287- Wash each spot twice with 5 μL of water using the on/off
pipette technique (see Note 4). - Allow the spots to dry. Add sinapic acid matrix by twice apply-
ing 0.7 μL to each spot (see Note 4).
For H50 chips: - Make up H50 binding buffer (10%ACN, 0.1%TFA in deion-
ized water) fresh. - Soak the chips twice in 50% methanol for 5 min and leave to
dry for 1 h. - Pre-wet each spot twice with H50 binding buffer for 2 min,
with blotting to remove buffer. - Add samples onto the spots (2 × 1 μL additions) and incubate
the chips in a humidity chamber for 30 min with gentle
shaking. - Wash each spot twice with 5 μL of H50 binding buffer for
2 min with shaking and blotting to remove buffer. - Allow the spots to dry. Add sinapic matrix by twice applying
0.7 μL to each spot (see Note 4). - Generate mass spectra using a SELDI-TOF MS (e.g., PSII)
Instrument (see Note 5). - For each experiment, optimal conditions are selected for a low
molecular weight analysis in the range 2000–50,000 Da and a
high molecular weight analysis in the range 10,000–200,000 Da
(Table 2 ). - Collect ten readings from each spot at positions 20–80 (steps
of 5) for low molecular weight analysis and 22–82 for high
molecular weight analysis (see Note 6). Once the optimal
parameters are established for a given chip surface, hold con-
stant for the entire experiment.
3.2 Generation
of Mass Spectra
Table 2
Parameters selected for mass spectral analysis of protein chips on
SELDI-TOF MS PSII instrument (Bio-Rad UK)Low MW High MWUpper limit: 50,000
Range: 2000–30,000
(Optimal) mass: 7000
Deflector: 2000
Laser intensity: 180a
Detector (sensitivity): 7a
Hardshot: 10
Warning shot = intensity + 10Upper limit: 200,000
Range: 20,000–150,000
(Optimal) mass: 50,000
Deflector: 10,000
Laser intensity: 200a
Detector (sensitivity): 8a
Hardshot: 10
Warning shot = intensity + 10
aThese parameters are adjusted for each chip surface, each experimentProteomic Protocol