Computational protocol: Serum α-1 Antitrypsin (AAT) antagonizes intrinsic apoptosis induction in neutrophils from patients with systemic inflammatory response syndrome

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[…] Proteomic analyses were performed by the Molecular Proteomics Laboratory at the Biologisch-Medizinisches Forschungszentrum (BMFZ) of Heinrich-Heine University Düsseldorf. Liquid chromatography electrospray ionization mass spectrometry was used for the identification of proteins from human serum sample. 5 μg of the sample was separated in a 4 to 12% bis-tris polyacrylamide gel (Life Technologies, Darmstadt, Germany). After a MS-compatible silver staining, the protein containing lane was cut out, destained, reduced and alkylated. The trypsin digestion was done overnight at 37°C. The peptides were extracted from the lane with a solution of 0.1% trifluoroacetic acid and acetonitrile (1:1 (v/v)). For the LC-MS/MS analysis the solvent was removed and 400 ng peptides were subjected to liquid chromatography.An UltiMate 3000 RSCLnano System (Dionex/Thermo Fisher Scientific, Idstein, Germany) was used to separate the peptides. After the injection of the sample the peptides were pre-concentrated on a C18-pepmap column (Acclaim PepMap 100; 2 cm length, 75 μm inner diameter, 3 μm particle size, 100 Å pore size; Dionex/Thermo Fisher Scientific, Idstein, Germany) with a flow rate of 6 μL/min. After 10 min the separation was started on an analytical C18 column (Acclaim PepMap 100-RSLC; 25 cm length, 75 μm inner diameter, 2 μm particle size, 100 Å pore size; Dionex/Thermo Fisher Scientific, Idstein, Germany). A 120 min gradient was used from 4 to 40% solvent B (A: 0.1% (v/v) formic acid in water B: 0.1% (v/v) formic acid, 84% (v/v) acetonitrile in water) with a flow rate of 300 nL/min.Mass spectrometry was performed on an Orbitrap Elite high resolution instrument (Thermo Fisher Scientific, Bremen, Germany). The peptides were directly eluted by nano electrospray ionization (voltage: 1.4 kV) using a nanosource interface. The MS instrument was operated in the positive mode with a mass range 350–1700 m/z and a resolution up to 60,000. The target value for the automatic gain control was 1,000,000 with the maximum fill time of 200 ms. The 20 most intense doubly and triply charged peptide ions (minimal signal intensity of 500) were isolated, transferred to the linear ion trap (LTQ) part of the instrument, and fragmented by collision-induced dissociation (mass range of 200–2000 m/z at a resolution of 5,400). The fragments of the peptides were analyzed with a maximal fill time of 300 ms and automatic gain control target value of 10,000, already fragmented peptides were automatically excluded for the fragmentation for 45 s.The generated raw-Files were analyzed using Proteome Discoverer (version 1.4.1.14, Thermo Fischer, Dreieich, Germany) connected to a Mascot server (version 2.4, Matrix sciences, London, UK) with default parameters for spectrum selection. The SwissProt database (version SwissProt_2014_06) was used for the peptide identification with the following parameters: mass tolerance of 10 ppm or 0.4 Da, trypsin specific cleavage with a maximum of one missed cleavage site, the oxidation of methionine as dynamic modification and carbamidomethyl as static modification. The false discovery rate (FDR) on peptide and protein level was below 1%. Known contaminations were removed from the protein list. A minimum of 2 unique peptides were required for identification as well as quantification. All proteins were quantified only based on unique peptides. […]

Pipeline specifications

Software tools Proteome Discoverer, Mascot Server
Application MS-based untargeted proteomics
Diseases Wounds and Injuries
Chemicals Staurosporine