diff --git a/_DominikNotes/2022-04-12_anja_stefanski.md b/_DominikNotes/2022-04-12_anja_stefanski.md index 139597f9cb07c5d48bed18984ec4747f4b4f3438..993a492228cc22f99185abe1cde20ced387f88b0 100644 --- a/_DominikNotes/2022-04-12_anja_stefanski.md +++ b/_DominikNotes/2022-04-12_anja_stefanski.md @@ -1,2 +1,78 @@ +# Meeting Anja Stefanski + +> 12.04.2022 - 10:30 - Webex + +- sample quality not soo good +- data analysis with proteome discoverer (thermo fisher) + - maxquant is good, but tricky for newbies +- identification or quantification + - quantification possible with triplicates + +## to dos + +### Dominik + +- send back sample list + - indicate order of fractions (F1-F4) + - species used + +### Anja + +- 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.