Ms data converter beta 1

Proteins in the secreted and sheared fractions were identified with MS. Nano-electrospray ionization MS/MS (nanoESI-MS/MS) analyses were performed on an EASY-nLC II system (Thermo Fisher Scientific) connected to a TripleTOF 5600+ mass spectrometer (AB SCIEX) operated under Analyst TF 1.6.1 control. The trypsin-digested samples were suspended in 0.1% formic acid, injected, trapped and desalted on a precolumn. The peptides were eluted and separated on a 15 cm analytical column (75 μm i.d.), pulled in-house (P2000 laser puller, Sutter Instrument). Trap and analytical column were packed with ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH). Peptides were eluted from the analytical column at a flow rate of 250 nl/min using a 30 min gradient from 5 to 35% of solution B (0.1% formic acid, 100% acetonitrile). The collected MS files were converted to Mascot generic format (MGF) using the AB SCIEX MS Data Converter beta 1.1 (AB SCIEX) and the “protein pilot MGF” parameters. The generated peak lists were searched using an in-house Mascot search engine (Matrix Science) against all C. acnes proteins in the UniProt database as well as against all tad plasmid-encoded proteins (the plasmid p09-9 was used). Search parameters were allowing one missed trypsin cleavage site and propionamide as a fixed modification with peptide tolerance and MS/MS tolerance set to 10 ppm and 0.2 Da, respectively.

Peak lists were generated from the collected MS files by the AB Sciex MS Data Converter beta 1.1 (AB Sciex). Peak lists were searched against a non-redundant UniProt database using an in-house Mascot search engine (Matrix Science). Search parameters were: one missed trypsin cleavage site; propionamide as a fixed modification and methionine oxidation as variable modification. Tolerance for MS and MS/MS were set to 10 ppm and 0.2 Da respectively. All search results were imported into MS Data Miner v. 1.2.1. from which a protein lists were extracted [58] (link).

The collected MS files were converted to Mascot generic format (MGF) using the AB SCIEX MS Data Converter beta 1.1 (AB SCIEX) and the "proteinpilot MGF" parameters. The peak lists were used to interrogate the Swiss-Prot (version 2012_04, 535,698 sequences) Rattus (7750 sequences) and Homo sapiens (20,250 sequences) databases using Mascot 2.3.02 (Matrix Science). Trypsin was employed and allowed one missed cleavage. Propionamide was chosen as a fixed modification and, oxidation of methionine was entered as a variable modification. The mass accuracy of the precursor and product ions were 10 ppm and 0.4 Da, respectively, and the instrument setting was specified as ESI-QUAD-TOF. The significance threshold (p) was set at 0.01 and with an ion score cut-off at 30. Mascot results were parsed using MS Data Miner v. 1.0 [18] (link), protein hits were automatically validated if they satisfied one of the following criteria (i), identification based on one or more unique peptides with ion score above or equal to 45, or (ii), identification based on two or more unique peptides with ion score above or equal to 25. Spectra for protein hits only identified with one peptide within aldosterone treated rats were manually validated by assignment of significant peaks and occurrence of uninterrupted y- or b-ion series of at least 3 consecutive amino acid residues.

Proteins in the secreted fraction were identified using nano-electrospray ionization MS/MS (nanoESI-MS/MS) analyses, performed on an eksigent nanoLC 415 system (SCIEX) connected to a TripleTOF 6600 mass spectrometer (SCIEX). The trypsin-digested samples were suspended in 0.1% formic acid, injected, trapped and desalted on a precolumn. The peptides were eluted and separated on a 15 cm analytical column (75 μm i.d.), pulled in-house (P2000 laser puller, Sutter Instrument). Trap and analytical column were packed with ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH). Peptides were eluted from the analytical column at a flow rate of 250 nl/min using a 30 min gradient from 5 to 35% of solution B (0.1% formic acid, 100% acetonitrile). The collected MS files were converted to Mascot generic format (MGF) using the AB SCIEX MS Data Converter beta 1.1 (AB SCIEX) and the “protein pilot MGF” parameters. The generated peak lists were searched using an in-house Mascot search engine (Matrix Science) against a customized S. saccharolyticus protein fasta database. Search parameters were allowing one missed trypsin cleavage site and carbamidomethyl as a fixed modification with peptide tolerance and MS/MS tolerance set to 10 ppm and 0.1 Da, respectively.