Spidroin-1 is one of the major ampullate silk proteins produced by spiders for use in the construction of the frame and radii of orb webs, and as a dragline to escape from predators. Only partial sequences of spidroin-1 produced by Nephila clavipes have been reported up to now, and there is no information on post-translational modifications (PTMs). A gel-based mass spectrometry strategy with ETD and CID fragmentation methods were used to sequence and determine the presence/location of any PTMs on the spidroin-1. Sequence coverage of 98.06%, 95.05%, and 98.37% were obtained for N. clavipes, Nephila edulis and for Nephila madagascariensis, respectively. Phosphorylation was the major PTM observed with 8 phosphorylation sites considered reliable on spidroin-1 produced by N. clavipes, 4 in N. madagascariensis and 2 for N. edulis. Dityrosine and 3,4-dihydroxyphenylalanine (formed by oxidation of the spidroin-1) were observed, although the mechanism by which they are formed (i.e. exposure to UV radiation or to peroxidases in the major ampullate silk gland) is uncertain. Herein we present structural information on the spidroin-1 produced by three different Nephila species; these findings may be valuable for understanding the physicochemical properties of the silk proteins and moreover, future designs of recombinantly produced spider silk proteins. Biotechnological significance The present investigation shows for the first time spidroin structure and post-translational modifications observed on the major ampullate silk spidroin-1. The many site specific phosphorylations (localized within the structural motifs) along with the probably photoinduction of hydroxylations may be relevant for scientists in material science, biology, biochemistry and environmental scientists. Up to now all the mechanical properties of the spidroin have been characterized without any consideration about the existence of PTMs in the sequence of spidroins. Thus, these findings for major ampullate silk spidroin-1 from Nephila spiders provide the basis for mechanical-elastic property studies of silk for biotechnological and biomedical potential applications. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Although the sodium/potassium transporting ATPase subunit alpha-3 (AT1A3) has been linked to memory mechanisms in rodents, regulation of this ATPase in terms of activity and complex levels by memory performance in a land maze has not been shown so far. It was therefore the aim of the study to link memory retrieval in the multiple T-Maze (MTM) to AT1A3 protein levels and activity. C57BL/6J mice were trained in the MTM and euthanized 6h following memory retrieval. Hippocampal membrane proteins were prepared by ultracentrifugation and run on blue native gel electrophoresis (BN-PAGE). Enzyme activity was evaluated using an in-gel method. AT1A3 protein was characterized using mass spectrometry (nano-LC-ESI-MS/MS). On BN-PAGE a single band was observed at 240 kDa, which corresponds to the dimeric form of the enzyme. Higher levels of AT1A3 complex were seen in trained mice. Also ATPase activity was higher in trained mice, and was observed both at 110 and at 240 kDa. Mass spectrometry unambiguously identified AT1A3 with 98.91% sequence coverage. A series of novel AT1A3 phosphorylation sites were detected. Taken together, it was shown that increased AT1A3 protein levels for the dimer as well as AT1A3 activity represented by the monomer and the dimer were paralleling memory training in the MTM. This may be relevant for understanding the role of the catalytic hydrolysis of ATP coupled with the exchange of sodium and potassium ions across the plasma membrane that generates the electrochemical gradient of sodium and potassium ions. Herein, we provide evidence for a possible role of AT1A3 in memory mechanisms and support previous findings using different animal models for memory formation.

Phosphatidylinositol-4,5-bisphosphate (PIP2 ) is a key regulator of many membrane proteins, including voltage-gated Kv7.2 channels. In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2 -binding domains in Kv7.2. Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2 . Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors. Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2 , thereby ensuring the tight regulation of the channel via G protein-coupled receptors.

The postsynaptic density (PSD) of excitatory synapses contains a highly organized protein network with thousands of proteins and is a key node in the regulation of synaptic plasticity. To gain new mechanistic insight into experience-induced changes in the PSD, we examined the global dynamics of the hippocampal PSD proteome and phosphoproteome in mice following four different types of experience. Mice were trained using an inhibitory avoidance (IA) task and hippocampal PSD fractions were isolated from individual mice to investigate molecular mechanisms underlying experience-dependent remodeling of synapses. We developed a new strategy to identify and quantify the relatively low level of site-specific phosphorylation of PSD proteome from the hippocampus, by using a modified iTRAQ-based TiSH protocol. In the PSD, we identified 3938 proteins and 2761 phosphoproteins in the sequential strategy covering a total of 4968 unique protein groups (at least two peptides including a unique peptide). On the phosphoproteins, we identified a total of 6188 unambiguous phosphosites (75%