posted on 2020-03-12, 18:35authored byZhenbin Zhang, Kyle M. Dubiak, Paul W. Huber, Norman J. Dovichi
We report a miniaturized filter aided
sample preparation method
(micro-FASP) for low-loss preparation of submicrogram proteomic samples.
The method employs a filter with ∼0.1 mm2 surface
area, reduces the total volume of reagents to <10 μL, and
requires only two sample transfer steps. The method was used to generate
25 883 unique peptides and 3069 protein groups from 1000 MCF-7
cells (∼100 ng protein content), and 13 367 peptides
and 1895 protein groups were identified from 100 MCF-7 cells (∼10
ng protein content). Single blastomeres from Xenopus laevis embryos at the 50-cell stage (∼200 ng yolk free protein/blastomere)
generated 20 943 unique peptides and 2597 protein groups; the
proteomic profile clearly differentiated left and right blastomeres
and provides strong support for models in which this asymmetry is
established early in the embryo. The parallel processing of 12 samples
demonstrates reproducible label free quantitation of 1 μg protein
homogenates.