Open AccessUltrasensitive proteome analysis using paramagnetic bead technology
Author(s) -
Hughes Christopher S,
Foehr Sophia,
Garfield David A,
Furlong Eileen E,
Steinmetz Lars M,
Krijgsveld Jeroen
Publication year - 2014
Publication title -
molecular systems biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 8.523
H-Index - 148
ISSN - 1744-4292
DOI - 10.15252/msb.20145625
Subject(s) - proteome , proteomics , biology , computational biology , multiplex , shotgun proteomics , profiling (computer programming) , flexibility (engineering) , quantitative proteomics , bioinformatics , computer science , genetics , statistics , mathematics , gene , operating system
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.