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 mm2 mouse kidney cortex
and medulla microdissections to show the method’s potential
for real-life small sample sources and to create kidney substructure-specific
proteomes.