD, we quantified CML and pentosidine in plasma proteins from AMD and control patients and compared their discriminatory accuracy with plasma CEP biomarkers. CEP biomarkers have been shown to enhance the AMD predictive accuracy of genomic AMD biomarkers (11). This report shows CML and pentosidine to be elevated in AMD plasma proteins and demonstrates their potential biomarker utility in assessing AMD risk and susceptibility especially in combination with CEP biomarkers.     

Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction,Molecular Transport,and Callose Biosynthesis

The plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species.The plasma membrane (PM)¹ is considered as one of the most interactive and dynamic supramolecular structures of the cell (1, 2). It forms a physical interface between the cytoplasm and the extracellular environment and is involved in many biological processes such as metabolite and ion transport, gaseous exchanges, endocytosis, cell differentiation and proliferation, defense against pathogens, etc. (3). Various combinations of biochemical and analytical approaches have been used to characterize the PM proteome in different organisms such as yeast, plants, and animals (4–8). Typically, PM proteins are either embedded in the phospholipid bilayer through transmembrane helices or less tightly bound to the membrane through reversible or irreversible surface interactions. In eukaryotic cells, some PM proteins are enriched in lateral lipid patches that form microdomains within the membrane (9, 10). These microdomains are considered to act as functional units that support and regulate specific biological processes associated with the PM (9, 10). Often referred to as “membrane (lipid) rafts” in animals and other organisms, they are typically described as being enriched in sphingolipids, sterols, and phospholipids that contain essentially saturated fatty acids (9–11). Early work on PM microdomains has suggested that their specific lipid composition confers resistance to certain concentrations of nonionic detergents, such as Triton X-100 and Nonidet P-40 (10, 11). Although this property has been exploited experimentally to isolate so-called detergent-resistant microdomains (DRMs), the relationship between DRMs and membrane rafts remains controversial (12). Indeed, the relation between the two is much debated, essentially because the use of Triton X-100 at 4 °C to prepare DRMs has been proposed to potentially induce the artificial formation of detergent-resistant structures whose composition may not fully reflect that of physiological membrane rafts (12). Nonetheless, DRM preparations represent an excellent system for the isolation and identification of groups of proteins—eventually associated in complexes—that tend to naturally interact with specific sets of lipids, thereby forming specialized functional units. Their biochemical characterization is therefore most useful in attempts to better understand the mode of interaction of specific proteins with sterols and sphingolipids and to gain insight into the protein composition and biological activity of subdomains from the PM.Plant DRMs have been understudied relative to their animal counterparts. Indeed, proteomic studies have been undertaken on DRM preparations from only a limited number of plant species. These include tobacco (13–15), Arabidopsis (16), barrel clover (Medicago truncatula) (17), rice (18), oat, and rye (19). These studies, essentially based on qualitative or semi-quantitative proteomics, led to the identification of hundreds of proteins involved in a large range of mechanisms, functions, and biochemical activities (15–19). Depending on the report considered, a variable proportion of the identified proteins can be intuitively linked to DRMs and potentially to PM microdomains. However, many proteins that are clearly not related to the PM and its microdomains co-purify with DRM. These include, for instance, soluble proteins from cytoplasmic metabolic pathways; histones; and ribosomal, chloroplastic, and mitochondrial proteins (15–19). Thus, there is a need to obtain a more restricted list of proteins that are specifically enriched in DRMs and that define specialized functional structures. One way to tackle this problem is through quantitative proteomics, eventually in combination with complementary biochemical approaches. Although quantitative techniques have been increasingly applied to the proteomic analysis of complex mixtures of soluble proteins, their exploitation for the characterization of membrane samples remains challenging. As a result, very few studies of plant DRMs have been based on truly quantitative methods. For instance, stable isotope labeling combined with the selective disruption of sterol-rich membrane domains by methylcyclodextrin was performed in Arabidopsis cell cultures (20). A similar approach was used to study compositional changes of tobacco DRMs upon cell treatment with the signaling elicitor cryptogenin (21). In another study, 64 Arabidopsis proteins were shown to be significantly enriched in DRMs in response to a pathogen-associated molecular pattern protein (22). Together, these few quantitative proteomics analyses suggest a role of plant membrane microdomains in signal transduction, as in mammalian cells.Although several reports describe the partial characterization of DRMs from higher plants (13–23), there are no data available to date on the protein composition of DRMs from a tree species. We have therefore employed a quantitative proteomic approach for the characterization of DRMs from cell suspension cultures of Populus trichocarpa. In addition, earlier work in our laboratory based on biochemical activity assays revealed the presence of cell wall polysaccharide synthases in DRMs from poplar (23), which suggests the existence of DRM populations specialized in cell wall biosynthesis. This concept was further supported by similar investigations performed on DRMs isolated from the oomycete Saprolegnia monoica (24). The comprehensive quantitative proteomic analysis performed here revealed enrichment in the poplar DRMs of specific carbohydrate synthases involved in callose polymerization. Consistent with the role of callose in plant defense mechanisms, additional proteins related to stress responses and signal transduction were found to be specifically enriched in the poplar DRMs, together with proteins involved in molecular transport. To date, our report is the only analysis available of the DRM proteome of a tree species based on quantitative proteomics. The specific biochemical properties of the 80 proteins significantly enriched in DRMs are described and examined in relation to their localization in membrane microdomains. The relationship between poplar DRMs and molecular transport, signal transduction, stress responses, and callose biosynthesis is discussed, with support from a hypothetical model that integrates the corresponding enriched proteins.     

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry

Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics.Protein glycosylation is a biologically significant and complex post-translational modification, involved in cell-cell and receptor-ligand interactions (1–4). In fact, clinical biomarkers and therapeutic targets are often glycoproteins (5–9). Comprehensive glycoprotein characterization, involving glycosylation site identification, glycan structure determination, site occupancy, and glycan isoform distribution, is a technical challenge particularly for quantitative profiling of complex protein mixtures (10–13). Both N- and O-glycans are structurally heterogeneous (i.e. a single site may have different glycans attached or be only partially occupied). Therefore, the MS¹ signals from glycopeptides originating from a glycoprotein are often weaker than from non-glycopeptides. In addition, the ionization efficiency of glycopeptides is low compared with that of non-glycopeptides and is often suppressed in the presence of non-glycopeptides (11–13). When the MS signals of glycopeptides are relatively high in simple protein digests then diagnostic sugar oxonium ion fragments produced by, for example, front-end collisional activation can be used to detect them. However, when peptides and glycopeptides co-elute, parent ion scanning is required to selectively detect the glycopeptides (14). This can be problematic in terms of sensitivity, especially for detecting glycopeptides in digests of complex protein extracts.Isolation of glycopeptides from proteolytic digests of complex protein mixtures can greatly enhance the MS signals of glycopeptides using reversed-phase LC-ESI-MS (RPLC-ESI-MS) or MALDI-MS (15–24). Hydrazide chemistry is used to isolate, identify, and quantify N-linked glycopeptides effectively, but this method involves lengthy chemical procedures and does not preserve the glycan moieties thereby losing valuable information on glycan structure and site occupancy (15–17). Capturing glycopeptides with lectins has been widely used, but restricted specificities and unspecific binding are major drawbacks of this method (18–21). Under reversed-phase LC conditions, glycopeptides from tryptic digests of gel-separated glycoproteins have been enriched using graphite powder medium (22). In this case, however, a second digestion with proteinase K is required for trimming down the peptide moieties of tryptic glycopeptides so that the glycopeptides (typically <5 amino acid residues) essentially resemble the glycans with respect to hydrophilicity for subsequent separation. Moreover, the short peptide sequences of the proteinase K digest are often inadequate for de novo sequencing of the glycopeptides.Glycopeptide enrichment under normal-phase LC (NPLC) conditions has been demonstrated using various hydrophilic media and different capture and elution conditions (23–28). NPLC allows either direct enrichment of peptides modified by various N-linked glycan structures using a ZIC®-HILIC column (23–27) or targeting sialylated glycopeptides using a titanium dioxide micro-column (28). However, NPLC is neither effective for enriching less hydrophilic glycopeptides, e.g. the five high mannose type glycopeptides modified by 7–11 monosaccharide units from a tryptic digest of ribonuclease b (RNase B), nor for enriching O-linked glycopeptides of bovine fetuin using a ZIC-HILIC column (23). The use of Sepharose medium for enriching glycopeptides yielded only modest recovery of glycopeptides (28). In addition, binding of hydrophilic non-glycopeptides with these hydrophilic media contaminates the enriched glycopeptides (23, 28).We have recently developed an ion-pairing normal-phase LC (IP-NPLC) method to enrich glycopeptides from complex tryptic digests using Sepharose medium and salts or bases as ion-pairing reagents (29). Though reasonably effective the technique still left room for significant improvement. For example, the method demonstrated relatively modest glycopeptide selectivity, providing only 16% recovery for high mannose type glycopeptides (29). Here we report on a new IP-NPLC method using acids as ion-pairing reagents and polyhydroxyethyl aspartamide (A) as the stationary phase for the effective isolation of tryptic glycopeptides. The method was developed and evaluated using a tryptic digest of RNase B and fetuin mixture. In addition, we demonstrate that O-linked glycopeptides can be effectively isolated from a fetuin tryptic digest by IP-NPLC after removal of the N-linked glycans by PNGase F.The new IP-NPLC method was used to enrich N-linked glycopeptides from the tryptic digests of protein extracts of wild-type (wt) and PglD mutant strains of Campylobacter jejuni NCTC 11168. C. jejuni has a unique N-glycosylation system that glycosylates periplasmic and inner membrane proteins containing the extended N-linked sequon, D/E-X-N-X-S/T, where X is any amino acid other than proline (30–32). The N-linked glycan of C. jejuni has been previously determined to be GalNAc-α1,4-GalNAc-α1,4-[Glcβ1,3]-GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac-β1 (BacGalNAc₅Glc residue mass: 1406 Da), where Bac is 2,4-diacetamido-2,4,6-trideoxyglucopyranose (30). In addition, the glycan structure of C. jejuni is conserved, unlike in eukaryotic systems (30–32). IP-NPLC recovered close to 100% of the bacterial N-linked glycopeptides with virtually no contamination of non-glycopeptides. Furthermore, we demonstrate for the first time that acetylation of bacillosamine is incomplete in the wt using IP-NPLC and label-free MS.     

The Deubiquitinating Enzyme USP10 Regulates the Post-endocytic Sorting of Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.The endocytosis, endocytic recycling, and endosomal sorting of numerous transport proteins and receptors are regulated by ubiquitination (1–6). Ubiquitin, an 8-kDa protein, is conjugated to target proteins via a series of steps that includes ubiquitin-activating enzymes (E1),² ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) (1). Proteins that are ubiquitinated in the plasma membrane are internalized and are either deubiquitinated and recycle back to the plasma membrane or, via interactions with the endosomal sorting complexes required for transport machinery, are delivered to the lysosome for degradation (1–7). Sorting of ubiquitinated plasma membrane proteins for either the lysosomal pathway or for the recycling pathway is regulated, in part, by the removal of ubiquitin by deubiquitinating enzymes (DUBs) (1–6). Thus, the balance between ubiquitination and deubiquitination regulates the plasma membrane abundance of several membrane proteins, including the epithelial sodium channel (ENaC), the epidermal growth factor receptor, the transforming growth factor-β receptor, and the cytokine receptor γ-c (8–14).CFTR is rapidly endocytosed from the plasma membrane and undergoes rapid and efficient recycling back to the plasma membrane in human airway epithelial cells, with >75% of endocytosed wild-type CFTR recycling back to the plasma membrane (15–18). A study published several years ago demonstrated that, although ubiquitination did not regulate CFTR endocytosis, ubiquitination reduced the plasma membrane abundance of CFTR in BHK cells by redirecting CFTR from recycling endosomes to lysosomes for degradation (19). However, neither the E3 ubiquitin ligase(s) responsible for the ubiquitination of CFTR nor the DUB(s) responsible for the deubiquitination of CFTR in the endocytic pathway have been identified in any cell type. Moreover, the effect of the ubiquitin status of CFTR on its endocytic sorting in human airway epithelial cells has not been reported. Thus, the goals of this study were to determine if the ubiquitin status regulates the post-endocytic sorting of CFTR in polarized airway epithelial cells, and to identify the DUBs that deubiquitinate CFTR.Approximately 100 DUBs have been identified in the human genome and are classified into five families based on sequence similarity and mechanism of action (1–6, 20, 21). To identify DUBs that regulate the deubiquitination of CFTR from this large class of enzymes, we chose an activity-based, chemical probe screening approach developed by Dr. Hidde Ploegh (4, 21, 22). This approach utilizes a hemagglutinin (HA)-tagged ubiquitin probe engineered with a C-terminal modification incorporating a thiol-reactive group that forms an irreversible, covalent bond with active DUBs. Using this approach we demonstrated in polarized human airway epithelial cells that ubiquitin-specific protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and thus its trafficking in the post-endocytic compartment. These studies demonstrate a novel function for USP10 in promoting the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.     

F-Actin Dynamics in Neurospora crassa

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.Actins are highly conserved proteins found in all eukaryotes and have an enormous variety of cellular roles. The monomeric form (globular actin, or G-actin) can self-assemble, with the aid of numerous actin-binding proteins (ABPs), into microfilaments (filamentous actin, or F-actin), which, together with microtubules, form the two major components of the fungal cytoskeleton. Numerous pharmacological and genetic studies of fungi have demonstrated crucial roles for F-actin in cell polarity, exocytosis, endocytosis, cytokinesis, and organelle movement (6, 7, 20, 34, 35, 51, 52, 59). Phalloidin staining, immunofluorescent labeling, and fluorescent-protein (FP)-based live-cell imaging have revealed three distinct subpopulations of F-actin-containing structures in fungi: patches, cables, and rings (1, 14, 28, 34, 60, 63, 64). Actin patches are associated with the plasma membrane and represent an accumulation of F-actin around endocytic vesicles (3, 26, 57). Actin cables are bundles of actin filaments stabilized with cross-linking proteins, such as tropomyosins and fimbrin, and are assembled by formins at sites of active growth, where they form tracks for myosin V-dependent polarized secretion and organelle transport (10, 16, 17, 27, 38, 47, 48). Cables, unlike patches, are absolutely required for polarized growth in the budding yeast Saccharomyces cerevisiae (34, 38). Contractile actomyosin rings are essential for cytokinesis in budding yeast, whereas in filamentous fungi, actin rings are less well studied but are known to be involved in septum formation (20, 28, 34, 39, 40).Actin cables and patches have been particularly well studied in budding yeast. However, there are likely to be important differences between F-actin architecture and dynamics in budding yeast and those in filamentous fungi, as budding yeasts display only a short period of polarized growth during bud formation, which is followed by isotropic growth over the bud surface (10). Sustained polarized growth during hyphal morphogenesis is a defining feature of filamentous fungi (21), making them attractive models for studying the roles of the actin cytoskeleton in cell polarization, tip growth, and organelle transport.In Neurospora crassa and other filamentous fungi, disruption of the actin cytoskeleton leads to rapid tip swelling, which indicates perturbation of polarized tip growth, demonstrating a critical role for F-actin in targeted secretion to particular sites on the plasma membrane (7, 22, 29, 56). Immunofluorescence studies of N. crassa have shown that F-actin localizes to hyphal tips as “clouds” and “plaques” (7, 54, 59). However, immunolabeling has failed to reveal actin cables in N. crassa and offers limited insights into F-actin dynamics. Live-cell imaging of F-actin architecture and dynamics has not been accomplished in N. crassa, yet it is expected to yield key insights into cell polarization, tip growth, and intracellular transport.We took advantage of a recently developed live-cell imaging probe for F-actin called Lifeact (43). Lifeact is a 17-amino-acid peptide derived from the N terminus of the budding yeast actin-binding protein Abp140 (5, 63) and has recently been demonstrated to be a universal live-cell imaging marker for F-actin in eukaryotes (43). Here, we report the successful application of fluorescent Lifeact fusion constructs for live-cell imaging of F-actin in N. crassa. We constructed two synthetic genes consisting of Lifeact fused to “synthetic” green fluorescent protein (sGFP) (S65T) (henceforth termed GFP) (12) or red fluorescent protein (TagRFP) (33) and expressed these constructs in various N. crassa strains. In all strain backgrounds, fluorescent Lifeact constructs clearly labeled actin patches, cables, and rings and revealed a direct association of F-actin structures with sites of cell polarization and active tip growth. Our results demonstrate the efficacy of Lifeact as a nontoxic live-cell imaging probe in N. crassa.     

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.