TITLE: Tissue lysis and digestion for MS analysis
AUTHORS: David Kotol

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Here, a workflow for sample preparation and quantification of brain and pancreatic tissue proteins with use of heavy labelled protein standards (QPrESTs) is described. The standards are added before the digestion ensuring high reproducibility and elimination of nuances apearing during proteolytical cleavage of proteins and further steps such as solid phase extraction. QPrESTs are heavy labelled recombinant protein fragments covering regions of proteins with low homology and span over 50-150 aminoacids. They were produced as a part of Human Protein Atlas project in auxotrofic E.coli and individually purified, serving as an excelent resource for MS-based protein quantification.</div></div>

[STEPS]
?. [Protein extraction]
Incubate adapter of the Tissue Lyser on dry ice for
?. [Protein extraction]
Add one 3mm bead to the tissue sample and incubate on dry ice for
?. [Protein extraction]
Disrupt the tissue using the Tissue Lyser for
?. [Protein extraction]
Add of Lysis Buffer (7M Urea, 2M Thiourea, 2% SDC)
250 µl
?. [Protein extraction]
Homogenize the tissue using the Tissue Lyser for
?. [Protein extraction]
Centrifuge for  at 20,000 × g at
?. [Protein extraction]
Precipitate the proteins with 4 volumes of ice-cold acetone and incubate in  over night
?. [Protein extraction]
Centrifuge at 20,000 × g at  for  and discard the supernatant
?. [Protein extraction]
Wash the pellet two times with  of ice-cold acetone followed by centrifugation at 20,000 × g at  for
400 µl
?. [Protein extraction]
Remove the acetone excess and dry the pellet
?. [Protein extraction]
Dissolve the pellet in of Pellet Solubilizer (7M Urea, 2M Thiourea)
200 µl
?. [Protein extraction]
Dilute 8 times with TEAB and measure protein concentration using Bio-Rad Protein assay according to the manufacturers instructions
100 Milimolar (mM)
?. [Protein Digestion]
Take volume that corresponds to of proteins and add QPrESTs mixture
15 µg
?. [Protein Digestion]
Add DTT to a final concentration of  and incubate  at
10 Milimolar (mM)
?. [Protein Digestion]
Add CAA to a final concentration ofand incubate in the dark at room temperature
50 Milimolar (mM)
?. [Protein Digestion]
Add  of Pierce Trypsin protease and incubate over night at
300 ng
?. [Protein Digestion]
Quench the digestion with TFA to a final concentration of 0.5% (v/v)
?. [Solid phase extraction using Strong Cation Exchange]
Prepare one StageTip per sample by inserting 3 layers of Empore Cation Extraction Discs in a 200uL tip
?. [Solid phase extraction using Strong Cation Exchange]
Activate membrane with 100% MeOH, spin at 1000 x g for
50 µl
?. [Solid phase extraction using Strong Cation Exchange]
Equilibrate StageTip with  Wash Buffer (30% MeOH, 0.1% FA),   spin at 1000 x g for
50 µl
?. [Solid phase extraction using Strong Cation Exchange]
Apply sample to the StageTip,  spin at 1000 x g for
?. [Solid phase extraction using Strong Cation Exchange]
Wash two times with Wash Buffer (30% MeOH, 0.1% FA),  spin at 1000 x g for
30 µl
?. [Solid phase extraction using Strong Cation Exchange]
Elute two times with  Elution Buffer (30% MeOH, 1.65% NH4OH), spin at 1000 x g for
20 µl
?. [Solid phase extraction using Strong Cation Exchange]
Vacuum dry forat  and store at until MS-analysis
?. [Solid phase extraction using Strong Cation Exchange]
For MS analysis, dissolve samples in Solvent A and inject 1.75 ug