Below
is the hardware-specific protocol:
A. Reversed Phase Protein Arrays
1. Bulk wash H50 ProteinChip arrays (Ciphergen) with 50%
acetonitrile (HPLC Grade; Aldrich, Milwaukee, WI, USA), 2
times, 5 minutes each.
2. Drying for 1 hour. Load onto a 192-well bioprocessor (Ciphergen).
Add 10% acetonitrile/ 0.1% trifluoroacetic acid (Fisher Scientific
International). Prepare 2:3 mixture of serum or plasma and
sample buffer consisting of 8M urea (Sigma, St. Louis, MO,
USA), 2% CHAPS ((3-[(3-Cholamidopropyl) dimethylammonio]-1-propansulfonat);
Sigma), pH 7.4 was prepared.
3. Robotic system (Biomek FX™, Beckman Coulter, Fullerton,
CA, USA) with 96-channel 200 µl head is used unless
otherwise specified.
4. 10 µl sample and 50 µl 10% acetonitrile/ 0.1%
trifluoroacetic acid are added to each array spot.
5. Incubation takes palce for an hour (60 cycles of repetititve
pipetting of the mixture up /down for 30 s each).
6. 75 µl 10% acetonitrile/ 0.1% trifluoroacetic acid
is used to wash the array spots for 3 times, 5 minutes each.
7. Array spots are washed with 75 µl water once for
five minutes.
8. The bioprocessor is allowed to dry for for 15 minutes
with the top removed.
B. Metal-Affinity and Cationic Protein Arrays
1. The metal-affinity (IMAC3) and cationic (CM10) protein
arrays are processed as in part A except for a few modifications
as follows:
2. For the IMAC3 arrays0.1M CuSO4 for 20 min was added to
charge the arrays. The IMAC3 arrays were then washed with
0.5 M NaCl / 0.1 M NaH2PO4.
4. For the CM10 arrays, 10mM HCl was used to activate the
array (for 5 min). The CM10 arrays were then washed with 20mM
NH4OAc / 0.1% Triton X-100 at pH 6.
C. Matrix Application
1. Sinapinic acid (SPA; Fluka, Buchs, Switzerland), was used
as the matrix molecule here. A saturated solution is made
with 50% acetonitrile/ 0.5% trifluoroacetic acid.
2. The solution is diluted one-to-one in 50% acetonitrile/0.5%
trifluoroacetic acid.
3. The arays are air dried. Afterwards, the robot system
uses 20 µl filter tips (Beckman Coulter) to add the
matrix to each array spot. SPecially, 2 times 1 µl is
added to H50 array spots, 1 times 1.2 µl for IMAC3 spots,
and two times 0.75 µl for CM10 array spots.
4. Lastly, the arrays are dried before analysis via mass
spectrometry.