PXD014182

PXD014182 is an original dataset announced via ProteomeXchange.

Title	An automated ‘cells-to-peptides’ sample preparation workflow for high-throughput, quantitative proteomic assays of microbes
Description	Mass spectrometry–based quantitative proteomic analysis has proven valuable for clinical and biotechnology related research and development. Driving this value have been improvements in the sensitivity, resolution, and robustness of mass analyzers. However, manual sample preparation protocols are often a bottleneck for sample throughput and can lead to poor reproducibility, especially for applications where thousands of samples per month must be analyzed. To alleviate these issues, we developed a ‘cells-to-peptides’ automated workflow for Gram-negative bacteria and fungi that includes cell lysis, protein precipitation, resuspension, quantification, normalization, and tryptic digestion. The workflow takes two hours to process 96 samples from cell pellets to the initiation of the tryptic digestion step and can process 384 samples in parallel. We measured the efficiency of protein extraction from various amounts of cell biomass and optimized the process for standard liquid chromatography-mass spectrometry systems. The automated workflow was tested by preparing 96 E. coli samples and quantifying over 600 peptides that resulted in a median coefficient of variation of 15.8%. Similar technical variance was observed for three other organisms as measured by highly-multiplexed LC-SRM-MS acquisition methods. These results show that this automated sample preparation workflow provides robust, reproducible proteomic samples for high-throughput applications.
HostingRepository	PanoramaPublic
AnnounceDate	2019-08-23
AnnouncementXML	Submission_2019-08-23_16:48:00.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Chris Petzold
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562; scientific name: Pseudomonas putida KT2440; NCBI TaxID: 160488; scientific name: Saccharomyces cerevisiae; NCBI TaxID: 4932; scientific name: Rhodotorula toruloides; NCBI TaxID: 5286;
ModificationList	Deamidated; Carbamidomethyl
Instrument	6460 Triple Quadrupole LC/MS

Revision	Datetime	Status	ChangeLog Entry
0	2019-06-07 15:30:34	ID requested
⏵ 1	2019-08-23 16:48:02	announced

Chen Y, Guenther JM, Gin JW, Chan LJG, Costello Z, Ogorzalek TL, Tran HM, Blake-Hedges JM, Keasling JD, Adams PD, Garc, í, a Mart, í, n H, Hillson NJ, Petzold CJ, Automated "Cells-To-Peptides" Sample Preparation Workflow for High-Throughput, Quantitative Proteomic Assays of Microbes. J Proteome Res, 18(10):3752-3761(2019) [pubmed]

submitter keyword: Automation, Sample preparation, Proteomics, Bacteria, Fungi, Microbes, Biotechnology, High-throughput

Chris Petzold
contact affiliation	Lawrence Berkeley National Laboratory
contact email	cjpetzold@lbl.gov
lab head
Chris Petzold
contact affiliation	Lawrence Berkeley National Laboratory
contact email	cjpetzold@lbl.gov
dataset submitter

Panorama Public dataset URI