Evidence Source
| Description | Human A2780 cells were grown in T75 flasks to 70% confluence. After few washes in PBS, the cells were lysed adding in flask NP40 lysis buffer (150 mM NaCl, 1% NP40, 50 mM Tris pH 8 and EDTA-free protease inhibitor). Cells were scraped off the walls and the lysate was centrifuged to remove debris. Protein samples were denaturated in 6 M Guanidine-HCl, 50 mM Tris-HCl (pH 8) and 4 mM DTT, reduced for 15 min at 95°C and finally alkylated by the addition of Iodacetamide (55mM) for 1 h at room temperature. Proteins were precipitated by the addition of four times the sample volume of cold acetone and centrifuge after 1 h at -20°C to remove salt and reagents. The protein pellet was resuspended in 50 mM ammonium bicarbonate. Proteins were digested by the addition of 1/100th (w/w) of sequencing grade modified porcine trypsin (Promega, Madison, WI) for 1 h at 37°C twice. Peptides were desalted by Sep-Pak (Waters, Milford, MA) solid phase extraction as recommended by the supplier. The retained material was eluted with 80% ACN, 0.1% TFA followed by evaporation to dryness using a vacuum centrifuge. The peptide mixture was resuspended in SCX-LC buffer A (5 mM KH2PO4, 30% ACN and 0.05% formic acid) and injected into Summit LC system (Dionex, Sunnyvale, CA) equipped with Polysulfoethyl A 200 x 2.1 mm 5 µm 200 Å column (PolyLC, Colombia, MD). Peptides were eluted with a gradient of increasing KCl (SCX-LC Buffer B: 350 mM KCl in SCX-LC Buffer A; 0 - 5 min, 0% B; 15 - 40 min, 5 - 26% B; 40 - 45 min, 26 - 35% B). Fractions were collected every 1-2 min for 50 min. A selection of the collected fractions, i.e. fractions eluted after 4min and up to 30 min that contain most of the N-terminus peptides, were concentrated using a vacuum centrifuge down to 50 µl and subjected to separation on an Ultimate 3000 nano-RP-LC (Dionex, Sunnyvale, CA). Five µl of the pre-concentrated sample were loaded onto a PepMap100 trap column at a flow of 20 µl/min of RP-LC buffer A (A: 5% ACN, 0.1% formic acid) and separated using a PepMap C18 75-μm i.d. x 15cm analytical column over a 100 min gradient (nano-RP-LC buffer B: 80% ACN, 0.1% formic acid; 8-25% B in 70 min then 25-50% B in 30 min) at a flow of 300 nl/min. The nano-RP-LC was coupled to a Q-Star XL (Applied Biosystems, Concorde, ON) and Analyst software was used for data dependant acquisition. Basic survey scan (1 sec) from 400-1200 Da, was followed by 4 EPI scan (1 sec). Acquired tandem mass spectra (MS/MS) were exported from Analyst using the script Mascot.dll 1.6b23 (Matrix Science, London, UK). MS/MS of same precursor mass within 0.1 Da range were combined together for 60 seconds to avoid generating multiple fragmentation pattern of the same precursor within a single analysis. |
|---|---|
Method |
SCX-fractionation |
Experimental system |
cell culture |
Perturbation |
|
Protease inhibitor(s) used |
EDTA-free protease inhibitor cocktail |
Directness |
direct |
Physiological relevance |
yes |
Evidence Code(s) |
|
Database / Laboratory |
- Lab: CNRS/ISV |
Raw data repository |
Evidence for:
1472
N-termini
0
C-termini
Publication(s) |
|
|---|