Protein extraction
200 mg of leaf material sampled as described before were ground to fine powder with mortar and pestle. The powder was incubated with pre-heated SDT lysis buffer (4% w/v SDS, 100mM TRIS/HCl pH 7.6, 0.1M DTT) at 95°C for 5min. Thereafter, the sample was sonicated in a sonication bath for 1min to shear the DNA and reduce viscosity of the sample. The sonicated sample was centrifuged at 5,000rpm for 10min at room temperature on a table centrifuge and the supernatant transferred to
tubes. This centrifugation step was repeated twice. After that, the protein concentration was determined by Bradford assay.
Proteolytic digestion and desalting
50µg of protein in the extracts were processed using the FASP method (Wisniewski 2009) as described in detail in Hartl et al. (2015). Cysteines were alkylated with chloroacetamide and successively hydrolyzed with LysC and trypsin. Trifluoroacetic acid (TFA) was added to a final concentration of 0.5%. Peptides were desalted and pre-fractionated prior LC-MS/MS into three fractions using the Empore Styrenedivenylbenzene Reverse Phase Sulfonate material (SDB-RPS, 3M) as described in detail in Kulak et al. (2014).
LC-MS/MS data acquisition
Dried peptides were redissolved in 2%ACN, 0.1% TFA for analysis and adjusted to a final concentration of 0.2µg/µl. Samples were analyzed using an EASY-nLC 1000 (Thermo Fisher) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher). Peptides were separated on 16cm frit-less silica emitters (New Objective, 0.75µm inner diameter), packed in-house with reverse-phase ReproDil-Pur C18 AQ 3µm resins (Dr. Maisch). Peptides (1µg) were loaded on the column and eluted for 130 min using a segmented linear gradient of 0% to 95% solvent B (solvent A: 5% ACN, 0.5% FA; solvent B 100% ACN, 0.5% FA) at a flow rate of 300nL/min. Mass spectra were acquired in the Orbitrap analyzer with a mass range of 300-1750 m/z at a resolution of 70.000 FWHM and a target value of 1x106 ions. Precursors were selected with an isolation window of 1.3 m/z. HCD fragmentation was performed at a normalized collision energy of 25. MS/MS spectra were acquired with a target value of 105 ions at a resolution of 17,500 FWHM and a fixed first mass of m/z 100. Peptides with a charge of +1 or with unassigned charge state were excluded from fragmentation for MS2, dynamic exclusion for 30s prevented repeated selection of precursors.
Data analysis
Raw data were processed using MaxQuant software (version 1.5.1.2, http://maxquant.org) (Cox and Mann, 2008) with label-free quantification (LFQ) and iBAQ enabled (Cox et al., 2014). MS/MS spectra were searched by the Andromeda search engine against the MIPS database (http://mips.helmholtz-muenchen.de). Sequences of 248 common contaminant proteins and decoy sequences were automatically added during the search. Trypsin specificity was required and a maximum of two missed cleavages allowed. Minimal peptide length was set to seven amino acids. Carbymidomethylation of cysteine residues was set as fixed, oxidation of methionine and protein N-terminal acetylation as variable modifications. Peptide-spectrum-matches and proteins were retained if they were below a false discovery rate of 1%. Subsequent quantitative statistical analysis was performed in Perseus (version 1.5.2.6,http://maxquant.org; Cox and Mann 2012). Hits were only retained if they were quantified in at least to of three replicates in any of the four conditions.
References for protein analysis
Cox, J. and Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol, 2008, 26, pp 1367-72.
Cox J., Hein M. Y., Luber C. A., Paron I., Nagaraj N., and Mann M., Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ. Mol Cell Proteomics, 2014, 13, pp 2513–2526.
Cox J and Mann M, 1D and 2D annotation enrichment: a statistical method integrating quantitative proteomics with complementary high-throughput data., BMC Bioinformatics. 2012;13 Suppl 16:S12.
doi: 10.1186/1471-2105-13-S16-S12. Epub 2012 Nov 5.
Hartl M, König AC, Finkemeier I. Identification of lysine-acetylated mitochondrial proteins and their acetylation sites. Methods Mol Biol. 2015
Kulak NA, Pichler G, Paron I, Nagaraj N, Mann M. 2014. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods. 11(3):319-24.
8 Acknowledgements
First, I would like to express my gratitude to my supervisor Maria von Korff for giving me the opportunity to do my PhD in her group, for her support throughout my PhD and my so-far academic career. Thank you, Maria, for adopting me, your open office door and advice about academia.
Further, I want to thank Seth Davis who initiated this PhD-project with me. Without you this project would not have been initiated.
A lot of gratitude also goes to Alison M. Smith. Her input was crucial at several stages of this project.
She is simply the best in plant metabolic biology.
Then I want to thank the whole von Korff group for an excellent and very nice working atmosphere.
Likewise, I want to thank all the people from the Coupland department for making the department so great.
I want to thank Andreas Weber and George Coupland for being my mentor and giving rich feedback on my work.
Special thanks go to my mum who is absolutely convinced that this is good work without really understanding much about it ;-).
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