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Analysis of the peptidome of the moss Physcomitrella patens
We performed the analysis of native peptides pool of gametophores, protonema and protoplast cells of the moss. To reduce endogenous proteolytic activity we used an acid extraction buffer with a mixture of plant protease inhibitors. All the actions were performed on ice. After the proper sample preparation, the extracted peptides were identified with use of tandem mass spectrometry on the basis of protein database uniref100, taxon Physcomitrella patens.
Sample Processing Protocol
For peptide extraction from moss tissues and protoplasts, the extraction buffer was 1 М acetic acid in 10% acetonitrile and 10 μL/mL of Protease Inhibitor Cocktail (Sigma-Aldrich). Protoplasts and intact chloroplasts were disrupted directly in the extraction buffer with a Ultra-Turrax T10 basic homogenizer (IKA, Staufen, Germany) using a S10N10G nozzle at a rotation speed of 3000 rpm at 4°C. The ground material was placed into cooled extraction buffer containing proteinase inhibitors and homogenized using a Dismembrator S ball mill (Sartorius, Göttingen, Germany) at 2600 rpm for 2 min with a mix of glass balls of 0.1, 0.3, and 1 mm diameter (Sartorius). After centrifugation, samples were immediately placed into a gel filtration column to extract and fractionate the peptides. Gel filtration was carried out on a 2.5 cm × 30 cm column filled with Sephadex G-25 superfine in 0.1 M acetic acid. The fractions containing peptides were lyophilized and resuspended in 5% acetonitrile-0.1% trifluoroacetic acid. Before recording the mass spectra, samples were desalted on reversed phase C18 microcolumns. The desalted peptide preparations were concentrated on a SpeedVac Concentrator vacuum centrifugal evaporator (Savant, Waltham, MA, USA) to a volume of 5μL and diluted with 3% acetonitrile in 0.1% trifluoroacetic acid to 20μL.
Data Processing Protocol
Moss native peptide identifications were performed on the basis of a single LC-MS run for each sample. The .wiff data files were analyzed with the ProteinPilot software 4.5 revision 1656 (ABSciex) using the search algorithm Paragon 220.127.116.11 revision 1654 and the default parameter set for protein identification with the following adjustments: uniref100_Physco_35213 protein sequence database no Cys alkylation, no digestion, TripleTOF5600 equipment, organism type not specified, search effort–thorough ID, detection protein threshold–unused protein score 0.05. Spectrum grouping was performed with default parameters using the ProGroup algorithm embedded in ProteinPilot. Peptide identification FDR statistical analysis was performed using the ProteomicS Performance Evaluation Pipeline Software (PSPEP) algorithm also embedded in the ProteinPilot software. Peptides with probability over 95% were selected for analysis. Additionally, spectra acquired with TripleTOF 5600+ and LTQ Orbitrap Velos were searched with Mascot Version: 2.2.07 (Matrix Science), using the following parameters: precursor mass tolerance 20 ppm, MS/MS mass tolerance 50 ppm, no fixed modifications. Peptides with Mascot scores above the threshold were selected for analysis.
Fesenko IA, Arapidi GP, Skripnikov AY, Alexeev DG, Kostryukova ES, Manolov AI, Altukhov IA, Khazigaleeva RA, Seredina AV, Kovalchuk SI, Ziganshin RH, Zgoda VG, Novikova SE, Semashko TA, Slizhikova DK, Ptushenko VV, Gorbachev AY, Govorun VM, Ivanov VT. Specific pools of endogenous peptides are present in gametophore, protonema, and protoplast cells of the moss Physcomitrella patens. BMC Plant Biol. 2015 Mar 15;15:87 PubMed: 25848929
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||29485||The analysis of the gametophores endogenous peptides||469||2400||1087||54628||1617||
|2||29486||The analysis of the gametophores endogenous peptides||142||851||217||47830||718||
|3||29487||The analysis of the gametophores endogenous peptides||76||461||151||19021||421||
|4||29488||The analysis of the gametophores endogenous peptides||254||1431||458||75926||1095||
|5||29489||The analysis of the protonema endogenous peptides||412||1119||633||40029||751||
|6||29490||The analysis of the protonema endogenous peptides||295||1963||670||38874||1597||
|7||29491||The analysis of the protoplasts endogenous peptides||1396||11439||3423||45473||6092||
|8||29492||The analysis of the protoplasts endogenous peptides||1219||10951||3240||38975||5553||
|9||29493||The analysis of the protoplasts endogenous peptides||1276||10335||3592||52069||5568||
|10||29494||The analysis of the protoplasts endogenous peptides||966||9660||2852||37542||5636||