Proteomics

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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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