Dataset Information

Alpha-1-Antitrypsin: A Novel Human High Temperature Requirement Protease A1 (HTRA1) Substrate in Human Placental Tissue

ABSTRACT: Substrate screening of the human protease HTRA1 in a placenta protein extract. The human serine protease high temperature requirement A1 (HTRA1) is highly expressed in the placental tissue, especially in the last trimester of gestation. This suggests that HTRA1 is involved in placental formation and function. With the aim of a better understanding of the role of HTRA1 in the placenta, candidate substrates were screened in a placenta protein extract using a gel-based mass spectrometric approach. Peptides were analysed using a nanoLC-ESI-IT-FTICR-MS instrument controlled by Xcalibur 2.07 software version (Thermo Scientific, Bremen). A by data-dependent acquisition method was used, where the five most intense precursor ions detected in the full MS scan (FTICR) were selected and fragmented in the IonTrap by collision induced dissociation (CID) (35% energy, 4 amu mass isolation width). Proteome Discoverer 1.3 and SEQUEST search engines (Thermo Scientific, Bremen) were used for data analysis. The MS/MS raw data were directly analysed with the Proteome Discoverer software using the following spectrum selector settings; minimum precursor mass 350 Da, maximum precursor mass 5000 Da, total intensity threshold 100, minimum peak count 10, signal-to-noise threshold 5 (FT-only). The identification searching parameters were: tryptic digestion, 2 missed cleavages, deamidated (N), oxidation (M) and propionamide (C) modifications, precursor mass tolerance 5 ppm and fragment mass tolerance 0.5 Da. The database UniProtKB/Swiss-Prot homo sapiens (September 2013, 20.267 Proteins) was chosen for protein identification. A list of contaminants was removed from this database, namely, Keratin, type I and type II cytoskeletal and trypsin. As discrimination criteria, protein identifications containing less than 2 peptides and SEQUEST scores lower than 40 were discarded.

INSTRUMENT(S): LTQ FT

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Violette Frochaux

LAB HEAD: Violette Frochaux

PROVIDER: PXD000473 | Pride | 2014-10-24

REPOSITORIES: Pride

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Alpha-1-antitrypsin: a novel human high temperature requirement protease A1 (HTRA1) substrate in human placental tissue.

Frochaux Violette V Hildebrand Diana D Talke Anja A Linscheid Michael W MW Schlüter Hartmut H

PloS one 20141020 10

The human serine protease high temperature requirement A1 (HTRA1) is highly expressed in the placental tissue, especially in the last trimester of gestation. This suggests that HTRA1 is involved in placental formation and function. With the aim of a better understanding of the role of HTRA1 in the placenta, candidate substrates were screened in a placenta protein extract using a gel-based mass spectrometric approach. Protease inhibitor alpha-1-antitrypsin, actin cytoplasmic 1, tropomyosin beta c ...[more]

PMID: 25329061

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Project description:Cerebella from wild-type and cGKI knockout mice, on 129/Sv background, were subjected to protein digestion using trypsin o/n and chemically labeled with dimethyl labeling. After labeling the samples were mixed and subjected to both strong cation exchange and phosphopeptide enrichment. The SCX fractions and the enriched phosphopeptides were then analyzed on an Orbitrap mass spectrometer. Data analysis: For each raw data file recorded by the mass spectrometer, peak lists were generated using Proteome Discoverer (version 1.3, Thermo Scientific, Bremen, Germany) using a standardized workflow. Peak lists, generated in Proteome Discoverer, were searched against a Swiss-Prot database (version 2.3.02, taxonomy Mus musculus, 32402 protein entries) supplemented with frequently observed contaminants, using Mascot (version 2.3.02 Matrix Science, London, UK). The database search was performed by using the following paramenters: a mass tolerance of 50 ppm for the precursor masses and +/-0.6 Da for CID/ETD fragment ions and +/-0.05 Da for HCD fragments. Enzyme speciﬁcity was set to Trypsin with 2 missed cleavages allowed. Carbarmidomethylation of cysteines was set as fixed modification, oxidation of methionine and dimethyl labeling (L, I, H) of lysine residues and N termini, and phosphorylation (S, T, Y) (for the phosphoproteome analysis) were used as variable modifications. Percolator was used to ﬁlter the PSMs for <1% false discovery-rate. Phosphorylation sites were localized by applying phosphoRS (pRS) (v2.0). Triplex dimethyl labeling was used as quantification method, with a mass precision for the 2 ppm for consecutive precursor mass scan. A retention time tolerance of 0.5 min was used to account effect of deuterium on retention time. To further filter for high quality data we used the following parameters: high confidence peptide spectrum matches, minimal Mascot score of 20, minimal peptide length of 6 and only unique rank 1 peptide. For the phosphopeptides analysis, the search rank 1 peptide was added to the previous filters. For the identification and quantitation of the proteins, only the unique peptides were considered.

			Action	DRS
	1D_A1.RAW	Raw
	1D_A1.pep.xml	Pepxml
	1D_A1.prot.xml	Xml
	1D_A2.RAW	Raw
	1D_A2.pep.xml	Pepxml