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 heterogeneous tissue samples, such as those obtained from laser capture microdissection, a modified BCA protein assay was developed that consumes and detects down to 15 ng of protein. As a proof-of-principle, the modular automated workflow was applied to 0.5 and 1 mm2mouse kidney cortex and medulla microdissections to show the method's potential for real-life small sample sources and to create kidney substructure-specific proteomes.
Fondazione Pisana per la Scienza ONLUS , Pisa 56121, Italy.
Erik Leonardus
EL
de Graaf
Fondazione Pisana per la Scienza ONLUS , Pisa 56121, Italy.|NEST, Scuola Normale Superiore , Pisa 56127, Italy.
Davide
D
Pellegrini
Fondazione Pisana per la Scienza ONLUS , Pisa 56121, Italy.|Leiden University Medical Center , Leiden 2333 ZA, The Netherlands.
Liam A
LA
McDonnell
D000818:Animals; D007668:Kidney / Q000737:chemistry / Q000648:ultrastructure*; D007672:Kidney Cortex / Q000737:chemistry; D007679:Kidney Medulla / Q000737:chemistry; D061346:Laser Capture Microdissection; D051379:Mice; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D018401:Sample Size; D013194:Staining and Labeling
D020543:Proteome
TMT; automated; cortex; high pH fractionation; kidney proteome; label free; medulla; microgram sample amount; modified BCA assay; preconditioning; protein assay