diff --git a/_DominikNotes/2022-04-12_anja_stefanski.md b/_DominikNotes/2022-04-12_anja_stefanski.md index 993a492228cc22f99185abe1cde20ced387f88b0..1359973f2f0f0156abab57e5ce02dcef68c67e70 100644 --- a/_DominikNotes/2022-04-12_anja_stefanski.md +++ b/_DominikNotes/2022-04-12_anja_stefanski.md @@ -13,7 +13,7 @@ ### Dominik -- send back sample list +- [x] send back sample list - indicate order of fractions (F1-F4) - species used @@ -22,57 +22,4 @@ - runs DA in standard pipeline (proteome discoverer) - ricinus proteome / reference from uniprot? If available - t-test, fold changes, ratios, normalised abundances for every peptide => plenty of excel tables - - possible output fasta of identified peptides / proteins? - - - - - - - - - -## Questions - -- Sample prep - - "silver stained according to MS-compatible protocol, reduced, alkylated and digested with trypsin" - - - -- MS / data acquisition - - specific settings? - - filtering? - - DDA - - "The 20 most intense doubly and triply charged peptide ions (minimal signal intensity 500) were isolated" - -- recommended software? - - -- core facility? - - analysis not included? Cost? - - cooperation (co-authorship) or service only (acknowledgements)? - - - - -- https://doi.org/10.1016/j.celrep.2020.107547 - -Alternatively, samples were separated by a 4%–12% polyacrylamide gel. After Coomassie brilliant blue staining, the protein con- -taining bands were excised and processed as described elsewhere (Poschmann et al., 2014). Briefly, bands were destained, washed, -reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin (Serva) in 50 mM NH4HCO3 overnight at 37C. -Peptides were extracted with 0.1% trifluoroacetic acid and subjected to liquid chromatography. For peptide separation over a -55 minute LC-gradient with 300 mL/min an Ultimate 3000 Rapid Separation liquid chromatography system (Thermo Scientific) equip- -ped with an Acclaim PepMap 100 C18 column (75 mm inner diameter, 25 cm length, 2 mm particle size from Thermo Scientific) was -used. MS analysis was carried out on a Q-Exactive plus mass spectrometer (Thermo Scientific) operating in positive mode and equip- -ped with a nano electrospray ionization source. Capillary temperature was set to 250C and source voltage to 1.4 kV. Survey scans -were carried out over a mass range from 200-2,000 m/z at a resolution of 70,000 (at 200 m/z). The target value for the automatic gain -control was 3,000,000 and the maximum fill time 50 ms. The 20 most intense peptide ions (excluding singly charged ions) were -selected for fragmentation. Peptide fragments were analyzed using a maximal fill time of 50 ms and automatic gain control target -value of 100,000 and a resolution of 17,500 (at 200 m/z). Already fragmented ions were excluded for fragmentation for 10 s. Acquired -spectra were searched using Mascot 2.4 within Proteome Discoverer version 1.4.1.14 against the SwissProt Homo sapiens proteome -dataset (release 2016_06, 20200 sequences). Carbamidomethyl at cysteines was set as fixed modification and phosphorylation at -serine, threonine and tyrosine and methionine oxidation were considered as variable modifications as well as tryptic cleavage spec- -ificity (cleavage behind K and R) with a maximum of two missed cleavage sites. Predefined values were used for other parameters -including a false discovery rate of 1% on peptide level, a main search precursor mass tolerance of 10 ppm and mass tolerance of -10 mmu for fragment spectra. - + - possible output fasta of identified peptides / proteins? \ No newline at end of file