
# 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.

