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Set of Novel Automated Quantitative Microproteomics Protocols for Small Sample Amounts and Its Application to Kidney Tissue Substructures

Here we assessed the ability of an automated sample preparation device equipped with disposable microcolumns to prepare mass-limited samples for high-sensitivity quantitative proteomics, using both label-free and isobaric labeling approaches. First, we compared peptide label-free quantification reproducibility for 1.5–150 μg of cell lysates and found that labware preconditioning was essential for reproducible quantification of <7.5 μg digest. Second, in-solution and on-column tandem mass tag (TMT) labeling protocols were compared and optimized for 1 μg of sample. Surprisingly, standard methods for in-solution and on-column labeling showed poor TMT labeling (50–85%); however, novel optimized and automated protocols restored efficient labeling to >98%. Third, compared with a single long gradient experiment, a simple robotized high-pH fractionation protocol using only 6 μg of starting material doubled the number of unique peptides and increased proteome coverage 1.43-fold. To facilitate the analysis of heter...

Authors:   Erik Leonardus de Graaf; Davide Pellegrini; Liam A. McDonnell
Journal:   Journal of Proteome Research
Year:   2016
DOI:   10.1021/acs.jproteome.6b00889
Publication date:   10-Nov-2016
Facts, background information, dossiers
  • labelling
  • tissue
  • solution
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