PXD000600

PXD000600 is an original dataset announced via ProteomeXchange.

Title	Temporal dynamics of protein synthesis and secretion during LPS activation of macrophages
Description	Macrophages provide the first line of host defense by their capacity to react to an array of cytokines and bacterial components, requiring tight regulation of protein expression and secretion to invoke a properly tuned innate immune response. To capture the dynamics of this system, we introduce a novel method combining pulsed SILAC labeling with pulse-labeling using the methionine-analogue azidohomoalanine (AHA), allowing the enrichment of newly synthesized proteins via click-chemistry followed by their identification and quantification by mass spectrometry. We show that this permits the analysis of proteome changes at a rapid time scale evidenced by the detection of 4852 newly synthesized proteins after only a 20-minute SILAC pulse. We have applied this methodology to study proteome response during macrophage activation in a time-course manner. We have combined this with full proteome and secretome analyses producing an integrative analysis of the first 3 hours of lipopolysaccharides (LPS)-induced macrophage activation. Data analysis: The mass spectrometric raw data were processed using MaxQuant (version 1.2.2.5) (1) and MS/MS spectra were searched using the Andromeda search engine (2) against mouse proteins in UniProt (53623 entries) (3), to which 265 frequently-observed contaminants as well as reversed sequences of all entries had been added. Enzyme specificity was set to trypsin/P and a maximum of two missed cleavages were allowed. Cysteine carbamidomethylation was used as fixed modification and methionine oxidation, protein N-terminal acetylation, as well as replacement of methionine by AHA (in case of AHA treatment experiments) were used as variable modifications. The minimal peptide length was set to 6 amino acids. Initial maximal allowed mass tolerance was set to 20 ppm for peptide masses, followed by 6 ppm in the main search and 0.5 Dalton for fragment ion masses. False discovery rates for peptide and protein identification were set to 1%. At least one unique peptide was required for protein identification. The protein identification was reported as an indistinguishable “protein group” if no unique peptide sequence to a single database entry was identified. For protein quantification a minimum of two ratio counts was set and the “requantify” and “match between runs” function enabled. For pSILAC samples a protein group was kept for the further analysis if the number of identified peptide species carrying an medium-heavy or heavy label divided by the total number of peptide species detected in the complete experimental setup was higher than 0.2. For all other samples proteins assigned to contaminants or reverse sequences were removed. Average protein ratios were reported, if they were quantified in two replicates each based on at least two ratio counts.
HostingRepository	PRIDE
AnnounceDate	2014-07-25
AnnouncementXML	Submission_2014-07-25_01:48:16.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Katrin Eichelbaum
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	monohydroxylated residue; acetylated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2013-11-23 14:38:13	ID requested
1	2014-03-07 05:12:20	announced
⏵ 2	2014-07-25 01:48:17	announced	Updated project metadata.

Eichelbaum K, Krijgsveld J, Rapid temporal dynamics of transcription, protein synthesis, and secretion during macrophage activation. Mol Cell Proteomics, 13(3):792-810(2014) [pubmed]

submitter keyword: macrophage activation/quantitative proteomics/protein synthesis/protein secretion/transcription

Katrin Eichelbaum
contact affiliation	EMBL
contact email	katrin.eichelbaum@gmx.de
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2014/03/PXD000600

PRIDE project URI

• PRIDE
  1. PXD000600
     1. Label: PRIDE project
     2. Name: Temporal dynamics of protein synthesis and secretion during LPS activation of macrophages