Many eukaryotic proteins share a sequence designated as the zona pellucida (ZP) domain. This structural element, present in extracellular proteins from a wide variety of organisms, from nematodes to mammals, consists of approximately 260 amino acids with eight conserved cysteine (Cys) residues and is located close to the C terminus of the polypeptide. ZP domain proteins are often glycosylated, modular structures consisting of multiple types of domains. Predictions can be made about some of the structural features of the ZP domain and ZP domain proteins. The functions of ZP domain proteins vary tremendously, from serving as structural components of egg coats, appendicularian mucous houses, and nematode dauer larvae, to serving as mechanotransducers in flies and receptors in mammals and nonmammals. Generally, ZP domain proteins are present in filaments and/or matrices, which is consistent with the role of the domain in protein polymerization. A general mechanism for assembly of ZP domain proteins has been presented. It is likely that the ZP domain plays a common role despite its presence in proteins of widely diverse functions.

Proteomics is the field of study that includes the analysis of proteins, from either a basic science prospective or a clinical one. Proteins can be investigated for their abundance, variety of proteoforms due to post-translational modifications (PTMs), and their stable or transient protein–protein interactions. This can be especially beneficial in the clinical setting when studying proteins involved in different diseases and conditions. Here, we aim to describe a bottom-up proteomics workflow from sample preparation to data analysis, including all of its benefits and pitfalls. We also describe potential improvements in this type of proteomics workflow for the future.

This article presents an overview of the literature and a review of recent advances in the analysis of stable and transient protein-protein interactions (PPIs) with a focus on their function within cells, organs and organisms. The significance of post-translational modifications within the PPIs is also discussed. We focus on methods to study PPIs and methods of detecting PPIs, with particular emphasis on electrophoresis-based and mass spectrometry (MS)-based investigation of PPIs, including specific examples. The validation of PPIs is emphasized and the limitations of the current methods for studying stable and transient PPIs are discussed. Perspectives regarding PPIs, with focus on bioinformatics and transient PPIs are also provided.

Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands leads to activation of signal transduction pathways and to formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well-understood. We used Blue Native PAGE (BN-PAGE) and mass spectrometry (MS) to analyze the transient protein-protein interactions that resulted from stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated (pY-IPs) from the cell lysates of both unstimulated (−) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles in EphB2 signaling of proteins such as FAK, WAVEs, and Nischarin.

The extracellular coat, or vitelline envelope (VE), of rainbow trout (Oncorhynchus mykiss) eggs consists of three proteins, called VEalpha (M(r) approximately 52 kDa), VEbeta (M(r) approximately 48 kDa), and VEgamma (M(r) approximately 44 kDa). Each of these proteins is related to mammalian egg zona pellucida (ZP) glycoproteins ZP1-3 and possesses an N-terminal signal sequence, a ZP domain, and a protease cleavage site near the C-terminus. VEalpha and VEbeta also have a trefoil domain. All three proteins possess a relatively large number of cysteine residues (VEalpha, 18; VEbeta, 18; VEgamma, 12), of which 8 are present in the ZP domain and 6 are present in the trefoil domain of VEalpha and VEbeta. Here, several types of mass spectrometry were employed, together with gel electrophoresis of chemical and enzymatic digests, to identify intramolecular disulfide linkages, as well as the N- and C-terminal amino acids of VEalpha, VEbeta, and VEgamma. Additionally, these methods were used to characterize two high molecular weight proteins (HMWPs; M(r) > 110 kDa) of rainbow trout VEs that are heterodimers of individual VE proteins. These analyses have permitted assignment of disulfide linkages and identification of N- and C-terminal amino acids for the VE proteins and determination of the protein composition of two forms of HMWPs. These experiments provide important structural information about fish egg VE proteins and filaments and about structural relationships between extracellular coat proteins of mammalian and nonmammalian eggs.