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.     

Identification of Bilirubin Binding Site in Type I Collagen

The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α₁(I) chain but not to α₂(I) chains which clearly designates that bilirubin shows greater affinity to α₁ chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible.     

Discovery of neuropeptides in the nematode Ascaris suum by database mining and tandem mass spectrometry

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to discover peptides in extracts of the large parasitic nematode Ascaris suum. This required the assembly of a new database of known and predicted peptides. In addition to those already sequenced, peptides were either previously predicted to be processed from precursor proteins identified in an A. suum library of expressed sequence tags (ESTs) or newly predicted from a library of A. suum genome survey sequences (GSSs). The predicted MS/MS fragmentation patterns of this collection of real and putative peptides were compared with the actual fragmentation patterns found in the MS/MS spectra of peptides fractionated by MS; this enabled individual peptides to be sequenced. Many previously identified peptides were found, and 21 novel peptides were discovered. Thus, this approach is very useful, despite the fact that the available GSS database is still preliminary, having only 1× coverage.     

Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation

Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.     

Purification and identification of antioxidant peptides from egg white protein hydrolysate

Egg white proteins were hydrolysed separately using five different proteases to obtain antioxidant peptides. The antioxidant activity of egg white protein hydrolysates was influenced by the time of hydrolysis and the type of enzyme. Of the various hydrolysates produced, papain hydrolysate obtained by 3-h hydrolysis (PEWPH) displayed the highest DPPH radical scavenging activity. PEWPH could also quench the superoxide anion and hydroxyl radicals, effectively inhibit lipid peroxidation and exhibit reducing power. Then, PEWPH was purified sequentially by ultrafiltration, gel filtration, RP-HPLC and two fractions with relatively strong antioxidant activity were subsequently subjected to LC-MS/MS for peptide sequence identification. The sequences of the two antioxidant peptides were identified to be Tyr-Leu-Gly-Ala-Lys (551.54?Da) and Gly-Gly-Leu-Glu-Pro-Ile-Asn-Phe-Gln (974.55?Da), and they were identified for the first time from food-derived protein hydrolysates. Last, the two purified peptides were synthesized and they showed 7.48- and 6.02-fold higher DPPH radical scavenging activity compared with the crude PEWPH, respectively. These results indicate that PEWPH and/or its isolated peptides may be useful ingredients in food and nutraceutical applications.     

The secreted salivary proteome of the pea aphid Acyrthosiphon pisum characterised by mass spectrometry

Nine proteins secreted in the saliva of the pea aphid Acyrthosiphon pisum were identified by a proteomics approach using GE‐LC‐MS/MS and LC‐MS/MS, with reference to EST and genomic sequence data for A. pisum. Four proteins were identified by their sequences: a homolog of angiotensin‐converting enzyme (an M2 metalloprotease), an M1 zinc‐dependant metalloprotease, a glucose‐methanol‐choline (GMC)‐oxidoreductase and a homolog to regucalcin (also known as senescence marker protein 30). The other five proteins are not homologous to any previously described sequence and included an abundant salivary protein (represented by ACYPI009881), with a predicted length of 1161 amino acids and high serine, tyrosine and cysteine content. A. pisum feeds on plant phloem sap and the metalloproteases and regucalcin (a putative calcium‐binding protein) are predicted determinants of sustained feeding, by inactivation of plant protein defences and inhibition of calcium‐mediated occlusion of phloem sieve elements, respectively. The amino acid composition of ACYPI009881 suggests a role in the aphid salivary sheath that protects the aphid mouthparts from plant defences, and the oxidoreductase may promote gelling of the sheath protein or mediate oxidative detoxification of plant allelochemicals. Further salivary proteins are expected to be identified as more sensitive MS technologies are developed.     

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

Induction and partial characterization of California halibut (Paralichthys californicus) vitellogenin

The egg yolk precursor protein, vitellogenin (Vg), was isolated by size exclusion and ion exchange chromatography from plasma of California halibut (Paralichthys californicus) treated with estrogen. MALDI TOF mass spectrometry (MS) analysis resulted in a molecular mass of 188 kDa. MS/MS de novo sequencing identified the protein as Vg by matching sequences of tryptic peptides to the known sequences of several other species. Matches were also made to two different forms of Vg in haddock, medaka, and mummichog, providing evidence that California halibut has more than one form of Vg. Native PAGE and Western blot with an antibody to turbot (Scophthalmus maximus) Vg confirmed the identity of the protein. Protein resolved on the SDS PAGE as a double band of approximately the same mass as determined with MALDI TOF, and two lower mass bands that were also immunoreactive. MALDI TOF and MS/MS de novo sequencing were useful for determining the molecular mass, identification, and exploring the multiplicity of Vg. The potential of using other MS methods to understand the structure and function of Vg is discussed.     

Identification of putative serum glycoprotein biomarkers for human lung adenocarcinoma by multilectin affinity chromatography and LC-MS/MS

Glycoproteins in human serum play fundamental roles in many biological processes, and also have clinical value as biomarkers for disease progression and treatment. In this study, we isolated glycoproteins from the sera of three healthy individuals and three lung adenocarcinoma patients using multilectin affinity chromatography. The recovered glycoproteins were subjected to treatment with peptide-N-glycosidase F (PNGase F) and in-gel digestion by trypsin. Tryptic peptides were analyzed by nano-LC coupled to ESI-MS/MS and the MS/MS spectra were processed by Bioworks 3.2 and an in-house bioinformatics tool, ProtAn. Approximately 90% of the proteins identified contained more than one potential glycosylation site. Comparison of the serum glycoproteome of healthy and adenocarcinoma individuals revealed 38 cancer-selective proteins. Among them, 60% have previously been reported as low abundance proteins in human sera. We identified several cancer-selective proteins that have been previously characterized as potential indicators of lung cancer in serum or plasma, including haptoglobin (HP), inter-alpha-trypsin inhibitor heavy chain 4 (ITI-H4), complement C3 precursor, and leucine-rich alpha-2-glycoprotein. In addition, plasma kallikrein (KLKB1) and inter-alpha-trypsin inhibitor heavy chain 3 (ITI-H3) were identified as being potentially elevated in the lung cancer group, and were validated by Western blot analysis. Furthermore, approximately 18 kDa plasma kallirein protein fragment was detected at high levels in 25 out of 28 adenocarcinoma patients, while one of the eight normal individuals showed moderate positive. The results suggest that KLKB1 represents a potential candidate serum biomarker of lung cancer.     

Proteomic and peptidomic analysis of the venom from Chinese tarantula Chilobrachys jingzhao

Chinese tarantula, Chilobrachys jingzhao is one of the most venomous spiders in southern China and its venom is a mixture of various compounds with diversified biological activities. The proteome of C. jingzhao venom was analyzed by proteomic techniques. Proteins with molecular weight of over 10 kDa, indicated by gel-filtration and SDS-PAGE, were analyzed using 2-DE and MALDI-TOF/TOF and LC/ESI-Q-TOF MS. More than 90 proteins were detected, with 47 confirmed by sequence similarity search using mass spectrum driven basic local alignment search tool (MS BLAST). On the other hand, peptides with MW lower than 10 kDa were separated by HPLC and identified by MALDI-TOF MS and Edman degradation sequencing. About 120 peptides were detected, 60 of which were fully or partially sequenced. Our results indicate that peptides with MW lower than 10 kDa are the major components in the crude venom of C. jingzhao. Like those of other tarantulas, these peptides are very likely to act on various ion channels. These results pave a way for further detailed structure-function correlation analysis of the individual toxins present in the venom of C. jingzhao.     

Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry

The 10(5) resolving power and MS/MS capabilities of Fourier-transform mass spectrometry provide electrospray ionization mass spectra containing >100 molecular and fragment ion mass values of high accuracy. Applying these spectra to the detection and localization of errors and modifications in the DNA-derived sequences of proteins is illustrated with the thiCEFSGH thiamin biosynthesis operon from Escherichia coli. Direct fragmentation of the multiply-charged intact protein ions produces large fragment ions covering the entire sequence; further dissociation of these fragment ions provides information on their sequences. For ThiE (23 kDa), the entire sequence was verified in a single spectrum with an accurate (0.3 Da) molecular weight (Mr) value, with confirmation from MS/MS fragment masses. Those for ThiH (46 kDa) showed that the Mr value (1 Da error) represented the protein without the start Met residue. For ThiF (27 kDa), MS/MS localized a sequence discrepancy to a 34 residue peptide. The first 107 residues of ThiC (74 kDa) were shown to be correct, with C-terminal heterogeneity indicated. For ThiG (predicted Mr = 34 kDa), ESI/FTMS showed two components of 7,310.74 (ThiS) and 26,896.5 Da (ThiG); MS/MS uncovered three reading frame errors and a stop codon for the first protein. MS/MS ions are consistent with 68 fragments predicted by the corrected ThiS/ThiG DNA sequences.     

Effect of cleavage enzyme, search algorithm and decoy database on mass spectrometric identification of wheat gluten proteins

While tandem mass spectrometry (MS/MS) is routinely used to identify proteins from complex mixtures, certain types of proteins present unique challenges for MS/MS analyses. The major wheat gluten proteins, gliadins and glutenins, are particularly difficult to distinguish by MS/MS. Each of these groups contains many individual proteins with similar sequences that include repetitive motifs rich in proline and glutamine. These proteins have few cleavable tryptic sites, often resulting in only one or two tryptic peptides that may not provide sufficient information for identification. Additionally, there are less than 14,000 complete protein sequences from wheat in the current NCBInr release. In this paper, MS/MS methods were optimized for the identification of the wheat gluten proteins. Chymotrypsin and thermolysin as well as trypsin were used to digest the proteins and the collision energy was adjusted to improve fragmentation of chymotryptic and thermolytic peptides. Specialized databases were constructed that included protein sequences derived from contigs from several assemblies of wheat expressed sequence tags (ESTs), including contigs assembled from ESTs of the cultivar under study. Two different search algorithms were used to interrogate the database and the results were analyzed and displayed using a commercially available software package (Scaffold). We examined the effect of protein database content and size on the false discovery rate. We found that as database size increased above 30,000 sequences there was a decrease in the number of proteins identified. Also, the type of decoy database influenced the number of proteins identified. Using three enzymes, two search algorithms and a specialized database allowed us to greatly increase the number of detected peptides and distinguish proteins within each gluten protein group.     

A comprehensive analysis of Trypanosoma brucei mitochondrial proteome

The composition of the large, single, mitochondrion (mt) of Trypanosoma brucei was characterized by MS (2‐D LC‐MS/MS and gel‐LC‐MS/MS) analyses. A total of 2897 proteins representing a substantial proportion of procyclic form cellular proteome were identified, which confirmed the validity of the vast majority of gene predictions. The data also showed that the genes annotated as hypothetical (species specific) were overpredicted and that virtually all genes annotated as hypothetical, unlikely are not expressed. By comparing the MS data with genome sequence, 40 genes were identified that were not previously predicted. The data are placed in a publicly available web‐based database (www.TrypsProteome.org). The total mitochondrial proteome is estimated at 1008 proteins, with 401, 196, and 283 assigned to the mt with high, moderate, and lower confidence, respectively. The remaining mitochondrial proteins were estimated by statistical methods although individual assignments could not be made. The identified proteins have predicted roles in macromolecular, metabolic, energy generating, and transport processes providing a comprehensive profile of the protein content and function of the T. brucei mt.     

Characterization by mass spectrometry of a recombinant hepatitis delta virus antigen and its proteolytic products.

The recombinant hepatitis delta virus antigen was obtained as a chimaeric protein fused to the C-terminus of the phage MS2 RNA polymerase. Following induction of the temperature-sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34-kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast-atom-bombardment mass spectrometry. The most represented degradation product, i.e. the 29-kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of delta antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis delta antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35-40 amino acids at the C-terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti-(hepatitis delta virus) antisera.     

Identification of three pertussis toxin substrates (41, 40 and 39 kDa proteins) in mammalian brain. Comparison of predicted amino acid sequences from G-protein alpha-subunit genes and cDNAs with partial amino acid sequences from purified proteins

We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human Gi2 alpha gene and rat Gi2 alpha cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat Gi1 alpha and Go alpha cDNAs, respectively.     

Partial characterization of vitellin and localization of vitellogenin production in the terrestrial isopod, Armadillidium vulgare

Vitellins were purified separately from ovaries and eggs of the isopod, Armadillidium vulgare. Ovarian vitellin consisted of at least six proteins with relative molecular masses of 205, 200, 185, 180, 122 and 112 kDa. The larger four proteins disappeared in eggs within a week after oviposition and a 59-kDa protein appeared thereafter. The amino-terminal amino acid sequences of these vitellin proteins were identical except for the ovarian 112 kDa, egg 112 kDa and 59 kDa proteins, and showed considerable similarity to those of known vitellogenins from other animals. Comparison of tryptic peptide maps of the 122 and 112 kDa proteins from eggs on reversed-phase HPLC and sequence identification of two randomly selected peaks having the same retention times indicated that two peptides were mostly similar in sequence. PCR-assisted cloning of the 5' region of a cDNA (591 bp) encoding vitellogenin revealed the presence of a signal peptide consisting of 16 amino acid residues and clarified the structural relationship among the protein components except for the ovarian 112 kDa and the egg 59 kDa proteins. Northern blot analysis revealed that the fat body is the main vitellogenin producing organ.     

Analysis of the adenovirus type 5 proteome by liquid chromatography and tandem mass spectrometry methods

We compared detection sensitivity and protein sequence coverage of the adenovirus type 5 proteome achievable by liquid chromatography and tandem mass spectroscopy (LC/MS/MS) using three sample preparation and clean up methods. Tryptic digestion was performed on either purified viral proteins or whole virus, and followed by shotgun sequencing using tandem mass spectrometry for peptide identification. We used a recombinant adenovirus type 5 as a test system. The methods included separation of adenoviral proteins by reversed-phase high-performance liquid chromatography followed by tryptic digestion and analysis by LC/MS/MS. Alternatively, the purified whole virus was digested with trypsin and the peptides separated either by one-dimensional (reversed-phase) or by two-dimensional (cation exchange and reversed-phase) chromatography and analyzed by tandem mass spectrometry. A total of 11 protein species were identified from 154 peptides. All of the major viral proteins were found. In addition, two minor proteins, the 23 kDa viral protease and the late L1 protein, were identified for the first time by chromatography based assays. The 23 kDa viral protease, present at only 10 copies per virus, and representing 0.2% of the protein content of the virus, was detected by the 2D LC/MS/MS analysis of the whole virus digest from a sample containing only 70 fmols of the protein. This demonstrates the high sensitivity and selectivity of the method. The 2D LC/MS/MS analysis of the whole virus digest was also able to detect all viral proteins with copy numbers at or above 10/virus particle, with broad coverage of the amino acid sequences. Coverage ranged from 2 to 54%, a majority between 20 and 35%, suggesting the possibility of using this analysis to assess the purity of the virus preparations. This broad coverage may also provide a useful approach to identify posttranslational modifications on the structural proteins of the adenovirus.     

Identification of two distinct proteins that are immunologically related to the alpha 1 subunit of the skeletal muscle dihydropyridine-sensitive calcium channel.

The alpha 1 subunit of the dihydropyridine-sensitive calcium channel is a protein which is critical for excitation-contraction coupling and L-type calcium current in skeletal muscle. Using antibodies generated against peptides from three regions of the deduced amino acid sequence of the alpha 1 subunit, we have identified two distinct proteins in rabbit skeletal muscle. Both proteins appeared to be recognized by antibodies against the amino (N) terminus of the alpha 1 subunit sequence. One protein was also recognized by antibodies against an internal (I) region of the predicted sequence but not by antibodies against the carboxyl (C) terminus. In contrast, the other protein was recognized by antibodies against the carboxyl terminus but not by the antibodies against the internal region. We have designated these proteins pNI and pNC based on their patterns of antibody recognition. No protein was detected which was recognized by all three antibodies. pNI is the protein commonly identified as the alpha 1 subunit of the dihydropyridine-sensitive calcium channel. Of note is that pNI, which apparently lacks sequences from the predicted carboxyl tail, is the protein present in preparations which we have previously demonstrated contain dihydropyridine-sensitive calcium channel activity. pNC is herein identified as a skeletal muscle protein that is immunologically related to the alpha 1 subunit of the dihydropyridine-sensitive calcium channel. Its function is unknown. In addition to their distinct patterns of antibody recognition, pNI and pNC were also distinguishable by several other properties. pNC migrated as a protein of approximately 160 kDa in 5% sodium dodecyl sulfate-polyacrylamide gels versus approximately 165 kDa for pNI. pNI was enriched in transverse tubule membranes, whereas pNC was found to be enriched in triad and junctional sarcoplasmic reticulum membrane fractions and was not found in transverse tubule membranes. Under conditions in which pNI bound to wheat germ agglutinin-Sepharose, pNC did not bind. The results demonstrate that there are two proteins in skeletal muscle which are immunologically related to the alpha 1 subunit of the dihydropyridine-sensitive calcium channel but which are distinguishable by several biochemical and immunological characteristics.     

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.