Miniaturized filter-aided sample preparation (MICRO-FASP) method for high throughput, ultrasensitive proteomics sample preparation reveals proteome asymmetry in Xenopus laevis embryos

Abstract

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 mm ² 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 3,069 protein groups from 1,000 MCF-7 cells (~100 ng protein content), and 13,367 peptides and 1,895 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 2,597 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.

Graphical Abstract