add meeting minutes

_DominikNotes/2022-04-12_anja_stefanski.md

 
+# 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.