TopFIND 4.0

Evidence Source

Description A. thaliana cells (derived from Columbia ecotype) were used and produced as described previously (55). Cultured cells were harvest by centrifugation. Cells equivalent to 10 mL are mixed with 20 mg of SDS, 150 mg of DTT, 100 mg of sodium carbonate, 5 mL of water and heated for 15 min at 95°C. A. thaliana plant tissues were frozen in liquid nitrogen and ground in a 2 ml microcentrifuge tube containing 3 mm and 5 mm iron beads for 1 min each, using a MM 300 mixer mill at 30 Hz (Qiagen). The resulting fine powder was dissolved in 1 mL of lysis buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 15mM MgCl2, 1 mM EDTA, 10 % glycerol, 1% Triton X-100, 2mM PMSF and protease inhibitor cocktail). The homogenates were incubated at 4 °C for 20 to 45 min with shaking. The supernatants were separated from the insoluble fraction by centrifugation at 15000g at 4 °C for 0.5 to 1 h. The resulting supernatants were used to measure protein concentration by the Bradford protocol (BioRad; Marnes-la-Coquette, France) and were stored at -80 °C unless used immediately. 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:

1338

N-termini

0

C-termini


Publication(s)