In order to obtain a systems-level understanding of a complex biological system, 
detailed proteome information is essential. Despite great progress in proteomics 
technologies, thorough interrogation of the proteome from quantity-limited 
biological samples is hampered by inefficiencies during processing. To address 
these challenges, here we introduce a novel protocol using paramagnetic beads, 
termed Single-Pot Solid-Phase-enhanced Sample Preparation (SP3). SP3 provides a 
rapid and unbiased means of proteomic sample preparation in a single tube that 
facilitates ultrasensitive analysis by outperforming existing protocols in terms 
of efficiency, scalability, speed, throughput, and flexibility. To illustrate 
these benefits, characterization of 1,000 HeLa cells and single Drosophila 
embryos is used to establish that SP3 provides an enhanced platform for 
profiling proteomes derived from sub-microgram amounts of material. These data 
present a first view of developmental stage-specific proteome dynamics in 
Drosophila at a single-embryo resolution, permitting characterization of 
inter-individual expression variation. Together, the findings of this work 
position SP3 as a superior protocol that facilitates exciting new directions in 
multiple areas of proteomics ranging from developmental biology to clinical 
applications.