In-depth proteomic analyses of Haliotis laevigata (greenlip abalone) nacre and prismatic organic shell matrix.

BACKGROUND: The shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years. A focus of this research has been the nacreous inner layer of the shell with its conspicuous arrangement of aragonite platelets, resembling in cross-section a brick-and-mortar wall. In comparison, the outer, less stable, calcitic prismatic layer has received much less attention. One of the first molluscan shell proteins to be characterized at the molecular level was Lustrin A, a component of the nacreous organic matrix of Haliotis rufescens. This was soon followed by the C-type lectin perlucin and the growth factor-binding perlustrin, both isolated from H. laevigata nacre, and the crystal growth-modulating AP7 and AP24, isolated from H. rufescens nacre. Mass spectrometry-based proteomics was subsequently applied to to Haliotis biomineralization research with the analysis of the H. asinina shell matrix and yielded 14 different shell-associated proteins. That study was the most comprehensive for a Haliotis species to date. METHODS: The shell proteomes of nacre and prismatic layer of the marine gastropod Haliotis laevigata were analyzed combining mass spectrometry-based proteomics and next generation sequencing. RESULTS: We identified 297 proteins from the nacreous shell layer and 350 proteins from the prismatic shell layer from the green lip abalone H. laevigata. Considering the overlap between the two sets we identified a total of 448 proteins. Fifty-one nacre proteins and 43 prismatic layer proteins were defined as major proteins based on their abundance at more than 0.2% of the total. The remaining proteins occurred at low abundance and may not play any significant role in shell fabrication. The overlap of major proteins between the two shell layers was 17, amounting to a total of 77 major proteins. CONCLUSIONS: The H. laevigata shell proteome shares moderate sequence similarity at the protein level with other gastropod, bivalve and more distantly related invertebrate biomineralising proteomes. Features conserved in H. laevigata and other molluscan shell proteomes include short repetitive sequences of low complexity predicted to lack intrinsic three-dimensional structure, and domains such as tyrosinase, chitin-binding, and carbonic anhydrase. This catalogue of H. laevigata shell proteins represents the most comprehensive for a haliotid and should support future efforts to elucidate the molecular mechanisms of shell assembly.

The calcified eggshell matrix proteome of a songbird, the zebra finch (Taeniopygia guttata).

BACKGROUND: The proteins of avian eggshell organic matrices are thought to control the mineralization of the eggshell in the shell gland (uterus). Proteomic analysis of such matrices identified many candidates for such a role. However, all matrices analyzed to date come from species of one avian family, the Phasianidae. To analyze the conservation of such proteins throughout the entire class Aves and to possibly identify a common protein toolkit enabling eggshell mineralization, it is important to analyze eggshell matrices from other avian families. Because mass spectrometry-based in-depth proteomic analysis still depends on sequence databases as comprehensive and accurate as possible, the obvious choice for a first such comparative study was the eggshell matrix of zebra finch, the genome sequence of which is the only songbird genome published to date. RESULTS: The zebra finch eggshell matrix comprised 475 accepted protein identifications. Most of these proteins (84 %) were previously identified in species of the Phasianidae family (chicken, turkey, quail). This also included most of the so-called eggshell-specific proteins, the ovocleidins and ovocalyxins. Ovocleidin-116 was the second most abundant protein in the zebra finch eggshell matrix. Major proteins also included ovocalyxin-32 and -36. The sequence of ovocleidin-17 was not contained in the sequence database, but a presumptive homolog was tentatively identified by N-terminal sequence analysis of a prominent 17 kDa band. The major proteins also included three proteins similar to ovalbumin, the most abundant of which was identified as ovalbumin with the aid of two characteristic phosphorylation sites. Several other proteins identified in Phasianidae eggshell matrices were not identified. When the zebra finch sequence database contained a sequence similar to a missing phasianid protein it may be assumed that the protein is missing from the matrix. This applied to ovocalyxin-21/gastrokine-1, a major protein of the chicken eggshell matrix, to EDIL3 and to lactadherin. In other cases failure to identify a particular protein may be due to the absence of this protein from the sequence database, highlighting the importance of better, more comprehensive sequence databases. CONCLUSIONS: The results indicate that ovocleidin-116, ovocleidin-17, ovocalyxin-36 and ovocalyxin-32 may be universal avian eggshell-mineralizing proteins. All the more important it is to elucidate the role of these proteins at the molecular level. This cannot be achieved by proteomic studies but will need application of other methods, such as atomic force microscopy or gene knockouts. However, it will also be important to analyze more eggshell matrices of different avian families to unequivocally identify other mineralization toolkit proteins apart from ovocleidins and ovocalyxins. Progress in this respect will depend critically on the availability of more, and more comprehensive, sequence databases. The development of faster and cheaper nucleotide sequencing methods has considerably accelerated genome and transcriptome sequencing, but this seems to concur with frequent publication of incomplete and fragmented sequence databases.

The Skeleton Forming Proteome of an Early Branching Metazoan: A Molecular Survey of the Biomineralization Components Employed by the Coralline Sponge Vaceletia Sp.

The ability to construct a mineralized skeleton was a major innovation for the Metazoa during their evolution in the late Precambrian/early Cambrian. Porifera (sponges) hold an informative position for efforts aimed at unraveling the origins of this ability because they are widely regarded to be the earliest branching metazoans, and are among the first multi-cellular animals to display the ability to biomineralize in the fossil record. Very few biomineralization associated proteins have been identified in sponges so far, with no transcriptome or proteome scale surveys yet available. In order to understand what genetic repertoire may have been present in the last common ancestor of the Metazoa (LCAM), and that may have contributed to the evolution of the ability to biocalcify, we have studied the skeletal proteome of the coralline demosponge Vaceletia sp. and compare this to other metazoan biomineralizing proteomes. We bring some spatial resolution to this analysis by dividing Vaceletia's aragonitic calcium carbonate skeleton into "head" and "stalk" regions. With our approach we were able to identify 40 proteins from both the head and stalk regions, with many of these sharing some similarity to previously identified gene products from other organisms. Among these proteins are known biomineralization compounds, such as carbonic anhydrase, spherulin, extracellular matrix proteins and very acidic proteins. This report provides the first proteome scale analysis of a calcified poriferan skeletal proteome, and its composition clearly demonstrates that the LCAM contributed several key enzymes and matrix proteins to its descendants that supported the metazoan ability to biocalcify. However, lineage specific evolution is also likely to have contributed significantly to the ability of disparate metazoan lineages to biocalcify.

Proteomic analysis of quail calcified eggshell matrix: a comparison to chicken and turkey eggshell proteomes.

BACKGROUND: Eggshell mineralization in commercially important species such as chicken, turkey or quail is of interest as a general model of calcium carbonate biomineralization. Knowledge of proteins and molecular mechanisms in eggshell assembly may also pave the way to manipulation of thickness of the calcified layer or other features. Comparison of eggshell matrix proteomes of different species may contribute to a better understanding of the mineralization process. The recent publication of the quail genome sequence now enables the proteomic analysis of the quail shell matrix and this comparison with those of chicken and turkey. RESULTS: The quail eggshell proteome comprised 622 identified proteins, 311 of which were shared with chicken and turkey eggshell proteomes. Forty-eight major proteins (iBAQ-derived abundance higher than 0.1 % of total identified proteome) together covered 94 % of total proteome mass. Fifteen of these are also among the most abundant proteins in chicken and turkey eggshell matrix. Only three proteins with a percentage higher than 1.0 % of the total had not previously been identified as eggshell matrix proteins. These were an uncharacterized member of the latexin family, an uncharacterized protease inhibitor containing a Kunitz domain, and gastric intrinsic factor. The most abundant proteins were ovocleidin-116, ovalbumin and ovocalyxin-36 representing approximately 31, 13 and 8 % of the total identified proteome, respectively. The major phosphoproteins were ovocleidin-116 and osteopontin. While osteopontin phosphorylation sites were predominantly conserved between chicken and quail sequences, conservation was less in ovocleidin-116. CONCLUSIONS: Ovocleidin-116 and ovocalyxin-36 are among the most abundant eggshell matrix proteins in all three species of the family Phasianidae analyzed so far, indicating that their presently unknown function is essential for eggshell mineralization. Evidence for other chicken eggshell-specific proteins in quail was inconclusive. Therefore measurement of additional eggshell proteomes, especially from species of different families and preferentially from outside the order Galliformes, will be necessary.

The Magellania venosa Biomineralizing Proteome: A Window into Brachiopod Shell Evolution.

Brachiopods are a lineage of invertebrates well known for the breadth and depth of their fossil record. Although the quality of this fossil record attracts the attention of paleontologists, geochemists, and paleoclimatologists, modern day brachiopods are also of interest to evolutionary biologists due to their potential to address a variety of questions ranging from developmental biology to biomineralization. The brachiopod shell is a composite material primarily composed of either calcite or calcium phosphate in close association with proteins and polysaccharides which give these composite structures their material properties. The information content of these biomolecules, sequestered within the shell during its construction, has the potential to inform hypotheses focused on describing how brachiopod shell formation evolved. Here, using high throughput proteomic approaches and next generation sequencing, we have surveyed and characterized the first shell-proteome and shell-forming transcriptome of any brachiopod, the South American Magellania venosa (Rhynchonelliformea: Terebratulida). We find that the seven most abundant proteins present in the shell are unique to M. venosa, but that these proteins display biochemical features found in other metazoan biomineralization proteins. We can also detect some M. venosa proteins that display significant sequence similarity to other metazoan biomineralization proteins, suggesting that some elements of the brachiopod shell-forming proteome are deeply evolutionarily conserved. We also employed a variety of preparation methods to isolate shell proteins and find that in comparison to the shells of other spiralian invertebrates (such as mollusks) the shell ultrastructure of M. venosa may explain the effects these preparation strategies have on our results.

The Lottia gigantea shell matrix proteome: re-analysis including MaxQuant iBAQ quantitation and phosphoproteome analysis.

BACKGROUND: Although the importance of proteins of the biomineral organic matrix and their posttranslational modifications for biomineralization is generally recognized, the number of published matrix proteomes is still small. This is mostly due to the lack of comprehensive sequence databases, usually derived from genomic sequencing projects. However, in-depth mass spectrometry-based proteomic analysis, which critically depends on high-quality sequence databases, is a very fast tool to identify candidates for functional biomineral matrix proteins and their posttranslational modifications. Identification of such candidate proteins is facilitated by at least approximate quantitation of the identified proteins, because the most abundant ones may also be the most interesting candidates for further functional analysis. RESULTS: Re-quantification of previously identified Lottia shell matrix proteins using the intensity-based absolute quantification (iBAQ) method as implemented in the MaxQuant identification and quantitation software showed that only 57 of the 382 accepted identifications constituted 98% of the total identified matrix proteome. This group of proteins did not contain obvious intracellular proteins, such as cytoskeletal components or ribosomal proteins, invariably identified as minor components of high-throughput biomineral matrix proteomes. Fourteen of these major proteins were phosphorylated to a variable extent. All together we identified 52 phospho sites in 20 of the 382 accepted proteins with high confidence. CONCLUSIONS: We show that iBAQ quantitation may be a useful tool to narrow down the group of functional biomineral matrix protein candidates for further research in cell biology, genetics or materials research. Knowledge of posttranslational modifications in these major proteins could be a valuable addition to previously published proteomes. This is true especially for phosphorylation, because this modification was already shown to modify mineralization processes in some instances.

Characterization of the pigmented shell-forming proteome of the common grove snail Cepaea nemoralis.

BACKGROUND: With a diversity of pigmented shell morphotypes governed by Mendelian patterns of inheritance, the common grove snail, Cepaea nemoralis, has served as a model for evolutionary biologists and population geneticists for decades. Surprisingly, the molecular mechanisms by which C. nemoralis generates this pigmented shelled diversity, and the degree of evolutionary conservation present between molluscan shell-forming proteomes, remain unknown. RESULTS: Here, using next generation sequencing and high throughput proteomics, we identify and characterize the major proteinaceous components of the C. nemoralis shell, the first shell-proteome for a pulmonate mollusc. The recent availability of several marine molluscan shell-proteomes, and the dataset we report here, allow us to identify 59 evolutionarily conserved and novel shell-forming proteins. While the C. nemoralis dataset is dominated by proteins that share little to no similarity with proteins in public databases, almost half of it shares similarity with proteins present in other molluscan shells. In addition, we could not find any indication that a protein (or class of proteins) is directly associated with shell pigmentation in C. nemoralis. This is in contrast to the only other partially characterized molluscan-shell pigmentation mechanism employed by the tropical abalone Haliotis asinina. CONCLUSIONS: The unique pulmonate shell-forming proteome that we report here reveals an abundance of both mollusc-specific and pulmonate-specific proteins, suggesting that novel coding sequences, and/or the extensive divergence of these sequences from ancestral sequences, supported the innovation of new shell types within the Conchifera. In addition, we report here the first evidence that molluscs use independently evolved mechanisms to pigment their shells. This proteome provides a solid foundation from which further studies aimed at the functional characterization of these shell-forming proteins can be conducted.

Effect of embryonic development on the chicken egg yolk plasma proteome after 12 days of incubation.

To better appreciate the dynamics of yolk proteins during embryonic development, we analyzed the protein quantitative changes occurring in the yolk plasma at the day of lay and after 12 days of incubation, by comparing unfertilized and fertilized chicken eggs. Of the 127 identified proteins, 69 showed relative abundance differences among conditions. Alpha-fetoprotein and two uncharacterized proteins (F1NHB8 and F1NMM2) were identified for the first time in the egg. After 12 days of incubation, five proteins (vitronectin, α-fetoprotein, similar to thrombin, apolipoprotein B, and apovitellenin-1) showed a major increase in relative abundance, whereas 15 proteins showed a significant decrease in the yolks of fertilized eggs. In unfertilized/table eggs, we observed an accumulation of proteins likely to originate from other egg compartments during incubation. This study provides basic knowledge on the utilization of egg yolk proteins by the embryo and gives some insight into how storage can affect egg quality.

The proteome of the calcified layer organic matrix of turkey (Meleagris gallopavo) eggshell.

BACKGROUND: Chicken eggshell mineralization is a prominent model for biomineralization not only because of its importance for avian reproduction but also because of the commercial interest associated with eggshell quality. An analysis and comparison of the protein constituents of eggshells of several species would contribute to a better understanding of the shell mineralization process. The recent publication of the turkey genome sequence now provides a basis for the in-depth analysis of the turkey eggshell proteome. RESULTS: Proteomic analysis of turkey acid-soluble and acid-insoluble organic eggshell matrix yielded 697 identified proteins/protein groups. However, intensity-based absolute quantification (iBAQ) results indicated that the 47 most abundant identified proteins already constituted 95% of the total turkey eggshell matrix proteome. Forty-four of these proteins were also identified in chicken eggshell matrix previously. Despite these similarities there were important and unexpected differences. While ovocleidin-116 and ovocalyxin-36 were major proteins constituting approximately 37% of the identified proteome, other members of the group of so-called eggshell-specific proteins were not identified. Thus ovocalyxin-21 and ovocalyxin-32 were missing among matrix proteins. Conversely, major turkey eggshell proteins were not detected in chicken, such as the bone protein periostin, the mammalian counterpart of which is involved in many aspects of bone metabolism and which represented 10-11% of the total identified proteome. CONCLUSIONS: Even members of the same avian family show important differences in eggshell matrix composition and more studies on the proteome and the transcriptome level will be necessary to identify a common toolkit of eggshell mineralization and to work out species differences among functional eggshell protein sets and their role in eggshell production.

In-depth proteomic analysis of a mollusc shell: acid-soluble and acid-insoluble matrix of the limpet Lottia gigantea.

BACKGROUND: Invertebrate biominerals are characterized by their extraordinary functionality and physical properties, such as strength, stiffness and toughness that by far exceed those of the pure mineral component of such composites. This is attributed to the organic matrix, secreted by specialized cells, which pervades and envelops the mineral crystals. Despite the obvious importance of the protein fraction of the organic matrix, only few in-depth proteomic studies have been performed due to the lack of comprehensive protein sequence databases. The recent public release of the gastropod Lottia gigantea genome sequence and the associated protein sequence database provides for the first time the opportunity to do a state-of-the-art proteomic in-depth analysis of the organic matrix of a mollusc shell. RESULTS: Using three different sodium hypochlorite washing protocols before shell demineralization, a total of 569 proteins were identified in Lottia gigantea shell matrix. Of these, 311 were assembled in a consensus proteome comprising identifications contained in all proteomes irrespective of shell cleaning procedure. Some of these proteins were similar in amino acid sequence, amino acid composition, or domain structure to proteins identified previously in different bivalve or gastropod shells, such as BMSP, dermatopontin, nacrein, perlustrin, perlucin, or Pif. In addition there were dozens of previously uncharacterized proteins, many containing repeated short linear motifs or homorepeats. Such proteins may play a role in shell matrix construction or control of mineralization processes. CONCLUSIONS: The organic matrix of Lottia gigantea shells is a complex mixture of proteins comprising possible homologs of some previously characterized mollusc shell proteins, but also many novel proteins with a possible function in biomineralization as framework building blocks or as regulatory components. We hope that this data set, the most comprehensive available at present, will provide a platform for the further exploration of biomineralization processes in molluscs.

The eggshell: structure, composition and mineralization.

The calcareous egg is produced by all birds and most reptiles. Current understanding of eggshell formation and mineralization is mainly based on intensive studies of one species - the domesticated chicken Gallus gallus. The majority of constituents of the chicken eggshell have been identified. In this article we review eggshell microstructure and ultrastructure, and the results of recent genomic, transcriptomic and proteomic analyses of the chicken eggshell matrix to draw attention to areas of current uncertainty such as the potential role of amorphous calcium carbonate and the specific nature of the molecules that initiate (nucleate) mammillary cone formation and terminate palisade layer calcification. Comparative avian genomics and proteomics have only recently become possible with the publication of the Taeniopygia guttata (zebra finch) genome. Further rapid progress is highly anticipated with the soon-to-be-released genomes of turkey (Meleagris gallopavo) and duck (Anas platyrhynchos). These resources will allow rapid advances in comparative studies of the organic constituents of avian eggshell and their functional implications.

In-depth analysis of the chicken egg white proteome using an LTQ Orbitrap Velos.

BACKGROUND: Hen's egg white has been the subject of intensive chemical, biochemical and food technological research for many decades, because of its importance in human nutrition, its importance as a source of easily accessible model proteins, and its potential use in biotechnological processes. Recently the arsenal of tools used to study the protein components of egg white has been complemented by mass spectrometry-based proteomic technologies. Application of these fast and sensitive methods has already enabled the identification of a large number of new egg white proteins. Recent technological advances may be expected to further expand the egg white protein inventory. RESULTS: Using a dual pressure linear ion trap Orbitrap instrument, the LTQ Orbitrap Velos, in conjunction with data analysis in the MaxQuant software package, we identified 158 proteins in chicken egg white with two or more sequence unique peptides. This group of proteins identified with very high confidence included 79 proteins identified in egg white for the first time. In addition, 44 proteins were identified tentatively. CONCLUSIONS: Our results, apart from identifying many new egg white components, indicate that current mass spectrometry technology is sufficiently advanced to permit direct identification of minor components of proteomes dominated by a few major proteins without resorting to indirect techniques, such as chromatographic depletion or peptide library binding, which change the composition of the proteome.

Antimicrobial activity of lipophilic avian eggshell surface extracts.

The avian eggshell cuticle is the waxy outermost layer of the mineralized eggshell in direct contact with the environment. In this study, lipophilic eggshell surface extracts from three domestic species were evaluated for their antimicrobial activity. Chicken and goose extracts demonstrated potent bactericidal activity against both Gram-positive and Gram-negative bacteria, while activity could not be detected for duck eggshell surface extracts. Using the chicken as a model species, evaluation of albumen, fecal material, and uropygial gland extracts eliminated these as a potential source of the observed activity. Results suggest that lipophilic components are incorporated into the egg during its formation and play a role in antimicrobial defense. This study represents the first successful extraction and evaluation of lipophilic antimicrobial components from the avian egg.

Proteomic analysis of sea urchin (Strongylocentrotus purpuratus) spicule matrix.

BACKGROUND: The sea urchin embryo has been an important model organism in developmental biology for more than a century. This is due to its relatively simple construction, translucent appearance, and the possibility to follow the fate of individual cells as development to the pluteus larva proceeds. Because the larvae contain tiny calcitic skeletal elements, the spicules, they are also important model organisms for biomineralization research. Similar to other biominerals the spicule contains an organic matrix, which is thought to play an important role in its formation. However, only few spicule matrix proteins were identified previously. RESULTS: Using mass spectrometry-based methods we have identified 231 proteins in the matrix of the S. purpuratus spicule matrix. Approximately two thirds of the identified proteins are either known or predicted to be extracellular proteins or transmembrane proteins with large ectodomains. The ectodomains may have been solubilized by partial proteolysis and subsequently integrated into the growing spicule. The most abundant protein of the spicule matrix is SM50. SM50-related proteins, SM30-related proteins, MSP130 and related proteins, matrix metalloproteases and carbonic anhydrase are among the most abundant components. CONCLUSIONS: The spicule matrix is a relatively complex mixture of proteins not only containing matrix-specific proteins with a function in matrix assembly or mineralization, but also: 1) proteins possibly important for the formation of the continuous membrane delineating the mineralization space; 2) proteins for secretory processes delivering proteinaceous or non-proteinaceous precursors; 3) or proteins reflecting signaling events at the cell/matrix interface. Comparison of the proteomes of different skeletal matrices allows prediction of proteins of general importance for mineralization in sea urchins, such as SM50, SM30-E, SM29 or MSP130. The comparisons also help point out putative tissue-specific proteins, such as tooth phosphodontin or specific spicule matrix metalloproteases of the MMP18/19 group. Furthermore, the direct sequence analysis of peptides by MS/MS validates many predicted genes and confirms the existence of the corresponding proteins.

Phosphoproteomes of Strongylocentrotus purpuratus shell and tooth matrix: identification of a major acidic sea urchin tooth phosphoprotein, phosphodontin.

BACKGROUND: Sea urchin is a major model organism for developmental biology and biomineralization research. However, identification of proteins involved in larval skeleton formation and mineralization processes in the embryo and adult, and the molecular characterization of such proteins, has just gained momentum with the sequencing of the Strongylocentrotus purpuratus genome and the introduction of high-throughput proteomics into the field. RESULTS: The present report contains the determination of test (shell) and tooth organic matrix phosphoproteomes. Altogether 34 phosphoproteins were identified in the biomineral organic matrices. Most phosphoproteins were specific for one compartment, only two were identified in both matrices. The sea urchin phosphoproteomes contained several obvious orthologs of mammalian proteins, such as a Src family tyrosine kinase, protein kinase C-delta 1, Dickkopf-1 and other signal transduction components, or nucleobindin. In most cases phosphorylation sites were conserved between sea urchin and mammalian proteins. However, the majority of phosphoproteins had no mammalian counterpart. The most interesting of the sea urchin-specific phosphoproteins, from the perspective of biomineralization research, was an abundant highly phosphorylated and very acidic tooth matrix protein composed of 35 very similar short sequence repeats, a predicted N-terminal secretion signal sequence, and an Asp-rich C-terminal motif, contained in [Glean3:18919]. CONCLUSIONS: The 64 phosphorylation sites determined represent the most comprehensive list of experimentally identified sea urchin protein phosphorylation sites at present and are an important addition to the recently analyzed Strongylocentrotus purpuratus shell and tooth proteomes. The identified phosphoproteins included a major, highly phosphorylated protein, [Glean3:18919], for which we suggest the name phosphodontin. Although not sequence-related to such highly phosphorylated acidic mammalian dental phosphoproteins as phosphoryn or dentin matrix protein-1, phosphodontin may perform similar functions in the sea urchin tooth. More than half of the detected proteins were not previously identified at the protein level, thus confirming the existence of proteins only known as genomic sequences previously.

Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family.

The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI approximately 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.

In-depth, high-accuracy proteomics of sea urchin tooth organic matrix.

BACKGROUND: The organic matrix contained in biominerals plays an important role in regulating mineralization and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin tooth, which is an important model for developmental biology and biomineralization, only few matrix components have been identified. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of genes potentially coding for matrix proteins, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy proteomic analysis. RESULTS: We identified 138 proteins in the matrix of tooth powder. Only 56 of these proteins were previously identified in the matrices of test (shell) and spine. Among the novel components was an interesting group of five proteins containing alanine- and proline-rich neutral or basic motifs separated by acidic glycine-rich motifs. In addition, four of the five proteins contained either one or two predicted Kazal protease inhibitor domains. The major components of tooth matrix were however largely identical to the set of spicule matrix proteins and MSP130-related proteins identified in test (shell) and spine matrix. Comparison of the matrices of crushed teeth to intact teeth revealed a marked dilution of known intracrystalline matrix proteins and a concomitant increase in some intracellular proteins. CONCLUSION: This report presents the most comprehensive list of sea urchin tooth matrix proteins available at present. The complex mixture of proteins identified may reflect many different aspects of the mineralization process. A comparison between intact tooth matrix, presumably containing odontoblast remnants, and crushed tooth matrix served to differentiate between matrix components and possible contributions of cellular remnants. Because LC-MS/MS-based methods directly measures peptides our results validate many predicted genes and confirm the existence of the corresponding proteins. Knowledge of the components of this model system may stimulate further experiments aiming at the elucidation of structure, function, and interaction of biomineral matrix components.

The sea urchin (Strongylocentrotus purpuratus) test and spine proteomes.

BACKGROUND: The organic matrix of biominerals plays an important role in biomineral formation and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin, which is an important model organism for developmental biology and biomineralization, only few matrix components have been identified and characterized at the protein level. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of possible matrix proteins at the gene level, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy, proteomic analysis. RESULTS: We identified 110 proteins as components of sea urchin test and spine organic matrix. Fourty of these proteins occurred in both compartments while others were unique to their respective compartment. More than 95% of the proteins were detected in sea urchin skeletal matrices for the first time. The most abundant protein in both matrices was the previously characterized spicule matrix protein SM50, but at least eight other members of this group, many of them only known as conceptual translation products previously, were identified by mass spectrometric sequence analysis of peptides derived from in vitro matrix degradation. The matrices also contained proteins implicated in biomineralization processes previously by inhibition studies using antibodies or specific enzyme inhibitors, such as matrix metalloproteases and members of the mesenchyme-specific MSP130 family. Other components were carbonic anhydrase, collagens, echinonectin, a alpha2-macroglobulin-like protein and several proteins containing scavenger receptor cysteine-rich domains. A few possible signal transduction pathway components, such as GTP-binding proteins, a semaphorin and a possible tyrosine kinase were also identified. CONCLUSION: This report presents the most comprehensive list of sea urchin skeletal matrix proteins available at present. The complex mixture of proteins identified in matrices of the sea urchin skeleton may reflect many different aspects of the mineralization process. Because LC-MS/MS-based methods directly measures peptides our results validate many predicted genes and confirm the existence of the corresponding proteins. Considering the many newly identified matrix proteins, this proteomic study may serve as a road map for the further exploration of biomineralization processes in an important model organism.

Proteomic analysis of the chicken egg vitelline membrane.

The avian vitelline membrane (VM) is a multilayered proteinaceous structure separating egg white from yolk. The innermost layer of the VM, deposited onto the oocyte plasma membrane in the ovary, corresponds to the mammalian zona pellucida (ZP). The outer layer is produced in the infundibulum, the first section of the oviduct. Using high-throughput, high-end LC-MS(n) 137 proteins were identified, only 13 of which were known previously to be components of the VM. Depending on the washing protocol, two largely overlapping, but not identical, sets of identified proteins were produced from water-washed and salt-washed VMs. Most of the components of the VM were known previously from other egg compartments, such as, for instance, the egg white proteins lysozyme C, ovalbumin, ovotransferrin, and ovomucin. Specific components of the VM not identified previously in other egg compartments included eight ZP proteins, oviductin protease, and two ATPases. The vitelline outer membrane protein (VMO) VMO II was identified as beta-defensin-11. The list of VM proteins presented in this report is by far the most comprehensive dataset available at present and complements proteomic analyses of chicken egg compartments published previously.

The chicken egg yolk plasma and granule proteomes.

Using 1-D SDS-PAGE, LC-MS/MS, and MS(3), we identified 119 proteins from chicken egg yolk, 86 of which were not identified in yolk previously. Proteins were roughly quantitated by calculating their exponentially modified protein abundance index (emPAI) to classify them as major or minor yolk components, and to estimate their distribution between yolk plasma and yolk granular fraction. The proteins with highest abundance were serum albumin, the vitellogenin cleavage products, apovitellenins, IgY, ovalbumin, and 12 kDa serum protein with cross-reactivity to beta2-microglobulin. In addition yolk contained many other serum and egg white proteins, the proteases nothepsin and thrombin, numerous protease inhibitors, and antioxidative enzymes, such as superoxide dismutase and glutathione peroxidase. Among the moderately abundant proteins were two alpha2-macroglobulin-like proteins different from egg white alpha2-macroglobulin, and the major biotin-binding protein of yolk. An unexpected identification was that of the eggshell matrix protein ovocleidin-116, which was previously thought to be eggshell-specific. The list of chicken egg yolk proteins provided in this report is by far the most comprehensive at present and may serve as a starting point for the characterization of less well-known yolk proteins.