Lys-C Trypsin Digestion on 20 Human Protein Mixture Test Sample

From Mswiki

April 7, 2007 Catherine

Lys-C + Trypsin Digestion on 20 Human Protein Mixture Test Sample

1. Dissolve protein in 60 ul 8 M urea, 100 mM Tris pH 8.5 (freshly made) 2. Add 0.3 ul 1 M TCEP (5 mM final conc.), incubate at RT for 20 mins. 3. Add 1.2 ul 500 mM IAA (10 mM final conc., freshly made), incubate at RT for 15 mins in the dark.

Lys-C digest 1. Add 1.5 ul Lys-C (0.5 ug/ul), (1/20 ~ 1/100) 2. Incubate in the dark for 3 ~ 4 hr at 37 C

Trypsin digest 1. Add 180 ul 100 mM Tris pH 8.5 to dilute urea by factor of 4 (2 M final conc.) 2. Add 2.4 ul 100 mM CaCl2 (1 mM final conc.) 3. Add 1.5 Trypsin (0.5 ug/ul), (1/20 ~ 1/100) 4. Incubate in the dark for over night at 37 C

Reaction stopped 1. Add 13.3 90% formic acid (5% final conc.) 2. Spin at top speed for 15 mins, transfer the supernatant to a new tube, freeze at -80 (or -20) degree.

LC-MS/MS Method

1. Sample loading column: 2.5 cm, 250 um i.d. fused silica column, packed with Aqua 5 u C18 material. 2. Analytical column: 15 cm, 100 um i.d. fused silica column, packed with Aqua 5 u C18 material. 3. LC gradient: Buffer B (%) 0.2 ml/min (300 nl/min at tip) 0-10 11 min 10-60 135 min 60-100 12 min 100 8 min 100-0 4 min 0 10 min 4. MS/MS data dependent method 1 full scan 400-1600 m/z (centroid mode) 1 us 50 ms max. ion injection time minimum MS signal: 1000

5 ms/ms scan “Most intense if no parent masses found” checked repeat count: 2 repeat duration: 30 s exclusion list size: 100 exclusion duration: 60 s

SEQUEST Search Method

None enzyme specific DTASelect2 --trypstat -p 4 --fp 0.05