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International audienc

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Specific Changes in the Local Flexibility of Plasminogen Activator Inhibitor 1 upon Binding to the Somatomedin B Domain of Vitronectin

The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding. Our data show that the PAI-1 core consisting of β-sheet B is rather protected against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB binding causes a pronounced and widespread stabilization of PAI-1 that is not confined to the binding interface with SMB. We further explored the local structural flexibility in a mutationally stabilized PAI-1 variant (14-1B) as well as the effect of stabilizing antibody Mab-1 on wild-type PAI-1. The three modes of stabilizing PAI-1 (SMB, Mab-1, and the mutations in 14-1B) all cause a delayed latency transition, and this effect was accompanied by unique signatures on the flexibility of PAI-1. Reduced flexibility in the region around helices B, C, and I was seen in all three cases, which suggests an involvement of this region in mediating structural flexibility necessary for the latency transition. These data therefore add considerable depth to our current understanding of the local structural flexibility in PAI-1 and provide novel indications of regions that may affect the functional stability of PAI-1

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Specific Changes in the Local Flexibility of Plasminogen Activator Inhibitor 1 upon Binding to the Somatomedin B Domain of Vitronectin

The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding. Our data show that the PAI-1 core consisting of β-sheet B is rather protected against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB binding causes a pronounced and widespread stabilization of PAI-1 that is not confined to the binding interface with SMB. We further explored the local structural flexibility in a mutationally stabilized PAI-1 variant (14-1B) as well as the effect of stabilizing antibody Mab-1 on wild-type PAI-1. The three modes of stabilizing PAI-1 (SMB, Mab-1, and the mutations in 14-1B) all cause a delayed latency transition, and this effect was accompanied by unique signatures on the flexibility of PAI-1. Reduced flexibility in the region around helices B, C, and I was seen in all three cases, which suggests an involvement of this region in mediating structural flexibility necessary for the latency transition. These data therefore add considerable depth to our current understanding of the local structural flexibility in PAI-1 and provide novel indications of regions that may affect the functional stability of PAI-1

Comprehensive Proteomic Analysis of <i>Trypanosoma cruzi</i> Epimastigote Cell Surface Proteins by Two Complementary Methods

<i>Trypanosoma cruzi</i> is a protozoan that causes Chagas’ disease, a neglected infectious illness that affects millions of people, mostly in Latin America. Here, the cell surface subproteome of the <i>T. cruzi</i> epimastigote life form was characterized. In order to prepare samples enriched in epimastigote plasma membrane protein, two distinct methodologies were optimized and evaluated. The first methodology was based on cell surface trypsinization (Shave) of intact living cells while the second approach used biotinylation of cell surface proteins followed by streptavidin affinity chromatography isolation of the labeled proteins. Both <i>T. cruzi</i> subproteomes were analyzed by LC-MS/MS. The results showed that the methodologies offered comprehensive and complementary information about the parasite’s plasma membrane subproteome

Performance of Isobaric and Isotopic Labeling in Quantitative Plant Proteomics

Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC–MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of <i>Ricinus communis</i> seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins

Performance of Isobaric and Isotopic Labeling in Quantitative Plant Proteomics

Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC–MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of <i>Ricinus communis</i> seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins

Performance of Isobaric and Isotopic Labeling in Quantitative Plant Proteomics

Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC–MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of <i>Ricinus communis</i> seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins

Performance of Isobaric and Isotopic Labeling in Quantitative Plant Proteomics

Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC–MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of <i>Ricinus communis</i> seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins

Moving Pieces in a Venomic Puzzle: Unveiling Post-translationally Modified Toxins from <i>Tityus serrulatus</i>

Besides being a public health problem, scorpion venoms have a potential biotechnological application since they contain peptides that may be used as drug leads and/or to reveal novel pharmacological targets. A comprehensive <i>Tityus serrulatus</i> venom proteome study with emphasis on the phosphoproteome and <i>N</i>-glycoproteome was performed to improve our knowledge on the molecular diversity of the proteinaceous toxins. We combined two peptide identification methodologies, <i>i.e.</i>, database search and <i>de novo</i> sequencing, to achieve a more comprehensive overview of the molecular diversity of the venoms. A total of 147 proteins were identified, including neurotoxins, enzymes, bradykinin-potentiating peptides, and molecules with antimicrobial and diuretic activities. Among those, three proteins were found to be phosphorylated, and one <i>N</i>-glycosylated. Finally, cleavage of toxin polypeptide chains seems to be a common post-translational modification in the venom since 80% of the identified molecules were, in fact, products of toxins proteolysis

Moving Pieces in a Venomic Puzzle: Unveiling Post-translationally Modified Toxins from <i>Tityus serrulatus</i>

Besides being a public health problem, scorpion venoms have a potential biotechnological application since they contain peptides that may be used as drug leads and/or to reveal novel pharmacological targets. A comprehensive <i>Tityus serrulatus</i> venom proteome study with emphasis on the phosphoproteome and <i>N</i>-glycoproteome was performed to improve our knowledge on the molecular diversity of the proteinaceous toxins. We combined two peptide identification methodologies, <i>i.e.</i>, database search and <i>de novo</i> sequencing, to achieve a more comprehensive overview of the molecular diversity of the venoms. A total of 147 proteins were identified, including neurotoxins, enzymes, bradykinin-potentiating peptides, and molecules with antimicrobial and diuretic activities. Among those, three proteins were found to be phosphorylated, and one <i>N</i>-glycosylated. Finally, cleavage of toxin polypeptide chains seems to be a common post-translational modification in the venom since 80% of the identified molecules were, in fact, products of toxins proteolysis