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      Ultrasensitive proteome analysis using paramagnetic bead technology

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          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.

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          Gene expression during the life cycle of Drosophila melanogaster.

          Molecular genetic studies of Drosophila melanogaster have led to profound advances in understanding the regulation of development. Here we report gene expression patterns for nearly one-third of all Drosophila genes during a complete time course of development. Mutations that eliminate eye or germline tissue were used to further analyze tissue-specific gene expression programs. These studies define major characteristics of the transcriptional programs that underlie the life cycle, compare development in males and females, and show that large-scale gene expression data collected from whole animals can be used to identify genes expressed in particular tissues and organs or genes involved in specific biological and biochemical processes.
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            The One Hour Yeast Proteome*

            We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS2 acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 h chromatographic method, the Q-OT-qIT hybrid collected an average of 13,447 MS1 and 80,460 MS2 scans (per run) to produce 43,400 (x̄) peptide spectral matches and 34,255 (x̄) peptides with unique amino acid sequences (1% false discovery rate (FDR)). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours.
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              Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis.

              Despite recent developments in bottom-up proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS-based protein extraction with rapid detergent removal, reactor-type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack-this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow-through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential proteomics tool.
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                Author and article information

                Journal
                Mol Syst Biol
                Mol. Syst. Biol
                msb
                Molecular Systems Biology
                BlackWell Publishing Ltd (Oxford, UK )
                1744-4292
                1744-4292
                01 October 2014
                30 October 2014
                : 10
                : 10
                : 1-10
                Affiliations
                European Molecular Biology Laboratory, Genome Biology Unit Heidelberg, Germany
                Author notes
                *Corresponding author. Tel: +49 6221 3878560; E-mail: jeroen.krijgsveld@ 123456embl.de

                Funding InformationMethods & Resources; Post-translational Modifications, Proteolysis & Proteomics

                Article
                10.15252/msb.20145625
                4299378
                25358341
                2a103498-a3ce-403a-ba1a-56367e2528b0
                © 2014 The Authors. Published under the terms of the CC BY 4.0 license

                This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 27 July 2014
                : 01 October 2014
                : 07 October 2014
                Categories
                Articles

                Quantitative & Systems biology
                mass spectrometry,paramagnetic beads,proteomics,quantification,sample preparation

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