Proteomics reveals localization of cuticular proteins in Anopheles gambiae.

Anopheles gambiae devotes over 2% of its protein coding genes to its 298 structural cuticular proteins (CPs). This paper provides new LC-MS/MS data on two adult structures, proboscises and palps, as well as three larval samples - 4th instar larvae, just their terminal segment, and a preparation enriched in their tracheae. These data were combined with our previously published results of proteins from five other adult structures, whole adults, and two preparations chosen for their relatively clean cuticle, the larval head capsules left behind after ecdysis and the pupal cuticles left behind after adult eclosion. Peptides from 28 CPs were recovered in all adult structures; 24 CPs were identified for the first time, 6 of these were members of the TWDL family. Most newly identified proteins came from the larval sources. Based solely on peptide recovery, from our data and from other investigators, most available on VectorBase, there were only 4 CPs that were restricted to a single adult structure. More were restricted to a single metamorphic stage, 14 in larvae, 0 in pupae and 32 in adults. Expression data from our earlier RT-qPCR studies reduces these numbers. Charting restriction of CPs to stage or structure is a step forward in establishing their specific roles.

Predicting the HILIC Retention Behavior of the N-Linked Glycopeptides Produced by Trypsin Digestion of Immunoglobulin Gs (IgGs).

The prediction of the retention behavior/time would facilitate the identification and characterization of glycoproteins, particularly the analytical challenges, such as the characterization of low-abundance glycoforms. This task is essential in the biotherapeutics industry, where the type and amount of glycosylation on recombinant IgG alter the efficacy, function, and immunogenicity. Models exist for the prediction of the hydrophilic interaction liquid chromatography retention of peptides and glycans. Here, we have devised a unified model to predict the retention behavior of glycopeptides from human IgGs and applied this to the analysis of glycopeptides from rabbit IgGs. The combined model is capable of accurately predicting the retention of native IgG glycopeptides on 2 completely different liquid chromatography-mass spectrometry systems.

Peptide retention prediction using hydrophilic interaction liquid chromatography coupled to mass spectrometry.

A model that predicts retention for peptides using a HALO® penta-HILIC column and gradient elution was created. Coefficients for each amino acid were derived using linear regression analysis and these coefficients can be summed to predict the retention of peptides. This model has a high correlation between experimental and predicted retention times (0.946), which is on par with previous RP and HILIC models. External validation of the model was performed using a set of H. pylori samples on the same LC-MS system used to create the model, and the deviation from actual to predicted times was low. Apart from amino acid composition, length and location of amino acid residues on a peptide were examined and two site-specific corrections for hydrophobic residues at the N-terminus as well as hydrophobic residues one spot over from the N-terminus were created.

Predicting the Retention Behavior of Specific O-Linked Glycopeptides.

O-Linked glycosylation is a common post-translational modification that can alter the overall structure, polarity, and function of proteins. Reverse-phase (RP) chromatography is the most common chromatographic approach to analyze O-glycosylated peptides and their unmodified counterparts, even though this approach often does not provide adequate separation of these two species. Hydrophilic interaction liquid chromatography (HILIC) can be a solution to this problem, as the polar glycan interacts with the polar stationary phase and potentially offers the ability to resolve the peptide from its modified form(s). In this paper, HILIC is used to separate peptides with O-N-acetylgalactosamine (O-GalNAc), O-N-acetylglucosamine (O-GlcNAc), and O-fucose additions from their native forms, and coefficients representing the extent of hydrophilicity were derived using linear regression analysis as a means to predict the retention times of peptides with these modifications.

Properties of the cuticular proteins of Anopheles gambiae as revealed by serial extraction of adults.

How cuticular proteins (CPs) interact with chitin and with each other in the cuticle remains unresolved. We employed LC-MS/MS to identify CPs from 5-6 day-old adults of Anopheles gambiae released after serial extraction with PBS, EDTA, 2-8M urea, and SDS as well as those that remained unextracted. Results were compared to published data on time of transcript abundance, localization of proteins within structures and within the cuticle, as well as properties of individual proteins, length, pI, percent histidine, tyrosine, glutamine, and number of AAP[A/V/L] repeats. Thirteen proteins were solubilized completely, all were CPRs, most belonging to the RR-1 group. Eleven CPs were identified in both soluble fractions and the final pellet, including 5 from other CP families. Forty-three were only detected from the final pellet. These included CPRs and members of the CPAP1, CPF, CPFL, CPLCA, CPLCG, CPLCP, and TWDL families, as well as several low complexity CPs, not assigned to families and named CPLX. For a given protein, many histidines or tyrosines or glutamines appear to be potential participants in cross-linking since we could not identify any peptide bearing these residues that was consistently absent. We failed to recover peptides from the amino-terminus of any CP. Whether this implicates that location in sclerotization or some modification that prevents detection is not known. Soluble CPRs had lower isoelectric points than those that remained in the final pellet; most members of other CP families had isoelectric points of 8 or higher. Obviously, techniques beyond analysis of differential solubility will be needed to learn how CPs interact with each other and with chitin.

Ethological principles predict the neuropeptides co-opted to influence parenting.

Ethologists predicted that parental care evolves by modifying behavioural precursors in the asocial ancestor. As a corollary, we predict that the evolved mechanistic changes reside in genetic pathways underlying these traits. Here we test our hypothesis in female burying beetles, Nicrophorus vespilloides, an insect where caring adults regurgitate food to begging, dependent offspring. We quantify neuropeptide abundance in brains collected from three behavioural states: solitary virgins, individuals actively parenting or post-parenting solitary adults and quantify 133 peptides belonging to 18 neuropeptides. Eight neuropeptides differ in abundance in one or more states, with increased abundance during parenting in seven. None of these eight neuropeptides have been associated with parental care previously, but all have roles in predicted behavioural precursors for parenting. Our study supports the hypothesis that predictable traits and pathways are targets of selection during the evolution of parenting and suggests additional candidate neuropeptides to study in the context of parenting.

The Separation and Quantitation of Peptides with and without Oxidation of Methionine and Deamidation of Asparagine Using Hydrophilic Interaction Liquid Chromatography with Mass Spectrometry (HILIC-MS).

Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein's function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications. Graphical Abstract ᅟ.

Distribution of cuticular proteins in different structures of adult Anopheles gambiae.

Anopheles gambiae devotes over 2% (295) of its protein coding genes to structural cuticular proteins (CPs) that have been classified into 13 different families plus ten low complexity proteins not assigned to families. Small groups of genes code for identical proteins reducing the total number of unique cuticular proteins to 282. Is the large number because different structures utilize different CPs, or are all of the genes widely expressed? We used LC-MS/MS to learn how many products of these genes were found in five adult structures: Johnston's organs, the remainder of the male antennae, eye lenses, legs, and wings. Data were analyzed against both the entire proteome and a smaller database of just CPs. We recovered unique peptides for 97 CPs and shared peptides for another 35. Members of 11 of the 13 families were recovered as well as some unclassified. Only 11 CPs were present exclusively in only one structure while 43 CPs were recovered from all five structures. A quantitative analysis, using normalized spectral counts, revealed that only a few CPs were abundant in each structure. When the MS/MS data were run against the entire proteome, the majority of the top hits were to CPs, but peptides were recovered from an additional 467 proteins. CP peptides were frequently recovered from chitin-binding domains, confirming that protein-chitin interactions are not mediated by covalent bonds. Comparison with three other MS/MS analyses of cuticles or cuticle-rich structures augmented the current analysis. Our findings provide new insights into the composition of different mosquito structures and reveal the complexity of selection and utilization of genes coding for structural cuticular proteins.

Noctilisin, a Venom Glycopeptide of Sirex noctilio (Hymenoptera: Siricidae), Causes Needle Wilt and Defense Gene Responses in Pines.

During oviposition, female Sirex noctilio (F.) (Siricidae) woodwasps inject their conifer hosts with a venom gland secretion. The secretion induces a variety of host physiological changes that facilitate subsequent lethal infection by a symbiotic fungus. A heat-stable factor that can migrate from the site of oviposition in the trunk through the xylem to needles in the crown of attacked pines was purified by size-fractionation and reversed-phase-high-performance liquid chromatography using activity assays based on defense gene induction as well as the needle wilt response in pine shoot explants. An 11-amino acid, posttranslationally modified peptide (SEGPROGTKRP) encoded by the most abundant transcript recovered from S. noctilio venom gland tissue comprised the backbone of the 1,850 Da active factor. Posttranslational modifications included hydroxylation of a Pro residue at position 6 as well as O-glycosylation of Ser and Thr residues at positions 1 and 8, respectively. The O-linked sugars were identical α-linked N-acetylgalactosamine residues modified at the C6 position by addition of phosphoethanolamine. In contrast to the native peptide, a synthetic version of the hydroxylated peptide backbone lacking the glycosyl side chains failed to induce pine defense genes or cause needle wilt in excised shoots. This peptide, hereafter called noctilisin, is related to the O-glycosylated short-chain proline-rich antimicrobial peptides exemplified by drosocin. The noctilisin structure contains motifs which may explain how it avoids detection by pine defense systems.