title: 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 (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.
authors.affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Hughes
authors.affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.
authors.forename: Sophia
authors.initials: S
authors.lastname: Foehr
authors.affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.
authors.forename: David A
authors.initials: DA
authors.lastname: Garfield
authors.affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.
authors.forename: Eileen E
authors.initials: EE
authors.lastname: Furlong
authors.affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.
authors.forename: Lars M
authors.initials: LM
authors.lastname: Steinmetz
authors.affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany jeroen.krijgsveld@embl.de.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Krijgsveld
mesh_terms: D000818:Animals; D004331:Drosophila melanogaster / Q000196:embryology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D060328:Magnetic Phenomena; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
chemical_list: D020543:Proteome
keywords: mass spectrometry; paramagnetic beads; proteomics; quantification; sample preparation