Project PXD000227

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Biological Dataset
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Summary

Title

Analysis of the peptidome of the moss Physcomitrella patens

Description

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 4.5.0.0 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.

Contact

Dmitry Alexeev, Proteome dept.
Vadim Govorun, IBCH RAS ( lab head )

Submission Date

13/05/2016

Publication Date

18/05/2016

Tissue

Not available

Cell Type

plant cell

Quantification

Not available

Experiment Type

Bottom-up proteomics

Assay count

10

Publication

    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

Assay

Showing 1 - 10 of 10 results
# 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