Ultrasensitive proteome analysis using paramagnetic bead technology

Abstract

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 ( SP 3). SP 3 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 SP 3 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 SP 3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications. image A new proteomic sample preparation protocol allows fast, efficient and ultra‐sensitive analyses. The method is illustrated by profiling proteomes from sub‐microgram amounts of material, including the first proteome screen of Drosophila development at a single‐embryo resolution. A novel protocol using paramagnetic beads, termed Single‐Pot Solid‐Phase‐enhanced Sample Preparation (SP3) is presented. SP3 enables protein and peptide enrichment, cleanup, digestion, chemical isotope labeling and fractionation in a single tube, without limitations arising from reagent compatibility. SP3 allows unmatched ultra‐sensitive proteome profiling from sub‐microgram amounts of material, as low as 1,000 HeLa cells or a single fly embryo. The first quantitative analysis of early Drosophila development at a single‐embryo resolution reveals dynamic trends in the developmental proteome.