DATA
Quantitative assay: Bradford : Proteored_Bradford_PM6.xls
3 replicates: called P1, P2, P3 : 3 independent digestions
2010_10_11_ALS_51_P1_R1.raw
2010_10_11_ALS_48_P2_R1.raw
2010_10_11_ALS_47_P3_R1.raw
Phosphopeptides enrichment: only one replicate on sample P3
2010_10_11_ALS_57_P3_phospho.raw
Parameters of EasyProt (Olav = Phenyx)) for three replicates
Z score adjusted to obtain FDR 1% for each request. z = 4.88 P1_R1 z = 4.80 P2_R1 z = 5.03 P3_R1
Parameters of EasyProt (Olav = Phenyx)) for phosphoenrichment fraction

Z score adjusted to obtain FDR 1% for each request. z = 4.84 P3_phospho
Protocol (3 replicates)
Digestion: to 9 ul (1.75 ug/ul) of the sample add:
1 ul H2O and 10 ul urea (12 M in 50 mM ammonium bicarbonate).
After 30 min at room temperature add 10 ul DTT (38mM in H2O).
After 1 hour at room temperature add 20 ul iodoacetamide 108 mM in 50 mM ammonium bicarbonate and leave for 1 hour at room temperature in the dark. Add 10 ul of 50 mM ammonium bicarbonate and 3 ul of Promega trypsine (50 ng/ul in 50 mM ammonium bicarbonate, ratio 105:1). Leave all the night at
37°C.
After spin column purification, the sample was analysed by MS.
Tryptic peptides (either 2 µg ) were trapped on a homemade 5 µm 200 Å Magic C18 AQ (Michrom) 0.1 × 20 mm pre-column. Following washing, they were separated on a homemade 5 µm 100 Å Magic C18 AQ (Michrom) 0.75 × 130 mm column with a gravity-pulled emitter. The analytical separation was run for 120 min using a gradient of
H2O/FA 99.9/0.1 (solvent A) and CH3CN/FA 99.9/0.1 (solvent B). The gradient was run as follows: 0 –1 min 95% A and 5% B, then to 65% A and 35% B at 85 min, and 20% A and 80% B at 95 min at a flow rate of 220 nL·min-1, followed by re-equilibration of the column.

Protocol (phosphor enrichment)
Phosphopeptides enrichment was perform following Larsen protocol (Rapid commun. mass. Spectrom.
2007, 21, 3635). Started from 7.5 µg of digested peptides (replicate P3) .