| Metadata |
| datasetIdentifier | PASS00738 |
| datasetType | SRM |
| submitter | Carina Ramallo Guevara <Carina.RamalloGuevara@rub.de> |
| submitter_organization | Ruhr-University Bochum |
| lab_head_full_name | PD Dr. Ansgar Poetsch |
| lab_head_email | Ansgar.Poetsch@rub.de |
| lab_head_organization | Plant biochemistry |
| lab_head_country | Germany |
| datasetTag | CRG01SRM |
| datasetTitle | Confirmation of age-related changes in abundance and oxidation for selected proteins of Podospora anserina mitochondria by SRM analysis |
| publicReleaseDate | 2015-12-31 00:00:00 |
| finalizedDate | 2015-09-15 04:20:10 |
| summary | The free radical theory of ageing is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Since the majority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the ageing model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidised and non-oxidised protein species. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during ageing as long as protein de-novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during ageing and rather suggest a protein damage homeostasis mechanism even at late age.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD001023.
For selected proteins, additional SRM experiments were performed to confirm the data obtained by iTRAQ quantification. |
| contributors | Carina Ramallo Guevara, Philipp,O., Hamann,A., Werner,A., Osiewacz,H., Rexroth,S., Rögner,M., Poetsch,A. Ansgar Poetsch |
| publication | Ramallo Guevara,C., Philipp,O., Hamann,A., Werner,A., Osiewacz,H., Rexroth,S., Rögner,M., Poetsch,A., Global protein oxidation profiling suggests efficient mitochondrial proteome homeostasis during ageing, MCP, in revision. |
| growth | The cultivation of the wild-type strain s and the isolation of crude mitochondrial fractions from P. anserina were performed as previously described (Rexroth et al, 2012). Cultures of six individuals serving as biological replicates were harvested at four different age stages (6 days, 9 days, 13 days and 16 days) resulting in a total of 24 mitochondrial samples. |
| treatment | For relative quantification of the protein abundance ratios using crude AQUA peptides, the four age stages of the six independent biological replicates were prepared in the same manner as described in experimental section: Experimental workflow for in-filter protein digestion and iTRAQ labelling, however without iTRAQ labelling step. 24 suitable peptides, 3 per protein, were selected from the iTRAQ data set based on sequence uniqueness, length between 5–20 amino acids, missed tryptic cleavage sites, and amino acid oxidation predisposition. Corresponding crude heavy peptides (non-tagged SpikeTides L) were synthesized from JPT Peptide Technologies containing C-terminal lysine or arginine stable isotopes to induce mass shifts of 8 or 10 Da per peptide. One hundred femtomole or one picomole of each heavy peptide was spiked into the tryptic digested samples prior to the desalting procedure.
For targeted quantification of the corresponding proteins with their oxidative modification sites, the six independent iTRAQ-labelled samples (see supplementary Table S1) were remeasured on the TSQ Vantage in SRM-mode. |
| extraction | The mitochondrial samples were processed according to the in-filter protein digestion (FASP II) procedure described by (Wiśniewski et al, 2009) with minor modifications. |
| separation | All desalted samples were resuspended in 2 % ACN in 0.1 % FA (1µg/µl) by sonication for 10 min prior to SRM analysis. Measurements were performed on a TSQ Vantage mass spectrometer coupled to a nanoACQUITY gradient UPLC pump system combined with an autosampler. The nanoACQUITY UPLC system was equipped with a reversed phase trapping column (UPLC trap sym C18) and a BEH130 C18 analytical column (1.7 µm, 75 μm μm x 150 mm) and a PicoTip Emitter (Silica TipTM, 10 µm i.d.).
Samples (5-μl injection) were loaded onto the column with 2% buffer B. Peptides were eluted from the column with a multistep gradient of buffer A and buffer B which was established as follows: for 0–5 min: 2% buffer B; for 5–10 min: 2–5% buffer B; for 10–71 min: 5–35% buffer B; for 71–77 min: 35–85% buffer B; and for 77–105 min: 2% buffer B. |
| digestion | Tryptic in-filter protein digestion (FASP II). |
| acquisition | Relative quantification of the protein abundance ratios using crude AQUA peptides was performed using a triple quadrupole mass spectrometer in SRM mode. Four transitions per peptide were selected based on maximum signal intensities observed during nLC-iTRAQ MS/MS and collision energy for each peptide was generated in-silico using Skyline software (version 2.6.0.7176). The SRM instrument method consisted of one SRM scan event over a 105 min run-time whereby 206 transitions were measured with a 10 s cycle time (0.05 ms scan time per transition). Fixed parameters were 0.2 fwhm Q1 for precursor ions and 0.7 fwhm Q3 resolution for product ions.
For targeted quantification of the corresponding proteins with their oxidative modification sites, the six independent iTRAQ-labelled samples (see supplementary Table S1) were remeasured on the TSQ Vantage in SRM-mode. The selected oxidised peptides, precursor ions, SRM transitions and collision energies used for this analysis are deposited here (PASS00738). Collision energy for each peptide was generated again by the skyline software and further experimentally refined for the iTRAQ-labelled peptides according to signal-to-noise measurements during SRM trials. The SRM instrument method consisted of one SRM scan event over 105 min run-time, whereby for all SRMs a scan width of 0.01 m/z and a scan time of 0.02 s was set, and fixed parameters were 0.2 fwhm Q1 and 0.7 fwhm Q3 resolution for peptide fragments and 0.3 fwhm Q3 resolution for iTRAQ reporter ions. Each biological replicate was analysed 3-times. |
| informatics | For SRM data normalisation of non-oxidised protein species, the total protein amount of each sample was determined by precursor ion quantification. For this normalisation, all 24 samples from SRM experiment were remeasured on a LTQ Orbitrap XL mass spectrometer, and for each sample the total protein area from all identified protein areas was summed to generate a normalization factor. More details are available in supplemental methods. Data analysis for age-related protein abundance changes was carried out by using Skyline 3.1.0.7382 software. All SRM-data were manually inspected to ensure correct peak identification whereby not accurately identified peptides based on selected transitions were excluded from data set. Furthermore two samples were excluded from the further data analysis, based on peculiarities of the samples exhibiting shifting of retention times for all peptides and a lower peptide and protein identification rate compared across all samples. The ratios between the peak areas of each light and heavy peptide were calculated using Skyline and exported to Excel for further statistical analysis. To account for differences in protein amount across the samples, the peptide peak ratios of the different samples were normalized based on the total protein area of each corresponding sample. As described above, the equal age ratios for each peptide of one of each biological replicate were calculated: (i), (ii), (iii). Subsequently we determined the inverses for all age ratios: (vi), (v), (vi) and logarithmised to base 2. The final protein age ratios were calculated as the median over all logarithmic distinct peptide ratios belonging to a protein. Additionally, the mean and the standard deviation were calculated in the same manner (supplementary Table S8).
Data analysis for age-related changes in protein oxidation of the corresponding proteins was performed by using Skyline 2.6.0.7176 software. Again all SRM-data were manually inspected to ensure correct peak identification and not accurately identified peptides by transition peaks were excluded from data set. Calculated areas of the iTRAQ reporter ions from each biological and technical replicate were summarised for each protein in an excel worksheet, and the same iTRAQ ratios per age ratio were computed: (i), (ii), (iii). All iTRAQ age ratios were normalised on the factors from respective biological replicate which were obtained during data analysis for unmodified protein species via Proteome Discoverer 1.3, and were therefore most reliable for normalisation. The inverses for all iTRAQ age ratios were determined: (vi), (v), (vi) followed by the logarithm to base 2. The median was computed over logarithmic iTRAQ age ratios from technical and biological replicates for final protein age ratios. Furthermore the mean and the standard deviation over logarithmic iTRAQ age ratios from technical and biological replicates was determined (supplementary Table S9). |
| instruments | Thermo Scientific TSQ Vantage |
| species | Podospora anserina |
| massModifications | static: C+57.021464, L+15.99492, M+15.9949, T+15.99492, W+15.99492, P-30.01057, N-Terminus+144.102063, K+144.102063, K+8.014199, R+10.008269. |
