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Keith L Williams - One of the best experts on this subject based on the ideXlab platform.
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Characterisation of individual N-and O-linked glycosylation sites using Edman Degradation
Techniques in Protein Chemistry, 2014Co-Authors: Andrew A Gooley, Anthony Pisano, Marion L. Loughnan, John W. Redmond, Nicolle H Packer, Alun Jones, Keith L Williams, Paul Francis AlewoodAbstract:Publisher Summary Solid-phase Edman Degradation provides the ideal chemical method for the purification of individual glycoforms as it is not limited by the problems associated with endoglycosidase specificity. This chapter describes the techniques for determining the sites of N and O-glycosylation as a part of Edman Degradation in the automated solid phase micro-sequencing of glycoproteins. Sites of O- and N-linked glycosylation can be positively identified during solid-phase Edman Degradation if polar solvents, such as anhydrous trifluoroacetic acid (TFA), are used to extract the cleaved amino acid from the reaction cartridge. The oligosaccharide is released by solid-phase Edman Degradation as a labeled reducing terminal sugar, the phenylthiohydantoin (PTH)-glycoamino acid. Although, there are several excellent methods for the analysis of N- and O-linked glycosylation sites, particularly, LC-ionspray MS analysis; however, solid-phase Edman Degradation of glycopeptides that contain a domain of clustered glycosylation sites is the method of choice for precise glycosylation site identification and characterization. Thus, solid-phase Edman Degradation in combination with the techniques of carbohydrate analysis, such as high performance anion exchange chromatography (HPAEC) and ionspray mass spectrometry, will allow a new approach to the characterization of heavily glycosylated proteins previously thought intractable for protein chemistry studies.
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A role for Edman Degradation in proteome studies
Electrophoresis, 1997Co-Authors: Andrew A Gooley, Jim Russell, Marc R. Wilkins, Jean-charles Sanchez, Denis F. Hochstrasser, Keith L WilliamsAbstract:Advances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two-dimensional electrophoresis. However, Edman Degradation remains the principal technique for protein identification and it presents a significant bottle-neck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman Degradation to rapidly generate the powerful protein attribute, an N-terminal sequence tag.
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Characterization of a single glycosylated asparagine site on a glycopeptide using solid-phase Edman Degradation
Glycoconjugate journal, 1994Co-Authors: Andrew A Gooley, Anthony Pisano, John W. Redmond, Paul Francis Alewood, Nicolle H Packer, Alun Jones, Malcolm S. Ball, Keith L WilliamsAbstract:The characterization of site-specific glycosylation is traditionally dependent on the availability of suitable proteolytic cleavage sites between each glycosylated residue, so that peptides containing individual glycosylation sites are recovered. In the case of heavily glycosylated domains such as the O-glycosylated mucins, which have no available protease sites, this approach is not possible. Here we introduce a new method to gain site-specific compositional data on the oligosaccharides attached to a single amino acid. Using a model glycopeptide from a mutant human albumin Casebrook, glycosylated PTH-Asn was recovered after sequential solid-phase Edman Degradation, subjected to acid hydrolysis and the sugars were identified by high performance anion exchange chromatography with pulsed amperometric detection. The PTH-Asn(Sac) derivative was further characterized by ionspray mass spectrometry. Comparison between an endoproteinase Glu-C glycopeptide and a tryptic glycopeptide showed that the oligosaccharide attached to Asn494 was stable after at least 10 cycles of Edman Degradation.
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Glycosylation sites identified by solid-phase Edman Degradation: O-linked glycosylation motifs on human glycophorin A
Glycobiology, 1993Co-Authors: Anthony Pisano, John W. Redmond, Keith L Williams, Andrew A GooleyAbstract:The human red blood cell sialoglycoprotein, glycophorin A (GpA), contains a 'mucin-like' extensively O-glycosylated extracellular domain which carries the MN blood group antigens. We have revised the sites of O-glycosylation in the extracellular domain of GpA by automated solid-phase Edman Degradation, which allowed positive identification and quantitation of O-glycosylated Ser and Thr residues, as well as the single N-glycosylation site. One N-linked and 16 O-linked sites were identified. Carbohydrate was absent on Ser1, Ser14, Ser15, Ser23, Thr28 and Thr58 in GpA. We propose that the glycosyltransferases present in erythrocytes recognize specific flanking sequences around potential O-glycosylation sites. All 16 O-glycosylation sites are explained on the basis of four motifs. Three motifs are associated with Thr-glycosylation: Xaa-Pro-Xaa-Xaa where at least one Xaa = Thr; Thr-Xaa-Xaa-Xaa where at least one Xaa = Thr; Xaa-Xaa-Thr-Xaa where at least one X = Arg or Lys. The fourth motif is associated with Ser-glycosylation: Ser-Xaa-Xaa-Xaa where at least one Xaa = Ser. These simple rules explain the glycosylation (or lack of it) on 21 of 22 Ser/Thr in the extracellular domain of GpA.
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Glycosylation sites identified by detection of glycosylated amino acids released from Edman Degradation: the identification of Xaa-Pro-Xaa-Xaa as a motif for Thr-O-glycosylation.
Biochemical and biophysical research communications, 1991Co-Authors: Andrew A Gooley, Brendan J. Classon, Rolf Marschalek, Keith L WilliamsAbstract:Here we report the use of automated Edman Degradation of covalently linked glycopeptides to identify positively the sites of O- and N-glycosylation. The O-glycosidic linkage of carbohydrate to the hydroxy amino acids Ser and Thr is a major form of post-translational modification. However, unlike Asn-linked glycosylation, which is identified by the consensus sequence Asn-Xaa-Thr/Ser, no simple motif conferring O-linkage to Thr and Ser has been described. After sequencing glycopeptides derived from two cell surface glycoproteins, a Thr-O-glycosylation motif of Xaa-Pro-Xaa-Xaa, where at least one Xaa = Thr(Sac), has been defined. This motif predicts the site(s) of Pro- associated Thr-O-glycosylation in O-glycosylated proteins, although it is clear that there are also other forms of Thr-O-glycosylation not associated with Pro.
Andrew A Gooley - One of the best experts on this subject based on the ideXlab platform.
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Characterisation of individual N-and O-linked glycosylation sites using Edman Degradation
Techniques in Protein Chemistry, 2014Co-Authors: Andrew A Gooley, Anthony Pisano, Marion L. Loughnan, John W. Redmond, Nicolle H Packer, Alun Jones, Keith L Williams, Paul Francis AlewoodAbstract:Publisher Summary Solid-phase Edman Degradation provides the ideal chemical method for the purification of individual glycoforms as it is not limited by the problems associated with endoglycosidase specificity. This chapter describes the techniques for determining the sites of N and O-glycosylation as a part of Edman Degradation in the automated solid phase micro-sequencing of glycoproteins. Sites of O- and N-linked glycosylation can be positively identified during solid-phase Edman Degradation if polar solvents, such as anhydrous trifluoroacetic acid (TFA), are used to extract the cleaved amino acid from the reaction cartridge. The oligosaccharide is released by solid-phase Edman Degradation as a labeled reducing terminal sugar, the phenylthiohydantoin (PTH)-glycoamino acid. Although, there are several excellent methods for the analysis of N- and O-linked glycosylation sites, particularly, LC-ionspray MS analysis; however, solid-phase Edman Degradation of glycopeptides that contain a domain of clustered glycosylation sites is the method of choice for precise glycosylation site identification and characterization. Thus, solid-phase Edman Degradation in combination with the techniques of carbohydrate analysis, such as high performance anion exchange chromatography (HPAEC) and ionspray mass spectrometry, will allow a new approach to the characterization of heavily glycosylated proteins previously thought intractable for protein chemistry studies.
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A role for Edman Degradation in proteome studies
Electrophoresis, 1997Co-Authors: Andrew A Gooley, Jim Russell, Marc R. Wilkins, Jean-charles Sanchez, Denis F. Hochstrasser, Keith L WilliamsAbstract:Advances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two-dimensional electrophoresis. However, Edman Degradation remains the principal technique for protein identification and it presents a significant bottle-neck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman Degradation to rapidly generate the powerful protein attribute, an N-terminal sequence tag.
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Characterization of a single glycosylated asparagine site on a glycopeptide using solid-phase Edman Degradation
Glycoconjugate journal, 1994Co-Authors: Andrew A Gooley, Anthony Pisano, John W. Redmond, Paul Francis Alewood, Nicolle H Packer, Alun Jones, Malcolm S. Ball, Keith L WilliamsAbstract:The characterization of site-specific glycosylation is traditionally dependent on the availability of suitable proteolytic cleavage sites between each glycosylated residue, so that peptides containing individual glycosylation sites are recovered. In the case of heavily glycosylated domains such as the O-glycosylated mucins, which have no available protease sites, this approach is not possible. Here we introduce a new method to gain site-specific compositional data on the oligosaccharides attached to a single amino acid. Using a model glycopeptide from a mutant human albumin Casebrook, glycosylated PTH-Asn was recovered after sequential solid-phase Edman Degradation, subjected to acid hydrolysis and the sugars were identified by high performance anion exchange chromatography with pulsed amperometric detection. The PTH-Asn(Sac) derivative was further characterized by ionspray mass spectrometry. Comparison between an endoproteinase Glu-C glycopeptide and a tryptic glycopeptide showed that the oligosaccharide attached to Asn494 was stable after at least 10 cycles of Edman Degradation.
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Glycosylation sites identified by solid-phase Edman Degradation: O-linked glycosylation motifs on human glycophorin A
Glycobiology, 1993Co-Authors: Anthony Pisano, John W. Redmond, Keith L Williams, Andrew A GooleyAbstract:The human red blood cell sialoglycoprotein, glycophorin A (GpA), contains a 'mucin-like' extensively O-glycosylated extracellular domain which carries the MN blood group antigens. We have revised the sites of O-glycosylation in the extracellular domain of GpA by automated solid-phase Edman Degradation, which allowed positive identification and quantitation of O-glycosylated Ser and Thr residues, as well as the single N-glycosylation site. One N-linked and 16 O-linked sites were identified. Carbohydrate was absent on Ser1, Ser14, Ser15, Ser23, Thr28 and Thr58 in GpA. We propose that the glycosyltransferases present in erythrocytes recognize specific flanking sequences around potential O-glycosylation sites. All 16 O-glycosylation sites are explained on the basis of four motifs. Three motifs are associated with Thr-glycosylation: Xaa-Pro-Xaa-Xaa where at least one Xaa = Thr; Thr-Xaa-Xaa-Xaa where at least one Xaa = Thr; Xaa-Xaa-Thr-Xaa where at least one X = Arg or Lys. The fourth motif is associated with Ser-glycosylation: Ser-Xaa-Xaa-Xaa where at least one Xaa = Ser. These simple rules explain the glycosylation (or lack of it) on 21 of 22 Ser/Thr in the extracellular domain of GpA.
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Glycosylation sites identified by detection of glycosylated amino acids released from Edman Degradation: the identification of Xaa-Pro-Xaa-Xaa as a motif for Thr-O-glycosylation.
Biochemical and biophysical research communications, 1991Co-Authors: Andrew A Gooley, Brendan J. Classon, Rolf Marschalek, Keith L WilliamsAbstract:Here we report the use of automated Edman Degradation of covalently linked glycopeptides to identify positively the sites of O- and N-glycosylation. The O-glycosidic linkage of carbohydrate to the hydroxy amino acids Ser and Thr is a major form of post-translational modification. However, unlike Asn-linked glycosylation, which is identified by the consensus sequence Asn-Xaa-Thr/Ser, no simple motif conferring O-linkage to Thr and Ser has been described. After sequencing glycopeptides derived from two cell surface glycoproteins, a Thr-O-glycosylation motif of Xaa-Pro-Xaa-Xaa, where at least one Xaa = Thr(Sac), has been defined. This motif predicts the site(s) of Pro- associated Thr-O-glycosylation in O-glycosylated proteins, although it is clear that there are also other forms of Thr-O-glycosylation not associated with Pro.
Matthias Mann - One of the best experts on this subject based on the ideXlab platform.
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The Rise of Mass Spectrometry and the Fall of Edman Degradation
Clinical chemistry, 2016Co-Authors: Matthias MannAbstract:Featured Article: Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Anal Chem 1996;68:850–8.2 Many of today's key proteins in biology and biomedicine were originally identified by protein chemical methods. Frederick Sanger obtained his first Nobel Prize for determining the sequence of insulin in the 1950s, and the Edman Degradation, in which one amino acid after another is cleaved off the end of a protein or peptide and identified by HPLC, reigned supreme into the 1990s. My background is in mass spectrometry (MS),3 in particular electrospray ionization (1), for which my advisor John Fenn won a Nobel Prize. On coming to the European Molecular Biology Laboratory (EMBL) in Heidelberg as a young group leader, I was determined to challenge what I saw as the old-fashioned chemical methods with high-tech and cool MS technology. Little did I know what I was up against, because this would require not only highly sensitive peptide sequencing by MS …
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Sequence patterns produced by incomplete enzymatic digestion or one‐step Edman Degradation of peptide mixtures as probes for protein database searches
Electrophoresis, 1996Co-Authors: Ole Nørregaard Jensen, Ole Vorm, Matthias MannAbstract:Mass spectrometric peptide mapping of proteins isolated by polyacrylamide gel electrophoresis is a rapid method for identifying proteins in sequence databases. A majority of tryptic peptide maps were found to contain pairs of peptide ion peaks separated by the molecular weight of the lysyl or arginyl residue. These peaks originate from amino acid sequence patterns such as Lys-Lys where trypsin has cleaved C-terminals to either one of the lysines. The peptide mass and the pattern define an N- or C-terminal sequence tag. Searching sequence databases by such a sequence tag results in only a moderate number of matches and significantly reduces the number of database matches when used in combination with a peptide mass map. Two N- or C-terminal sequence tags alone unambiguously identify a protein in most cases. The technique discussed here is simple, does not require additional measurments, and increases the percentage of protein samples that can be identified by their mass maps alone. N-Terminal peptide sequence tags for database searching can also be generated by manual one-step Edman Degradation of the unseparated peptide mixture.
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sequence patterns produced by incomplete enzymatic digestion or one step Edman Degradation of peptide mixtures as probes for protein database searches
Electrophoresis, 1996Co-Authors: Ole Nørregaard Jensen, Ole Vorm, Matthias MannAbstract:Mass spectrometric peptide mapping of proteins isolated by polyacrylamide gel electrophoresis is a rapid method for identifying proteins in sequence databases. A majority of tryptic peptide maps were found to contain pairs of peptide ion peaks separated by the molecular weight of the lysyl or arginyl residue. These peaks originate from amino acid sequence patterns such as Lys-Lys where trypsin has cleaved C-terminals to either one of the lysines. The peptide mass and the pattern define an N- or C-terminal sequence tag. Searching sequence databases by such a sequence tag results in only a moderate number of matches and significantly reduces the number of database matches when used in combination with a peptide mass map. Two N- or C-terminal sequence tags alone unambiguously identify a protein in most cases. The technique discussed here is simple, does not require additional measurments, and increases the percentage of protein samples that can be identified by their mass maps alone. N-Terminal peptide sequence tags for database searching can also be generated by manual one-step Edman Degradation of the unseparated peptide mixture.
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Edman Degradation and MALDI sequencing enables N- and C-terminal sequence analysis of peptides
Techniques in Protein Chemistry, 1995Co-Authors: Roland Kellner, Gert Talbo, Tony Houthaeve, Matthias MannAbstract:Publisher Summary This chapter describes the combined use of automated Edman sequencing and MALDI sequencing for the determination of proteolytic peptide fragments in the low picomole range. Automated Edman Degradation or MALDI sequencing can in principle yield the complete sequence of peptides. However, the amounts of peptide for sequencing in demanding biological problems are very low. The chapter suggests that both methods need to be operated at their highest performance possible and inherent limitations must be considered. A combined application of both methods is feasible, because of the very low sample consumption for MALDI MS. The example ambiguous sequence calls described in the chapter can be clarified by the complementary information. The application on proteolytic peptide fragments results often in metastable ions from the C-terminal part. In this way, the N-terminal sequence is achieved by Edman Degradation, the molecular weight determines the overall size of the fragment, and ambiguous amino acid residues are identified by MALDI sequencing.
Vladimir Y Butnev - One of the best experts on this subject based on the ideXlab platform.
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identification of twelve o glycosylation sites in equine chorionic gonadotropin β and equine luteinizing hormone β by solid phase Edman Degradation
Biology of Reproduction, 2001Co-Authors: George R Bousfield, Vladimir Y ButnevAbstract:The O-glycosylation sites for equine LHb (eLHb) and eCGb were identified by solid-phase Edman Degradation of four glycopeptides derived from the C-terminal region. Both subunits were O-glycosylated at the same 12 positions, rather than the 4‐6 sites anticipated. These sites were partially glycosylated, with carbohydrate attachment ranging from 20% to 100% for eCGb and from 10% to 100% for eLHb. When the C-terminal peptide containing all but one of the O-linked oligosaccharides was removed by mild acid hydrolysis of either eLHb or eCGb, hybrid hormones could be obtained by reassociating eLHa, eFSHa ,o r eCGa with the truncated b subunit derivatives. These hybrid hormones were identical in LH receptor-binding activity when des(121-149)eLHb or des(121-149)eCGb were combined with the same a subunit preparation. Thus, O-glycosylation appears to be responsible for the b subunit contribution to the substantial difference in LH receptor-binding activity between eLH and eCG. Comparison of the equid LH/CGb sequences with those available for the primate CGb subunits indicated a greater conservation of glycosylation patterns in the former. anterior pituitary, hormone action, LH, placenta
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Identification of Twelve O-Glycosylation Sites in Equine Chorionic Gonadotropin β and Equine Luteinizing Hormone β by Solid-Phase Edman Degradation
Biology of reproduction, 2001Co-Authors: George R Bousfield, Vladimir Y ButnevAbstract:The O-glycosylation sites for equine LHb (eLHb) and eCGb were identified by solid-phase Edman Degradation of four glycopeptides derived from the C-terminal region. Both subunits were O-glycosylated at the same 12 positions, rather than the 4‐6 sites anticipated. These sites were partially glycosylated, with carbohydrate attachment ranging from 20% to 100% for eCGb and from 10% to 100% for eLHb. When the C-terminal peptide containing all but one of the O-linked oligosaccharides was removed by mild acid hydrolysis of either eLHb or eCGb, hybrid hormones could be obtained by reassociating eLHa, eFSHa ,o r eCGa with the truncated b subunit derivatives. These hybrid hormones were identical in LH receptor-binding activity when des(121-149)eLHb or des(121-149)eCGb were combined with the same a subunit preparation. Thus, O-glycosylation appears to be responsible for the b subunit contribution to the substantial difference in LH receptor-binding activity between eLH and eCG. Comparison of the equid LH/CGb sequences with those available for the primate CGb subunits indicated a greater conservation of glycosylation patterns in the former. anterior pituitary, hormone action, LH, placenta
Peter Roepstorff - One of the best experts on this subject based on the ideXlab platform.
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isoforms of a cuticular protein from larvae of the meal beetle tenebrio molitor studied by mass spectrometry in combination with Edman Degradation and two dimensional polyacrylamide gel electrophoresis
Protein Science, 2008Co-Authors: Sophie Haebel, Charlotte Harken Jensen, Svend Olav Andersen, Peter RoepstorffAbstract:Simultaneous sequencing, using a combination of mass spectrometry and Edman Degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants.
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Sequence determination of three cuticular proteins and isoforms from the migratory locust, Locusta migratoria, using a combination of Edman Degradation and mass spectrometric techniques.
Biochimica et biophysica acta, 2003Co-Authors: Dario E. Kalume, Svend Olav Andersen, Sylvie Kieffer, Kate Rafn, Lene Skou, Peter RoepstorffAbstract:The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman Degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.
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Combined plasma-desorption mass spectrometry and Edman Degradation applied to simultaneous sequence determination of isoforms of structural proteins from the cuticle of Locusta migratoria.
European journal of biochemistry, 1993Co-Authors: Lea Andreasen, Svend Olav Andersen, Peter Højrup, Peter RoepstorffAbstract:The primary structures of two basic low-molecular-mass proteins, Lm-67 and Lm-70 from the pharate cuticle of the migratory locust, Locusta migratoria, were determined. The sequencing strategy was based on combined use of plasma--desorption mass spectrometry (PDMS) and automatic Edman Degradation of the proteins and their enzymically derived peptides. The mass-spectral data showed the presence of two proteins in each preparation. For protein preparation Lm-67, this was indicated by the mass spectrum of the intact protein. For protein preparation Lm-70, the presence of two variants only became evident by mass-spectrometric analysis of the enzymically derived peptides. Both proteins show strong similarity to other exocuticular proteins from L. migratoria.
