Esophageal Adenocarcinoma Methods and Protocols

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ples, tumor tissue lysates, and fractionated samples prepared for

protein identification purposes.

The technique involves:

1. A sample preparation step to denature the proteins.


2. Application of the samples to a selected protein-binding chip
   surface in the presence of a selected equilibration buffer, fol-
   lowed by washing to remove unbound materials.


3. Addition of a matrix to trigger ionization in response to appli-
   cation of the laser to the chip surface.


4. Generation of spectra in the SELDI-TOF MS instrument
   under appropriately selected conditions for laser bombard-
   ment and detection of ionized proteins.


5. Detection of peaks (mass-to-charge ratios, m/z) in the resul-
   tant spectra and clustering of peaks common to different
   samples.


6. Detection of peaks that give a statistically significant difference
   between groups under test.


7. Identification of the protein in a significant peak.


8. For CM10, Q10, and IMAC chips, dilute serum or plasma
   samples 1:5 in U9 buffer, incubate for 30 min, and centrifuge
   prior to analysis (see Note 1).


9. Use neat serum or plasma samples for H50 tubes.


10. For tissue lysates, dilute to a final concentration of 100 μg/mL
    protein on the chip surface.


11. Assay samples in accordance with supplier’s (Bio-Rad) recom-
    mendations. Include the all-in-one protein standard for at least
    one spot on each chip surface being used. Each protein chip
    surface has an array of eight spots where protein-binding takes
    place. Clamp the chips in a reaction housing that contains a
    (microtiter plate-like) block, which allows reagents and sam-
    ples to be exposed to the chip surface. Perform incubations
    using a microtiter plate two-way rotator shaker. Protocols
    appropriate for each chip surface are detailed below.


12. Equilibrate the chips using equilibration buffer with triton
    (2 × 150 μL buffer and 10 min incubations) taking care to
    avoid bubbles and ensure liquid is in contact with the chip
    surface.


13. Add the pre-treated samples to the required final dilution (see
    Note 3) in 100 μL of buffer plus triton and incubate the chips
    for 30 min.


14. Perform a wash with equilibration buffer with triton
    (1 × 150 μL buffer and 5 min incubation), followed by two

3.1 Sample
Equilibration
on Protein Chip
Surface

3.1.1 CM10 and Q10
Chip Protocol

Proteomic Protocol