Differential Gel-Based Proteomic Approach for Cancer Biomarker Discovery Using Human Plasma - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 235

database. The search parameter settings should be as follows:
Homo sapiens , variable modifi cation, oxidized at methionine
residues (+16 Da), carbamidomethylated at cysteine residues
(+57 Da), maximum allowed missed cleavage = 1, MS toler-
ance = 1.2 Da, MS/MS tolerance = 0.6 Da, and charge
states = 2+ and 3+. Filter the matched peptides with a signifi -
cance threshold of p < 0.05 and set the minimum threshold to
30 Mascot peptide score. For further details see ref. 12 .

4. Notes

1. The lysis buffer must not contain DTT or BPB because DTT

interferes with CyDye labeling and BPB obstructs the CyDye
color checking during the labeling process.

2. The equilibrium solution must be made in the dark without

the addition of distilled water, and TBP must be added freshly
to the equilibrium solution prior to 2DE. The single-step treat-
ment of TBP and acrylamide is used for effi cient reduction and
alkylation of cystine/cysteine residues ( 19 ).

3. Healthy donors and the HCC case control patients tested neg-

ative for HIV-1 and HIV-2 antibodies, HIV-1 antigen (HIV-1),
hepatitis B surface antigen (HBsAg), hepatitis B core antigen
(anti-HBc), hepatitis C virus (anti-HCV), HTLV-I/II antibody
(anti-HTLV-I/II), and syphilis. The HCC patients' clinical
and pathologic data were gathered at Yonsei University College
of Medicine and are as follows: 70 years of age, male, and
cancer grade = HCC stage II with 10% necrosis of liver tissue.
Authorization for use of plasma for research purposes was
obtained from the Institutional Review Board (IRB).

4. Bound proteins are eluted from the MARS column with Buffer

B at a fl ow rate of 1 mL/min for 3.5 min. The MARS column
is regenerated by equilibrating with Buffer A for 8 min at a
fl ow rate of 1 mL/min.

5. After TCA treatment, the pellets must be resuspended in ace-

tone into very small particles for the following reasons. First,
any residual TCA remaining in the pellet may increase the
amount of NaOH solution necessary to adjust to pH 8.5 for
CyDye labeling, and NaOH interferes with IEF. Second, resus-
pension maximizes the surface of the particles and subsequent
exposure to the labeling reaction. Third, this method mini-
mizes protein loss.

6. The 50 g of the internal pooled standard sample is prepared

by combining 25

μ

μ g each of the two samples prior to Cy2

labeling.