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Stem Cell Derived Secretome Renders Therapeutic Potential in Steroid Induced and Genetic Glaucoma Models

ABSTRACT: TCA-precipitated proteins were urea-denatured, reduced, alkylated and digested with endoproteinase Lys-C followed by modified trypsin. Peptide mixtures were loaded onto 250 um fused silica microcapillary columns packed with strong cation exchange resin and 5-um C18 reverse phase, and then connected to a 100 um fused silica microcapillary column packed with 5-um C18 reverse phase. Loaded microcapillary columns were placed in-line with a Quaternary Agilent 1100 series HPLC pump and a LTQ Orbitrap Elite mass spectrometer equipped with a nano-LC electrospray ionization source. Fully automated 10-step MudPIT runs were carried out on the electrosprayed peptides. Tandem mass (MS/MS) spectra were interpreted using ProluCID against a database consisting of 79096 nonredundant Human proteins (NCBI, 2016-06-10 release), 193 usual contaminants. To estimate false discovery rates (FDR)s, the amino acid sequence of each non-redundant proteinentry was randomized to generate a virtual library. This resulted in a total library of 158578 non-redundant sequences against which the spectra were matched. All cysteines were considered as fully carboxamidomethylated, while methionine oxidation was searched as a differential modification. DTASelect and swallow, an in-house developed software, were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here all controlled FDRs were less than 5%. All 4 data sets were contrasted against their merged data set, respectively, using Contrast v 1.9 and in house developed sandmartin v0.0.1. Our in-house developed software, NSAF7 v0.0.1, was used to generate spectral count-based label free quantitation results.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Laurence Florens

PROVIDER: MSV000085652 | MassIVE | Tue Jun 30 11:27:00 BST 2020

SECONDARY ACCESSION(S): PXD020121

REPOSITORIES: MassIVE

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Project description:MudPIT Analysis Four biological replicates of FLAG-Hoxa1, and corresponding negative controls, and FLAG-Hoxb1, and corresponding negative controls, were prepared from a chromatin enriched fraction isolated from mouse KH2ES cells and subjected to FLAG affinity purification. The eluted proteins were TCA-precipitated and analyzed by MudPIT. TCA-precipitated proteins were urea-denatured, reduced, alkylated and digested with endoproteinase Lys-C (Roche) followed by modified trypsin (Promega). Peptide mixtures were loaded onto 250 um fused silica microcapillary columns packed with strong cation exchange resin (Luna, Phenomenex) and 5-um C18 reverse phase (Aqua, Phenomenex), and then connected to a 100 um fused silica microcapillary column packed with 5-um C18 reverse phase (Aqua, Phenomenex). Loaded microcapillary columns were placed in-line with a Quaternary Agilent 1100 series HPLC pump and a LTQ linear ion trap mass spectrometer equipped with a nano-LC electrospray ionization source (ThermoScientific, San Jose, CA). Fully automated 10-step MudPIT runs were carried out on the electrosprayed peptides. Tandem mass (MS/MS) spectra were interpreted using ProluCID (v. 1.3.3) against a database consisting of 78014 nonredundant Mus musculus proteins (NCBI, 2016-06-23 release), 193 usual contaminants (human keratins, IgGs, and proteolytic enzymes). To estimate false discovery rates (FDR)s, the amino acid sequence of each non-redundant protein entry was randomized to generate a virtual library. This resulted in a total library of 116008 non-redundant sequences against which the spectra were matched. All cysteines were considered as fully carboxamidomethylated (+57 Da statically added), while methionine oxidation was searched as a differential modification. DTASelect (v. 1.9) and swallow, an in-house developed software, were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here all controlled FDRs were less than 5%. All 16 data sets were contrasted against their merged data set, respectively, using Contrast v 1.9 and in house developed sandmartin v0.0.1. Our in-house developed software, NSAF7 v0.0.1, was used to generate spectral count-based label free quantitation results.

Project description:Sample Preparation: To identify proteins that are recruited by Hoxb1 to two newly identified DNA motifs, namely Motif_2 and LONG2x, nuclear extracts from 24hrs Retinoic acid- and Doxycycline-induced KH2 cell line with inducible Hoxb1 were added to DNA fragments immobilized on magnetic beads. Five experimental conditions were used: motif_2 (15bp) DNA, long2X (45bp) DNA, Hoxb1-ARE DNA as positive control, and 2 negative controls, random DNA and beads-only. In all cases, proteins were incubated at 30C for 30min to allow binding, followed by 2 sequential washes with buffer containing 0.1mg/ml BSA. Proteins were eluted by 2% SDS, 50mM Tris-HCl pH8.8 at 70C for 10 min, then precipitated by 20% TCA. MudPIT Analysis TCA-precipitated proteins were urea-denatured, reduced, alkylated and digested with endoproteinase Lys-C (Roche) followed by modified trypsin (Promega). Peptide mixtures were loaded onto 250 um fused silica microcapillary columns packed with strong cation exchange resin (Luna, Phenomenex) and 5-um C18 reverse phase (Aqua, Phenomenex), and then connected to a 100 um fused silica microcapillary column packed with 5-um C18 reverse phase (Aqua, Phenomenex). Loaded microcapillary columns were placed in-line with a Quaternary Agilent 1100 series HPLC pump and a LTQ linear ion trap mass spectrometer equipped with a nano-LC electrospray ionization source (ThermoScientific, San Jose, CA). Fully automated 10-step MudPIT runs were carried out on the electrosprayed peptides. Tandem mass (MS/MS) spectra were interpreted using ProluCID (v. 1.3.3) against a database consisting of 61441 non-redundant M. musculus proteins (NCBI, 2020-11-03 release), 426 usual contaminants (human keratins, IgGs, and proteolytic enzymes). To estimate false discovery rates (FDR)s, the amino acid sequence of each non-redundant protein entry was randomized, resulting in a total search space of 123728 non-redundant sequences. All cysteines were considered as fully carboxamidomethylated (+57 Da statically added), while methionine oxidation was searched as a differential modification. DTASelect (v. 1.9) and swallow, an in-house developed software, were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here all controlled FDRs were less than 5%. All 16 data sets were contrasted against their merged data set, respectively, using Contrast v 1.9 and in house developed sandmartin v0.0.1. Our in-house developed software, NSAF7 v0.0.1, was used to generate spectral count-based label free quantitation results.

Project description:[NOTE: PF3D7_1470600 previously named RAP14 is now named RAP21]. HA-affinity purification - Purified late asexual parasites from parental and HA-tagged PF3D7_0105200 (RAP01-HA) and PF3D7_1470600 (RAP21-HA) lines were suspended in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 % Triton X-100, 1 mM AEBSF and EDTA-free protease inhibitor cocktail (Roche). After lysis by passing through a 26G needle 25 times, the soluble extracts were treated with 100 units of DNase I (NEB) for 10 min at room temperature and centrifuged at 14,000g for 15 min at 4C. The lysates were precleared with Dynabeads Protein A (Invitrogen) for 1h at 4C. The Rb anti-HA antibody (1:100, Abcam ab9110) was added in each sample and incubated overnight at 4C. Dynabeads Protein A were used to precipitate antibody-protein complexes and were washed with buffer A (1 % Triton X-100, 1 mM EDTA in PBS), buffer B (wash buffer A, 0.5 M NaCl) and buffer C (1 mM EDTA in PBS). Proteins were eluted using 0.1 M glycine, pH 2.8, and neutralized using 2 M Tris-HCl, pH 8.0. Then proteins were precipitated in 20% TCA followed by cold acetone washes. Multidimensional Protein Identification Technology - TCA-precipitated proteins were urea-denatured, reduced, alkylated, and digested with endoproteinase Lys-C (Roche) followed by modified trypsin (Promega). Peptide mixtures were loaded onto 100-um fused silica microcapillary columns packed with 5-um C18 reverse phase (Aqua, Phenomenex), strong cation exchange resin (Luna, Phenomenex), and 5-um C18 Aqua. Loaded microcapillary columns were placed in-line with a Quaternary Agilent 1100 series HPLC pump and a LTQ linear ion trap mass spectrometer equipped with a nano-LC electrospray ionization source (ThermoScientific, San Jose, CA). Fully automated 11-step MudPIT runs were carried out on the electrosprayed peptides. Tandem mass (MS/MS) spectra were interpreted using ProluCID against a database consisting of 5527 non-redundant (NR) Plasmodium falciparum 3D7 proteins (PlasmoDB-42 release), 36661 NR human proteins (NCBI, 2018-03-30 release), 419 usual contaminants (human keratins, IgGs, and proteolytic enzymes). To estimate false discovery rates (FDRs), the amino acid sequence of each NR protein entry was randomized, which resulted in a total search space of 85246 NR sequences. All cysteines were considered as fully carboxamidomethylated (+57 Da statically added), while methionine oxidation was searched as a differential modification. DTASelect and swallow, an in-house developed software (v.0.0.1, https://github.com/tzw-wen/kite), were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here, all controlled FDRs were less than 1.2%. Three immunoprecipitations were performed each for RAP01-HA and RAP21-HA along with 4 negative controls experiments.

Project description:Sample preparation, protein digestion, and MS data acquisition are described in MSV000086790. Post-translational Modification Searches | Peak files were extracted using RawDistiller and searched using ProLuCID (v. 1.3.3) against a database consisting of 5,846 S. cerevisiae non-redundant proteins (downloaded from NCBI 11-01-2016), 193 usual contaminants (such as human keratins, IgGs, and proteolytic enzymes), and, to estimate false discovery rates (FDRs), 6039 randomized amino acid sequences derived from each non-redundant protein entry. All cysteines were considered as fully carboxamidomethylated (+57.0215 Da statically added). The MS/MS dataset for the samples only digested with trypsin was further searched for modified peptides in a recursive manner. A total of eight differential modification searches were set up to query for oxidized methionines (+15.9949 Da) in combination with: 1) acetylated lysines, serines, and threonines (+42.0106 Da); 2) formylated arginines and lysines (+27.9949 Da); 3) hydroxylated aspartates, tyrosines, histidines, and prolines (+15.9949); 4) mono- (+14.0157 Da) and 5) dimethylated (+28.0314 Da) arginines and lysines; 6) trimethylated (+42.0470) lysines; 7) phosphorylated (+79.9663 Da) serines, threonines, and tyrosines; and 8) ubiquitinated lysines (+114.0429 Da). Mass tolerances of 7 ppm for precursor and 800 ppm for fragment ions were used. MS/MS datasets generated for trypsin-digested peptides were searched with KR peptide-end requirements. After this round of searches, an in-house software, sqt-merge (Zybailov et al 2005), was used to combine the ProLuCID output files (.sqt files) allowing only the best matches out of the eight differential queries to be ranked first based on cross-correlation scores (XCorr) and for normalized differences between XCorrs (DeltaCn) to be recalculated accordingly. DTASelect v1.9 and swallow v. 0.0.1 (https://github.com/tzw-wen/kite) were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here, all controlled FDRs were less than 1%.

Project description:MudPIT analysis of Plasmodium polysomes. TCA precipitated pellet from Plasmodium falciparum polysomes (~50ug) was solubilized in TRIS-HCl pH8.5 and Urea; reduced and alkylated. The protein suspension was digested overnight using Endoproteinase Lys-C followed by a second overnight digestion at 37°C using Trypsin. Formic acid (5% final) was added to stop the reactions. Sample was loaded on split-triple-phase fused-silica micro-capillary column and placed in-line with linear Velos pro ion-trap mass spectrometer (Thermo Scientific), coupled with quaternary Agilent 1260 series HPLC. Fully automated 10-step chromatography run (for a total of 20 hours) was carried out on the sample. Each full MS scan (400-1600 m/z) was followed by ten data-dependent MS/MS scans. The number of the micro scans was set to 1 both for MS and MS/MS. The dynamic exclusion settings used were as follows: repeat count 2; repeat duration 30s; exclusion list size 500 and exclusion duration 90 sec, the minimum signal threshold was set to 500. The MS/MS dataset was searched using SEQUEST (v.27; rev. 9) against a database of 72358 sequences, consisting of 5487 P. falciparum non-redundant proteins (downloaded from PlasmoDB on 2012-07-12), 30536 H. sapiens non-redundant proteins (downloaded from NCBI on 2012-08-27), 177 usual contaminants (such as human keratins, IgGs, and proteolytic enzymes), and, to estimate false discovery rates, 36179 randomized amino acid sequences derived from each non-redundant protein entry. To account for alkylation by CAM, 57 Da were added statically to cysteine residues and to account for the oxidation of methionine residues to methionine sulfoxide (that can occur as an artifact during sample processing) 16Da were added as a differential modification to methionine residue. Peptide/spectrum matches were sorted, selected using DTASelect/CONTRAST (v 1.9). Proteins had to be detected by 1 peptide with 2 independent spectra, leading to FDRs at the protein and spectral levels of 2.89% and 0.26%, respectively.

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Stem Cell Derived Secretome Renders Therapeutic Potential in Steroid Induced and Genetic Glaucoma Models

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