Filter aided, single tip (FAST) method for high throughput, ultrasensitive proteomics sample preparation reveals proteome asymmetry in Xenopus embryos

We report a filter aided, single tip (FAST) method for ultrasensitive proteomic sample preparation. To minimize sample loss and contamination, the method reduces the surface area of the filter to ~0.1 mm2, the total volume of reagents to < 10 μL, and the number of sample transfer steps to two. 25,883 unique peptides and 3,069 protein groups were identified from 1,000 MCF-7 cells (~100 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.