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• W2016804077 abstract "Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In our studies we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus. Analysis of the physical structure of egg case silk using scanning electron microscopy demonstrates the presence of small and large diameter fibers. By using the strong protein denaturant 8 m guanidine hydrochloride to solubilize the fibers, we demonstrated by SDS-PAGE and protein silver staining that an abundant component of egg case silk is a 100-kDa protein doublet. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called ecp-1, which encodes for one of the protein components of the 100-kDa species. BLAST searches of the NCBInr protein data base using the primary sequence of ECP-1 revealed similarity to fibroins from spiders and silkworms, which mapped to two distinct regions within the ECP-1. These regions contained the conserved repetitive fibroin motifs poly(Ala) and poly(Gly-Ala), but surprisingly, no larger ensemble repeats could be identified within the primary sequence of ECP-1. Consistent with silk gland-restricted patterns of expression for fibroins, ECP-1 was demonstrated to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands. ECP-1 monomeric units were also shown to assemble into higher aggregate structures through the formation of disulfide bonds via a unique cysteine-rich N-terminal region. Collectively, our findings provide new insight into the components of egg case silk and identify a new class of silk proteins with distinctive molecular features relative to traditional members of the spider silk gene family. Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In our studies we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus. Analysis of the physical structure of egg case silk using scanning electron microscopy demonstrates the presence of small and large diameter fibers. By using the strong protein denaturant 8 m guanidine hydrochloride to solubilize the fibers, we demonstrated by SDS-PAGE and protein silver staining that an abundant component of egg case silk is a 100-kDa protein doublet. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called ecp-1, which encodes for one of the protein components of the 100-kDa species. BLAST searches of the NCBInr protein data base using the primary sequence of ECP-1 revealed similarity to fibroins from spiders and silkworms, which mapped to two distinct regions within the ECP-1. These regions contained the conserved repetitive fibroin motifs poly(Ala) and poly(Gly-Ala), but surprisingly, no larger ensemble repeats could be identified within the primary sequence of ECP-1. Consistent with silk gland-restricted patterns of expression for fibroins, ECP-1 was demonstrated to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands. ECP-1 monomeric units were also shown to assemble into higher aggregate structures through the formation of disulfide bonds via a unique cysteine-rich N-terminal region. Collectively, our findings provide new insight into the components of egg case silk and identify a new class of silk proteins with distinctive molecular features relative to traditional members of the spider silk gene family. Araneoid spiders contain specialized glands that produce up to seven different silk fibers and glues that have been shown to have distinct physical and chemical properties (1Forlix R.F. Biology of Spiders,2nd Ed. Oxford University Press, New York1996: 1-336Google Scholar). Experimental evidence has demonstrated that these secretions are likely encoded by different members that belong to the silk gene family (2Guerette P.A. Ginzinger D.G. Weber B.H. Gosline J.M. Science. 1996; 272: 112-115Crossref PubMed Scopus (374) Google Scholar, 3Gatesy J. Hayashi C. Motriuk D. Woods J. Lewis R. Science. 2001; 291: 2603-2605Crossref PubMed Scopus (428) Google Scholar, 4Hayashi C.Y. Exs (Basel). 2002; 92: 209-223Google Scholar). With respect to the seven different sets of silk glands in a typical araneoid, cDNAs encoding fibroins have been characterized from five glandular types: major ampullate (manufactures dragline silk and frame silk) (2Guerette P.A. Ginzinger D.G. Weber B.H. Gosline J.M. Science. 1996; 272: 112-115Crossref PubMed Scopus (374) Google Scholar, 3Gatesy J. Hayashi C. Motriuk D. Woods J. Lewis R. Science. 2001; 291: 2603-2605Crossref PubMed Scopus (428) Google Scholar, 5Xu M. Lewis R.V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7120-7124Crossref PubMed Scopus (611) Google Scholar, 6Hinman M.B. Lewis R.V. J. Biol. Chem. 1992; 267: 19320-19324Abstract Full Text PDF PubMed Google Scholar, 7Beckwitt R. Arcidiacono S. J. Biol. Chem. 1994; 269: 6661-6663Abstract Full Text PDF PubMed Google Scholar, 8Arcidiacono S. Mello C. Kaplan D. Cheley S. Bayley H. Appl. Microbiol. Biotechnol. 1998; 49: 31-38Crossref PubMed Scopus (149) Google Scholar, 9Lawrence B.A. Vierra C.A. Moore A.M. Biomacromolecules. 2004; 5: 689-695Crossref PubMed Scopus (70) Google Scholar), minor ampullate (synthesizes capture spiral silk) (2Guerette P.A. Ginzinger D.G. Weber B.H. Gosline J.M. Science. 1996; 272: 112-115Crossref PubMed Scopus (374) Google Scholar, 10Colgin M.A. Lewis R.V. Protein Sci. 1998; 7: 667-672Crossref PubMed Scopus (149) Google Scholar), flagelliform (makes core fiber of the capture spiral) (2Guerette P.A. Ginzinger D.G. Weber B.H. Gosline J.M. Science. 1996; 272: 112-115Crossref PubMed Scopus (374) Google Scholar, 11Hayashi C.Y. Lewis R.V. J. Mol. Biol. 1998; 275: 773-784Crossref PubMed Scopus (324) Google Scholar), aciniform (expresses proteins involved in wrapping silk) (12Hayashi C.Y. Blackledge T.A. Lewis R.V. Mol. Biol. Evol. 2004; 21: 1950-1959Crossref PubMed Scopus (180) Google Scholar), and tubuliform (produces egg case silk) (2Guerette P.A. Ginzinger D.G. Weber B.H. Gosline J.M. Science. 1996; 272: 112-115Crossref PubMed Scopus (374) Google Scholar). No fibroin cDNAs, genes, or protein sequences have been described for aggregate (sticky glue) and piriform (attachment disc silks) tissues. The published cDNAs of all the araneoid silks display similar structural characteristics. The fibroin mRNAs are long relative to typical transcripts (ranging from ∼4–16 kb) (13Hayashi C.Y. Shipley N.H. Lewis R.V. Int. J. Biol. Macromol. 1999; 24: 271-275Crossref PubMed Scopus (519) Google Scholar), which code for fibroins with relatively high molecular masses ranging from 275 to 320 kDa (14Candelas G. Candelas T. Ortiz A. Rodriguez O. Biochem. Biophys. Res. Commun. 1983; 116: 1033-1038Crossref PubMed Scopus (40) Google Scholar, 15Mello C.M. Senecal K. Yeung B. Vouros P. Kaplan D. Kaplan D. Wade W.W. Farmer B. Viney Silk Polymers: Materials Science and Biotechnology. 544. American Chemical Society, Washington, D. C.1994: 67-79Google Scholar). The fibroin mRNAs code for highly internally repetitive modules as well as for a conserved, non-repetitive C-terminal region. Although the repetitive regions of different silk gene paralogues have been shown to be quite divergent, sequence similarities in the 3′ C-terminal region have been one of the hallmarks of fibroin identification and classification (2Guerette P.A. Ginzinger D.G. Weber B.H. Gosline J.M. Science. 1996; 272: 112-115Crossref PubMed Scopus (374) Google Scholar, 3Gatesy J. Hayashi C. Motriuk D. Woods J. Lewis R. Science. 2001; 291: 2603-2605Crossref PubMed Scopus (428) Google Scholar, 4Hayashi C.Y. Exs (Basel). 2002; 92: 209-223Google Scholar, 7Beckwitt R. Arcidiacono S. J. Biol. Chem. 1994; 269: 6661-6663Abstract Full Text PDF PubMed Google Scholar). The divergence of the internal repetitive sequences that characterize each fibroin has been suggested to be responsible in part for the differing mechanical properties of the spun silk fibers produced by the different glands (16Gosline J.M. Guerette P.A. Ortlepp C.S. Savage K.N. J. Exp. Biol. 1999; 202: 3295-3303Crossref PubMed Google Scholar). Despite the divergence within the repetitive regions of the paralogues, some common molecular features exist within the silk proteins, including four amino acid motifs that are found in various combination and numbers (3Gatesy J. Hayashi C. Motriuk D. Woods J. Lewis R. Science. 2001; 291: 2603-2605Crossref PubMed Scopus (428) Google Scholar, 13Hayashi C.Y. Shipley N.H. Lewis R.V. Int. J. Biol. Macromol. 1999; 24: 271-275Crossref PubMed Scopus (519) Google Scholar). The four motifs include the following: 1) polyalanine (An) stretches; 2) alternating glycine and alanine couplets (GA)n; 3) three amino acids composed of two glycines followed by a variable amino acid (GGX)n; and 4) glycine-proline-glycine modules (GPGXn). These modules described above are assembled in different numbers and combinations to form larger ensemble repeats that are iterated many times throughout the internal region of the fibroins. In a few instances some fibroins have been reported to contain only certain motifs (12Hayashi C.Y. Blackledge T.A. Lewis R.V. Mol. Biol. Evol. 2004; 21: 1950-1959Crossref PubMed Scopus (180) Google Scholar). Although much emphasis has been placed on understanding the components of dragline and capture silk, little information has been reported regarding the molecular and biochemical features of egg case silk. Compared with other araneoid silks, tubuliform silks display different mechanical properties. The difference in mechanical properties may be explained in part by different amino acid compositions of the constituent proteins, with tubuliform silks containing substantially higher levels of serine relative to the silk from the minor ampullate, major ampullate; and flagelliform glands (17Vollrath F. Knight D.P. Nature. 2001; 410: 541-548Crossref PubMed Scopus (1292) Google Scholar). Because tubuliform silks have different chemical compositions and mechanical properties relative to other araneoid silks, we hypothesized that tubuliform silks contain unique proteins assembled into the egg case silk fiber. In our search for genes encoding egg case silk proteins, we have isolated a novel cDNA that codes for a tubuliform-restricted protein. Based upon the pattern of expression and the abundance of the protein in egg case fibers, we have named this gene product ECP-1 (egg case protein-1). The primary sequence of ECP-1 represents the only completed sequence for egg case silk proteins. Analysis of the predicted primary sequence of ECP-1 reveals similarity to published fibroin proteins from spiders and silkmoths. By SDS-PAGE analysis of egg case silk protein samples, we also demonstrate ECP-1 monomers are assembled into higher ordered structures and comprise abundant components of egg case silk. Our findings suggest that ECP-1 represents a new class of silk proteins belonging to the spider gene silk family. Scanning Electron Microscopy—Egg case silk was coated to a thickness of ∼14–20 nm with gold alloy in a Pelco SC-7 auto-sputter coater with an FTM-2 film thickness monitor. Samples were examined on a Hitachi S-2600 S.E. operated with an accelerator voltage of 3 kV. The diameter of both strands was measured to the nearest 0.01 μm at three distant places along the S.E. sample. Tests were done at ambient temperature and humidity, which ranged from 22 to 25 °C and 30–36%, respectively. Characterization of Egg Case Proteins by Differential Solubilization—Individual egg cases from female black widow spiders were cut open using sterilized scissors to remove the eggs. The egg case was discarded if any eggs were broken during the isolation procedure. Silk from each egg case was extracted with 8 m GdnHCl 1The abbreviations used are: GdnHCl, guanidine hydrochloride; MALDI, matrix-assisted laser desorption ionization; TOF, time-offlight; MS/MS, tandem mass spectrometry; ECP, egg case protein; RT-qPCR, real time quantitative PCR; CAD, collision-activated dissociation; RACE, rapid amplification of cDNA ends. (3 ml of solution per mg of silk) for 10 min with agitation. The viscous supernatant solution obtained was removed and conserved for dialysis. The solid residue was then extracted with 1 ml of water, which left the solid white core fiber. The water extract solution was also conserved for dialysis. A second, fresh 8 m GdnHCl solution (same volume to weight ratio as the initial solution) was then added to the silk residue, and the mixture was agitated continuously with a vertical shaker. Samples of this supernatant were collected after 10 min and after 5, 15, 25, and 40 h. Each of the silk protein solutions (solutions obtained from initial GdnHCl and water extracts, and multiple samples from the second GdnHCl extraction) was dialyzed against three changes of 100 ml of 50 mm Tris-HCl (pH 7.8) for 4 h each, using 1000 molecular weight cut-off dialysis tubing (Sigma). The proteins from the dialyzed samples were separated on SDS-PAGE with a 4–20% polyacrylamide gradient gel (Bio-Rad). Separated proteins were visualized by silver staining (ProteoSilver™ Plus silver stain kit, Sigma). The results are shown in Fig. 2, lanes 2–8. Broad range molecular weight markers were used to determine protein sizes (Bio-Rad). Tryptic Digestions of Egg Case Protein and Mass Spectrometric Analysis—Sequencing grade trypsin (trypsin gold, Promega) was dissolved in 50 mm acetic acid at a concentration of 1 mg/ml. This solution was then diluted with 50 mm NH4HCO3 (pH 7.8) to give a 20 μg/ml stock solution of trypsin. In-solution digests of the 40-h extract of egg case silk were prepared by mixing 100 μl of the ECP protein solution with 10 μl of trypsin solution and incubating at 37 °C overnight. The digest was then completely dried using a vacuum centrifuge and redissolved in 10 μl of 0.1% aqueous trifluoroacetic acid. Peptides were extracted and desalted with a C18 Zip-Tip (Millipore) according to the manufacturer's instructions. In order to test whether any ECP species were also included with the peripheral proteins from the first GdnHCl extraction, we prepared an in-gel tryptic digest of the 100-kDa bands from this extract following a published protocol (18Jiménez C.R. Huang L. Qiu Y. Burlingame A.L. Current Protocols in Protein Science: In-gel Digestion of Proteins for MALDI-MS. Fingerprint Mapping. John Wiley & Sons, Inc., New York1998: 16.4.1-16.4.5Google Scholar). Briefly, the 100-kDa bands in Fig. 2, lane 2, were excised from the gel. After destaining, the pieces were minced into fine particles with sealed pipette tips, washed with 25 mm NH4HCO3, 50% acetonitrile, and then dried in a vacuum centrifuge. The dried pieces were rehydrated and reduced with 10 mm dithiothreitol, alkylated with 55 mm iodoacetamide, dried again, and then rehydrated and digested overnight with 250 ng of trypsin. Peptides were extracted with two changes of 30 μl of 50% acetonitrile, 5% formic acid solution. The extracts were combined and then reduced in volume to about 10 μl to concentrate the sample, after which the solutions were desalted with C18 Zip-Tips. In-gel digestion was also carried out for the ECPs in the 40-h extracts (100-kDa bands in Fig. 2, lane 8). In order to enhance peptide sequence coverage, 300 μl of the 40-h extracts were concentrated to 40 μl using PAGE Prep™ Protein Clean-up and Enrichment kit (Pierce), 20 μl of which were loaded on a separate 4–20% gradient gel. The 100-kDa bands were excised and subjected to in-gel trypsin digestion. The three digests (the in-solution and in-gel digests of ECPs and the in-gel digest of the 100-kDa bands from the first GdnHCl wash) were analyzed with a MALDI tandem TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Foster City, CA). One-half microliter of each desalted digest solution was mixed with an equal volume of a 10 mg/ml solution of α-cyano-4-hydroxycinnamic acid in 50/50 acetonitrile, 0.1% aqueous trifluoroacetic acid and spotted on the MALDI target. For the in-solution digest, two negative controls were used as follows: a mixture of 100 μl of 50 mm Tris-HCl (pH 7.8) and 10 μl of trypsin solution, and a mixture of 100 μl of ECP sample and 10 μl of 50 mm NH4HCO3 (no trypsin) were treated in the same way as the actual digests. For the in-gel digests, a piece of blank gel was digested as described above. Monoisotopic masses of all significant peptides generated from the tryptic digests were measured in positive mode. Several peptides were selected for analysis by MS/MS following high energy CAD (1 keV lab frame). De novo peptide sequences were derived from the MS/MS spectra by manual interpretation. cDNA Library Construction—Fifteen black widow spiders were dissected, and their silk-producing glands were isolated. The isolated glands included the major ampullate, minor ampullate, tubuliform, flagelliform, aciniform, and pyriform glands. Total RNA was isolated from the glands using an RNeasy maxi kit (Qiagen). Poly(A) RNA was purified using the PolyATtract mRNA isolation system (Promega) as described previously (9Lawrence B.A. Vierra C.A. Moore A.M. Biomacromolecules. 2004; 5: 689-695Crossref PubMed Scopus (70) Google Scholar). cDNAs were generated using a HybriZAP 2.1 cDNA synthesis kit and the cDNA library created using the HybriZAP 2.1 XR library construction kit according to the manufacturer's instructions (Stratagene). Cloning of the ecp-1 Gene—The peptide sequences LLESDGFGPIIR and QGQQGFSETLSQSDSR were used to synthesize degenerate oligonucleotides corresponding to the underlined regions. PCRs containing the forward primer 5′-CAAGGWCAACAAGGWTTY-3′ (encodes for QGQQGF; Y = T or C; W = A or T) and the reverse primer 5′-NGGNCCRAANCCRTC-3′ (specifies DGFGP; R = AorG; N = A, G, T, or C) successfully amplified a 525-bp fragment of ECP-1 from a cDNA library produced from black widow silk glands (corresponded to amino acids 353–527 Fig. 4). The 525-bp fragment was sequenced as described previously (19Mitchell B. Mugiya M. Youngblom J. Funes-Duran M. Miller R. Ezpeleta J. Rigby N. Vierra C. Biochim. Biophys. Acta. 2000; 1492: 320-329Crossref PubMed Scopus (11) Google Scholar). To amplify the 5′ end of the ECP-1 cDNA, the forward primer (anchor) from the pGAL4-AD library vector (5′-CGATGATGAAGATACCCCACC-3′) was used with the gene-specific reverse primer 5′-CGTATTGTAATCCAGAGGAACC-3′ (nucleotides 1506–1527, corresponding to amino acids 497–503). The product from the pGAL4-AD forward and reverse primer resulted in an ∼1.4-kb product. A similar approach was used to obtain the 3′ end of the cDNA. The reverse primer from the pGAL4-AD library vector 5′-GCACAGTTGAAGTGAACTTGC-3′ and the forward primer 5′-GAGGAAGAGGATTCGGTGTTACA-3′ (nucleotides 1405–1427, corresponding to residues 464–470) amplified a product that was ∼1.7 kb. Both 5′- and 3′-RACE products were purified using the QIAquick gel extraction kit according to the manufacturer's instruction (Qiagen) and sequenced as described above. Amino Acid Composition of the Core Silk Filament—The core silk fibers were subjected to amino acid analysis at the Protein Chemistry Laboratory of Texas A & M University as described previously (20Casem M.L. Turner D. Houchin K. Int. J. Biol. Macromol. 1999; 24: 103-108Crossref PubMed Scopus (55) Google Scholar). Briefly, vapor phase hydrolysis of the core proteins by 6 m HCl was employed to generate the constituent amino acids. Amino acids obtained were derivatized with o-phthalaldehyde and 9-fluoromethylchloroformate. The derivatized amino acids were separated by reversed phase high performance liquid chromatography with UV detection. Real Time PCR Analysis with SYBR Green Detection—Reverse transcription reactions were carried out as described previously (21Nguyen L. Round J. O'Connell R. Geurts P. Funes-Duran M. Wong J. Jongeward G. Vierra C.A. Nucleic Acids Res. 2001; 29: 4423-4432Crossref PubMed Scopus (8) Google Scholar). Typically, 2 μl of a reverse transcription reaction were used for real time PCR analysis using the DyNAmo SYBR Green qPCR kit (MJ Research). Real time PCR fluorescence detection was performed in 96-well plates using an Opticon II instrument (MJ Research). Amplification products were monitored by SYBR Green detection and routinely checked using dissociation curve software and 1% agarose gel electrophoresis. Reactions were performed in triplicate. Oligonucleotides used for the analysis of ECP-1 were the forward and reverse primers 5′-GAATCCAGTAGTGCCTCCCAATT-3′ (nucleotides 1110–1132) and 5′-TTGTGAACTCTCCTCCTTGACT-3′ (nucleotides 1293–1314), respectively. Primers were selected using the Beacon Designer 2.0 software (PREMIER Biosoft International). Physical Structure of Black Widow Egg Case Fibers—To understand more regarding the physical structure of egg case silk (Fig. 1A), we used a scanning electron microscope to examine the physical structure of egg cases collected from black widow spiders (Fig. 1B). The scanning electron micrograph shows that egg case silk is composed of fibers of two different diameters (Fig. 1C). The larger diameter fibers, which represent the major component of egg case silk, are produced by the tubuliform gland (22Sekiguchi K. Sci. Rep. Tokyo Kyoiku Daigaku Sect. B. 1955; 8: 23-32Google Scholar). The diameter of the large fibers was 4–5 μm, whereas the diameters the smaller fibers were on the order of 500 nm. The smaller diameter fibers, which constitute a minor component, are likely synthesized by the aciniform gland (23Foradori M.J. Kovoor J. Moon M.J. Tillinghast E.K. J. Morphol. 2002; 252: 218-226Crossref PubMed Scopus (20) Google Scholar). Identification of Egg Case Proteins—To examine the biochemical features and the number of proteins assembled into egg case silk, we dissolved egg cases collected from black widow spiders in 8 m GdnHCl. Analysis of the egg case extracts by SDS-PAGE followed by silver staining revealed a broad distribution of molecular masses, ranging from ∼10 to >300 kDa (Fig. 2, lane 2). Analysis of egg cases taken from six different black widow spiders generated similar protein patterns (data not shown). The broad distribution of molecular weight proteins is likely a result of translational pausing of fibroin mRNAs (14Candelas G. Candelas T. Ortiz A. Rodriguez O. Biochem. Biophys. Res. Commun. 1983; 116: 1033-1038Crossref PubMed Scopus (40) Google Scholar), protein degradation, and/or the presence of glue proteins. To identify major structural proteins in the silk fiber, we performed a solubilization time course assay with GdnHCl. Egg case silk was repeatedly extracted with 8 m GdnHCl over a 40-h time period. Many egg case proteins dissolved immediately upon the first exposure to the GdnHCl solution (Fig. 2, lane 2). A distinct color change was observed after the first GdnHCl extraction and water wash, with the egg case changing from a yellowish hue to white. The treatment of silk with GdnHCl or other types of chaotropic agents has been reported to solubilize efficiently the hydrophobic proteins. SDS-PAGE analysis of the water wash following the first GdnHCl extraction of the silk produced a protein doublet with an apparent molecular mass of ∼100 kDa (Fig. 2, lane 3). This protein doublet had the same apparent size as the strong 100-kDa species observed in the initial GdnHCl extract (compare Fig. 2, lanes 2 and 3). No other proteins were detected in the water wash (Fig. 2, lane 3). Because silk fibroins are not water-soluble, it is very probable that these species represent the diluted sample from the initial treatment because the 100-kDa doublet was the strongest band in the initial GdnHCl extraction. When the residue after the water wash (about 70% of the silk mass) was extracted with 8 m GdnHCl for a second time, the remaining core fibroins and associated proteins displayed reduced solubility. Samples extracted for less than 15 h failed to generate sufficient proteins for detection by silver staining following SDS-PAGE (Fig. 2, lanes 4–6). However, after 25 h, a faint protein doublet was detected (Fig. 2, lane 7). This protein doublet became relatively stronger at 40 h (Fig. 2, lane 8), and these species were named egg case proteins or ECPs. No higher molecular weight proteins were detected by the SDS-PAGE at this point. Trypsin Digestion of ECPs and Mass Spectrometric Sequence Analysis—We then performed in-solution tryptic digestions of the protein doublet described in the secondary 40-h GdnHCl extract fraction (Fig. 2, lane 8). This digest was found to contain numerous peptides whose molecular masses were determined by MALDI-MS analysis (Fig. 3A). Seven peptides were sequenced using high energy collision-activated dissociation (CAD), those with precursor ion masses (MH+, monisotopic) of 855.4, 1316.7, 1502.7, 1613.8, 1623.8, 1754.8, and 3424.5. The product ion spectra from the peptides of MH+ 1316.7, 1613.8, and 3424.5 are shown in Fig. 3, D–F, respectively. The N termini of the 1613.8 and 3424.5 peptides could not be determined, but a significant amount of sequence information was derived. The primary sequences of the remaining peptides were also determined (Table I). The sequences of the peptides at m/z 1613.8 and 1623.8 were nearly identical, having only a single amino acid difference (Table I). The selection of ions for sequence analysis was deliberately biased toward peptide masses that were repeatedly observed from different black widow spiders (data not shown). Analysis of the derived peptide sequences using the algorithm BLAST revealed no significant similarity to any polypeptides in the NCBInr protein data base.Fig. 3MALDI-TOF and MALDI tandem TOF analyses of tryptic fragments generated from the 100-kDa protein species. A, peptide map obtained by in-solution tryptic digestion of the 100-kDa protein doublet extracted for 40 h from the egg case silk from L. hesperus. B, peptide map from in-gel digest of 100-kDa proteins in 40-h extract. C, peptide map from in-gel digest of 100-kDa proteins in initial GdnHCl extract. D, high energy CAD (MS/MS) spectrum of precursor ion with m/z 1316.7. The sequence of this peptide was found to be LLESDGFGPIIR. The two leucine residues at the N terminus could be any combination of leucine and isoleucine. The identities of the two isoleucine residues close to the C terminus were confirmed by the presence of w2a, w3a, and w3b ions. E, high energy CAD spectrum of precursor ion with m/z 1613.8. The partial sequence of this peptide was found to be SGAQGSSGLQYGR. The N terminus sequence of this peptide could not be determined because of the loss of the large fragment under high energy CAD. F, high energy CAD spectrum of precursor ion with m/z 3424.5. The partial sequence of this peptide was found to be GNFGSANDAESFAASESESFAGQSAAGSR. Because of the loss of a large fragment under high energy CAD, some of the N-terminal residues could not be sequenced.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3MALDI-TOF and MALDI tandem TOF analyses of tryptic fragments generated from the 100-kDa protein species. A, peptide map obtained by in-solution tryptic digestion of the 100-kDa protein doublet extracted for 40 h from the egg case silk from L. hesperus. B, peptide map from in-gel digest of 100-kDa proteins in 40-h extract. C, peptide map from in-gel digest of 100-kDa proteins in initial GdnHCl extract. D, high energy CAD (MS/MS) spectrum of precursor ion with m/z 1316.7. The sequence of this peptide was found to be LLESDGFGPIIR. The two leucine residues at the N terminus could be any combination of leucine and isoleucine. The identities of the two isoleucine residues close to the C terminus were confirmed by the presence of w2a, w3a, and w3b ions. E, high energy CAD spectrum of precursor ion with m/z 1613.8. The partial sequence of this peptide was found to be SGAQGSSGLQYGR. The N terminus sequence of this peptide could not be determined because of the loss of the large fragment under high energy CAD. F, high energy CAD spectrum of precursor ion with m/z 3424.5. The partial sequence of this peptide was found to be GNFGSANDAESFAASESESFAGQSAAGSR. Because of the loss of a large fragment under high energy CAD, some of the N-terminal residues could not be sequenced.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IPeptide sequences obtained from the 100-kDa protein doublet following tryptic digestion and MALDI tandem TOF mass spectrometryM + H+De novo sequenceLocation of peptide sequence855.4EFGSYPR614-6201316.7LLESDGFGPIIRNA1502.7TAGVSQSFGTAFSSR338-3521613.8(AAGF) SGAQGSSGLQYGR488-5051623.8SGAQGSPGLQYGRNA1754.8QGQQGFSETLSQSDSRNA3424.5(AVSSGIT) GNFGSANDAESFAASESESFAGQSAAGSR382-417 Open table in a new tab Most of the peptides from the in-solution digest of the 40-h extract (Fig. 3A) were also found in the in-gel digest of the 100-kDa proteins in the 40-h extract (F" @default.
• W2016804077 created "2016-06-24" @default.
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• W2016804077 date "2005-06-01" @default.
• W2016804077 modified "2023-10-17" @default.
• W2016804077 title "Egg Case Protein-1" @default.
• W2016804077 cites W1505366472 @default.
• W2016804077 cites W1542008397 @default.
• W2016804077 cites W1557763633 @default.
• W2016804077 cites W1658845344 @default.
• W2016804077 cites W1982331202 @default.
• W2016804077 cites W1987048516 @default.
• W2016804077 cites W1988320083 @default.
• W2016804077 cites W1992516996 @default.
• W2016804077 cites W2000550628 @default.
• W2016804077 cites W2001378989 @default.
• W2016804077 cites W2001389196 @default.
• W2016804077 cites W2003824294 @default.
• W2016804077 cites W2010794422 @default.
• W2016804077 cites W2012057679 @default.
• W2016804077 cites W2029033315 @default.
• W2016804077 cites W2031417134 @default.
• W2016804077 cites W2058985073 @default.
• W2016804077 cites W2076083059 @default.
• W2016804077 cites W2077990286 @default.
• W2016804077 cites W2088084607 @default.
• W2016804077 cites W2091003242 @default.
• W2016804077 cites W2104167472 @default.
• W2016804077 cites W2132668802 @default.
• W2016804077 cites W2150295226 @default.
• W2016804077 cites W2161746138 @default.
• W2016804077 cites W2161826643 @default.
• W2016804077 cites W2168231365 @default.
• W2016804077 doi "https://doi.org/10.1074/jbc.m412316200" @default.
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