Three biological exosome samples (20 μg/sample) were separated on 4–15% mini-protein TGX precast protein gels (Bio-Rad) and subsequently stained with Coomassie Blue, then each sample lane was excised and digested with trypsin and spiked with 0.2 pmol of ADH peptides (YEAST Alcohol dehydrogenase 1) at the Mass Spectrometry Facility at the University of Massachusetts Medical School. The samples were then injected into Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific) in technical triplicates for label-free quantitation (LFQ) analysis. The data were searched against Swiss-Prot Mouse protein database using Mascot search engine through Proteome Discoverer software. The data was exported and normalized as intensity-based absolute quantification (iBAQ) quantitative values in Scaffold (version Scaffold_4.10, Proteome software). The selected parameters for protein identification were the following: Protein Threshold >95%; minimum 3 peptides per candidate protein; Peptide Threshold >90%; >1 × 105 iBAQ value in at least one of samples. The iBAQ value of the housekeeping protein ADH was used for normalization of biological replicates.
Adh peptides
Manufactured by Bio-Rad
ADH peptides are synthetic peptides derived from the alcohol dehydrogenase (ADH) enzyme. The core function of ADH peptides is to serve as biochemical tools for research and analysis purposes, such as in the study of alcohol metabolism and enzymatic activity.
Automatically generated - may contain errors
2 protocols using adh peptides
1
Label-free Quantification of Exosomal Proteins
2
Label-Free Proteomics of Exosome Samples
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Three biological exosome samples (20 μg/sample) were separated on 4–15% mini-protein TGX precast protein gels (Bio-Rad) and subsequently stained with Coomassie Blue, then each sample lane was excised and digested with trypsin and spiked with 0.2 pmol of ADH peptides (YEAST Alcohol dehydrogenase 1) at the Mass Spectrometry Facility at the University of Massachusetts Medical School. The samples were then injected into Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific) in technical triplicates for label-free quantitation (LFQ) analysis. The data was searched against Swiss-Prot Mouse protein database using Mascot search engine through Proteome Discoverer software. The data was exported and normalized as intensity-based absolute quantification (iBAQ) quantitative values in Scaffold (version Scaffold_4.10, Proteome software). The selected parameters for protein identification were the following: Protein Threshold > 95%; minimum 3 peptides per candidate protein; Peptide Threshold > 90%; > 1 × 105 iBAQ value in at least one of samples. The iBAQ value of the housekeeping protein ADH was used for normalization of biological replicates.
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