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Quantitative proteomics with Regen V standardization of PTEN deletion-induced optic nerve regeneration mouse models and controls.

ABSTRACT: A quantitative proteomic profiling was performed on PTEN deletion-induced optic nerve regeneration mouse models and controls. At postnatal day 28 (week 4), PTENloxP/loxP mice were subjected to intravitreal injection (2-3ul) of adeno-associated viruses expressing Cre (AAV2-Cre) or control placenta alkaline phosphatase (AAV2-PLAP). Optic Nerve crush was performed 2 weeks post AAV injections at three times points (0, 7, 14 days post injury) and collected for proteomics analysis. A protein extraction was carried out by homogenization of the tissue in extraction buffer (TEAB, NaCl and SDS). During extraction, three internal peptide standards, named as Regen III, were spiked to measure extraction efficiency. Protein amounts were estimated and normalized across experimental samples. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. TMT (Tandem Mass Tag) labelling was carried out using 1 sets of 18 tags from a 18plex TMT kit for quantification. After combination and drying of all peptide samples, each TMT sample was spiked with two additional isobarically labelled human peptides to be used as an ionization control and cross-sample quantification standard (Regen II). Overall, Regen V standards (refers to a combination of Regen III and Regen II, for extraction and ionization normalization) were used to compare and normalize against any future axon regeneration sample cohort. Untargeted liquid chromatography-mass spectrometry was performed on an Easy-nLC 1000 liquid chromatograph coupled to a QExactive mass spectrometer (LC-MS/MS). Data analysis was performed using Proteome Discoverer 3.0 and Graph Pad Prism 10. After animals were euthanized, optic nerves were collected by dissection. There were 6 experimental conditions and three biological replicates for each condition for a total of 18 nerves. Protein extraction was carried out by homogenizing the optic nerve tissue in TEAB, NaCl and SDS. Three synthetic bacterial peptide standards (Regen III) were added to the samples during the extraction to measure extraction efficiency. Each standard was spiked into samples for a total concentration of 18uM per standard. After extraction was carried out, protein amounts were estimated using dot blot densitometry and ImageJ and normalized to 50ug/ul. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. All samples were labelled using 1 set of 18 tags from a 18plex TMT (Tandem Mass Tag) kit for quantification. After combination and drying of all peptide samples, the samples were fractionated via Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher). Each combined TMT sample was spiked with two additional human peptide standards containing isobaric labels (Regen II). The final concentration of Regen II was 54uM. These standards were spiked in directly before mass spectrometry analysis to be used as an ionization control. Additionally, all five standards, known as Regen V (Regen III + Regen II), serve as a normalization method that may be used to compare protein abundance data across multiple datasets. Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The mouse proteome was downloaded from UniProt and used as the target database for protein identification. Two additional local databases were created. One contains the Regen V internal standard peptide sequences, and the other contains BSA peptide sequences. The identification of the Regen V and BSA standards was run in a separate Proteome Discoverer workflow targeted to identify the internal standards separately from experimental proteins. Max missed cleavage site was set to 2 and minimum peptide length to 6. Precursor Mass Tolerance was set to 10ppm and Fragment Mass Tolerance to 0.02 Da. Post-translational modifications for experimental proteins included oxidation, acetylation, carbamidomethylation and TMTpro. Post translational modifications of the Regen II, (biotin, carbon-13 and nitrogen-15) were added during this step to ensure correct identification of the modified standards. The Normalization was set to total peptide amount and confidence to low.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Sanjoy Bhattacharya

PROVIDER: MSV000093512 | MassIVE | Mon Nov 27 10:03:00 GMT 2023

SECONDARY ACCESSION(S): PXD047286

REPOSITORIES: MassIVE

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Project description:In this labeled quantitative proteomics dataset we profile the proteomic changes in a transgenic line of 1 year old Xenopus laevis Tg(islet2bgfp) frogs that were left untreated (naive) or had a monocular surgery of either a left optic crush injury (crush) or a sham surgery. Matching controls of uninjured optic nerves were also collected for a control factor. The Tg(islet2bgfp) frogs were allowed to recover for 12 and 27 days post optic nerve crush before euthanasia and optic nerve collection. A protein extraction was carried by homogenization of the tissue in extraction buffer (TEAB, NaCl, SDS) via Precellys. During extraction, three internal peptide standards, named as Regen III, were spiked to measure extraction efficiency. Protein amounts were estimated and normalized across experimental samples. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. TMT (Tandem Mass Tag) labelling was carried out using 3 sets of 13 tags from a 16plex TMT kit for quantification. The samples were fractionated into 8 fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific). After fractionation and drying of all peptide samples, each TMT sample was spiked with two additional isobarically labelled human peptides to be used as an ionization control and cross-sample quantification standard (Regen II). The combination of Regen III and Regen II (Regen V) were used to compare and normalize against a future axon regeneration cohort. Untargeted liquid chromatography-mass spectrometry was performed on an Easy-nLC 1000 liquid chromatograph coupled to a QExactive mass spectrometer (LC-MS/MS). Data analysis was performed using Proteome Discoverer 3.0 and Graph Pad Prism 10. After animals were euthanized, optic nerves were collected by dissection. There were six experimental conditions and six biological replicates for each condition for a total of 36 nerves. Protein extraction was carried out by homogenizing the optic nerve tissue in TEAB, NaCl and SDS. Three synthetic bacterial peptide standards (Regen III) were added to the samples during the extraction to measure extraction efficiency. Each standard was spiked into samples for a total concentration of 48uM per standard. After extraction was carried out, protein amounts were estimated using dot blot densitometry and ImageJ and normalized to 50ug/ul. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. Prior to labelling, three samples of mass spec grade pre-digested bovine serum albumin (BSA) were prepared at a concentration of 150pmol. These samples were included to account for differences in labelling efficiency between TMT batches. Three TMT batches were used to label the 36 experimental conditions, so three additional standards were prepared to be used for normalization. All samples, including the three BSA standards, were labelled using 3 sets of 13 tags from a 16plex TMT (Tandem Mass Tag) kit for quantification. Each BSA standard was labelled with a different tag. After combination and drying of all peptide samples, each combined TMT sample (including one BSA-labelled standard) was spiked with two additional human peptide standards containing isobaric labels (Regen II). The final concentration of Regen II was 54uM. These standards were spiked in directly before mass spectrometry analysis to be used as an ionization control. Additionally, all five standards, known as Regen V (Regen III + Regen II), serve as a normalization method that may be used to compare protein abundance data across multiple datasets. Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The mouse proteome was downloaded from UniProt and used as the target database for protein identification. Two additional local databases were created. One containing the Regen V internal standard peptide sequences, and the other containing BSA peptide sequences. The identification of the Regen V and BSA standards was run in a separate Proteome Discoverer workflow targeted to identify the internal standards separately from experimental proteins. Max missed cleavage site was set to 2 and minimum peptide length to 6. Precursor Mass Tolerance was set to 10ppm and Fragment Mass Tolerance to 0.02 Da. Post-translational modifications for experimental proteins included oxidation, acetylation, carbamidomethylation and TMTpro. Post translational modifications of the Regen II, (biotin, carbon-13 and nitrogen-15) were added during this step to ensure correct identification of the modified standards. The Normalization was set to total peptide amount and confidence to low.

Project description:This labeled quantitative proteomics dataset was collected from a transgenic channel rhodopsin mouse model (Chr2) subjected to light stimulation after traumatic optic nerve crush (ONC) was performed. Mouse models expressing channel rhodopsin were subjected to therapeutic light stimulation promoting axon regeneration of retinal ganglion cell (RGC) axons post optic nerve crush. Experimental mice and wild type control mice were euthanized, and optic nerves were collected. A protein extraction was carried out by careful mincing of the tissue in extraction buffer (TEAB, NaCl and SDS). During extraction, three internal peptide standards, named as Regen III, were spiked to measure extraction efficiency. Protein amounts were estimated and normalized across experimental samples. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. TMT (Tandem Mass Tag) labelling was carried out using 3 sets of 12 tags from a 16plex TMT kit for quantification. After combination and drying of all peptide samples, each TMT sample was spiked with two additional isobarically labelled human peptides to be used as an ionization control and cross-sample quantification standard (Regen II). Overall Regen V standards (refers to a combination of Regen III and Regen II, for extraction and ionization normalization) were used to compare and normalize against any future axon regeneration sample cohort. Untargeted liquid chromatography-mass spectrometry was performed on an Easy-nLC 1000 liquid chromatograph coupled to a QExactive mass spectrometer (LC-MS/MS). Data analysis was performed using Proteome Discoverer 3.0 and Graph Pad Prism 10. After animals were euthanized, optic nerves were collected by dissection. There were 12 experimental conditions and three biological replicates for each condition for a total of 36 nerves. Protein extraction was carried out by mincing the optic nerve tissue in TEAB, NaCl and SDS. Three synthetic bacterial peptide standards (Regen III) were added to the samples during the extraction to measure extraction efficiency. Each standard was spiked into samples for a total concentration of 36uM per standard. After extraction was carried out, protein amounts were estimated using dot blot densitometry and ImageJ and normalized to 70ug/ul. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. Prior to labelling, three samples of mass spec grade pre-digested bovine serum albumin (BSA) were prepared at a concentration of 150pmol. These samples were included to account for differences in labelling efficiency between TMT batches. Three TMT batches were used to label the 36 experimental conditions, so three additional standards were prepared to be used for normalization. All samples, including the three BSA standards, were labelled using 3 sets of 13 tags from a 16plex TMT (Tandem Mass Tag) kit for quantification. Each BSA standard was labelled with a different tag. After combination and drying of all peptide samples, each combined TMT sample (including one BSA-labelled standard) was spiked with two additional human peptide standards containing isobaric labels (Regen II). The final concentration of Regen II was 54uM. These standards were spiked in directly before mass spectrometry analysis to be used as an ionization control. Additionally, all five standards, known as Regen V (Regen III + Regen II), serve as a normalization method that may be used to compare protein abundance data across multiple datasets. Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The mouse proteome was downloaded from UniProt and used as the target database for protein identification. Two additional local databases were created. One containing the Regen V internal standard peptide sequences, and the other containing BSA peptide sequences. The identification of the Regen V and BSA standards was run in a separate Proteome Discoverer workflow targeted to identify the internal standards separately from experimental proteins. Max missed cleavage site was set to 2 and minimum peptide length to 6. Precursor Mass Tolerance was set to 10ppm and Fragment Mass Tolerance to 0.02 Da. Post-translational modifications for experimental proteins included oxidation, acetylation, carbamidomethylation and TMTpro. Post translational modifications of the Regen II, (biotin, carbon-13 and nitrogen-15) were added during this step to ensure correct identification of the modified standards. The Normalization was set to total peptide amount and confidence to low.

Project description:Zebrafish (Danio Rerio) have the capacity for successful adult optic nerve regeneration, unlike mammals. Optic nerve regeneration is frequently studied using optic nerve crush (ONC). For ONC, animals were deeply anesthetized in 0.033% tricaine methane-sulfonate (MS-222). The right optic nerve was exposed by gently removing the connective tissue on the dorsal half of the eye and rotating the eye ventrally out of the orbit with a pair of number 5 forceps. A nerve crush was then performed using number 5 forceps to crush the nerve ~0.5 to 1 mm from the optic nerve head for 5 seconds. Success of crush was assessed by identifying the generation of a translucent stripe in the nerve that completely separated two areas of white myelination with no bleeding. Fish were then revived in fresh aquatic system water in individual tanks. After 1 hour the tanks were returned to the fish system and animals were maintained normally with daily feeding until 3 days post injury. Female and male (6 month to 1 year old) Zebrafish optic nerves (right side/OD) were crushed and collected three days after. The associated retinas and tecta were also collected under the same conditions. Contralateral, uninjured optic nerves, retinas and tecta were collected as controls. For tissue collection, animals were euthanized by overdose of MS-222 (>400mg/L) followed by dissection. The tissue was collected from the optic nerve head to the optic chiasm. The tissues were immediately frozen on dry ice. Optic nerve samples were pooled for each category (female crush, female control, male crush, male control) and pooled at n = 31 to obtain sufficient protein concentrations for analysis (pooled optic nerves served as one sample). Retina and tectum samples were pooled using the same categories (female crush, female control, male crush, male control) at n = 10-12 (pooled tissue served as one sample). Protein extraction was carried out by homogenizing the optic nerve tissue in TEAB, NaCl and SDS. Three synthetic peptide standards (Regen III) were added to the samples during the extraction to measure extraction efficiency. Each standard was spiked into samples for a total concentration of 48uM per standard. After extraction was carried out, protein amounts were estimated using dot blot densitometry and ImageJ and normalized to 50ug/ul. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. All samples were labelled using 2 sets of 14 tags from a 18plex TMT (Tandem Mass Tag) kit (A52045: Thermo Fisher Scientific, Waltham, MA) for quantification. After combination and drying of all peptide samples, the samples were fractionated into 9 fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (84868: Thermo Fisher Scientific, Waltham, MA). After fractionation and drying of all peptide samples, each fractionated TMT sample was spiked with two additional peptide standards (Regen II) containing isobaric labels. The final concentration of the post extraction spiked peptides was 54uM per plex. These standards (Regen II) were spiked directly before mass spectrometry analysis to be used as an ionization control. Additionally, all five standards serve as a normalization method that may be used to compare protein abundance data across multiple datasets. Untargeted liquid chromatography-mass spectrometry was performed on an Easy-nLC 1000 liquid chromatograph coupled to a QExactive mass spectrometer (LC-MS/MS). Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The Danio rerio proteome was downloaded from UniProt and used as the target database for protein identification. Max missed cleavage site was set to 2 and minimum peptide length to 6 . Precursor Mass Tolerance was set to 10ppm and Fragment Mass Tolerance to 0.02 Da. Post-translational modifications for experimental proteins included oxidation, acetylation, carbamidomethylation and TMTpro. The Normalization was set to total peptide amount and confidence to low. Two additional local databases were created for the pre- and post- extraction peptides and ran separately from experimental protein identification.

Project description:In this labeled quantitative proteomics dataset, we profile the proteomic changes in the tectum, retina, chiasm, and optic nerve in transgenic lines of 1 year old Xenopus laevis Tg(islet2bgfp). The frogs had monocular surgery of either a left optic crush injury (crush) or sham surgery (sham) and the matching controls of uninjured optic nerves were collected as a control factor. The Tg(islet2bgfp) frogs were allowed to recover for 12 and 27 days post optic nerve crush before euthanasia and tissue collection. Protein extraction was carried by homogenization of the tissue in extraction buffer (TEAB, NaCl, SDS) via Precellys. During extraction, three internal peptide standards were spiked to measure extraction efficiency. Protein amounts were estimated and normalized across experimental samples. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. TMT (Tandem Mass Tag) labelling was carried out using 6 sets of 17 tags from a 18plex TMT kit for quantification. The samples were fractionated into 9 fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific). After fractionation and drying of all peptide samples, each TMT sample was spiked with two additional isobarically labelled peptides to be used as an ionization control and cross-sample quantification standard. The combination internal peptides were used to compare and normalize against a future axon regeneration cohort. Untargeted liquid chromatography-mass spectrometry was performed on an Easy-nLC 1000 liquid chromatograph coupled to a QExactive mass spectrometer (LC-MS/MS). Data analysis was performed using Proteome Discoverer 3.0 and Graph Pad Prism 10. After animals were euthanized, optic nerves were collected by dissection. There were six experimental conditions and six biological replicates per tissue for a total of 102 samples. Protein extraction was carried out by homogenizing the optic nerve tissue in TEAB, NaCl and SDS. Three synthetic peptide standards were added to the samples during the extraction to measure extraction efficiency. Each standard was spiked into samples for a total concentration of 48uM per standard. After extraction was carried out, protein amounts were estimated using dot blot densitometry and ImageJ and normalized to 50ug/ul. Samples were reduced using TCEP, alkylated with iodoacetamide and digested overnight with trypsin. All samples were labelled using 6 sets of 17 tags from a 18plex TMT (Tandem Mass Tag) kit for quantification. After combination and drying of all peptide samples, each combined TMT sample was spiked with two additional peptide standards containing isobaric labels. The final concentration of the post extraction peptides was 54uM per plex or 324uM for all six plex's. These standards were spiked in directly before mass spectrometry analysis to be used as an ionization control. Additionally, all five standards serve as a normalization method that may be used to compare protein abundance data across multiple datasets. Raw mass spectrometry data files were analyzed using Proteome Discoverer 3.0. The frog proteome was downloaded from UniProt and used as the target database for protein identification. Max missed cleavage site was set to 2 and minimum peptide length to 6. Precursor Mass Tolerance was set to 10ppm and Fragment Mass Tolerance to 0.02 Da. Post-translational modifications for experimental proteins included oxidation, acetylation, carbamidomethylation and TMTpro. The Normalization was set to total peptide amount and confidence to low.

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Quantitative proteomics with Regen V standardization of PTEN deletion-induced optic nerve regeneration mouse models and controls.

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