Materials
All the chemicals were purchased from MilliporeSigma (St. Louis, MO). Isoelectric Focusing (IEF) unit that is capable of running IEF at different length is from Bio-Rad (PROTEAN IEF Cell). Spectrophotometer Plate Reader capable of reading 384-well plates with a wide wavelength selection and fluorescence reading is the SPECTRAMax Plus from Molecular Devices (Sunnydale, CA). Semi-Blot unit for protein transfer such as Bio-Rad’s Trans-Blot SD Semi-Dry Transfer Cell. AlbuVoid™ serum protein enrichment beads was from Biotech Support Group (Monmouth Junction, NJ). Protein Elution Plate (PEP) is a product of Array Bridge (St. Louis, MO).
AlbuVoid™ treatment for low abundance serum protein enrichment
200 mg of AlbuVoid™ beads were used to process 0.8 ml of human serum (contains about 40 mg total serum protein). The breast cancer patient serum and the matching normal people serum were collected at Zibo Central Hospital in China after the approval from the Hospital Ethics Committee with reference number of 20140102. Serum samples from normal people or breast cancer patient were pooled with equal volume (100 μl each) respectively and either used directly in the analysis or enriched for low abundance serum proteins with AlbuVoid™ according to the manufacturer’s instruction before loading to the IEF. The enriched low abundance serum proteins were eluted with 0.8 ml elution solution containing 8 M urea, 2% CHAPS in 25 mM phosphate buffer, pH 8.0. The protein concentration was determined by BCA before 2-D gel electrophoresis.
Isoelectric focusing (IEF) and 2-D gel electrophoresis
To prepare for the IEF separation, Bio-Lyte Ampholyte (Bio-Rad #1631112) was added to the serum solution directly or AlbuVoid™ elute above with a final concentration of 0.5%. Rehydration was using 0.4 ml sample solution with nonlinear pH 3–10 11 cm IPG strip (Bio-Rad ReadyStrip #1632016) overnight with a total loading of 1 mg protein/IPG strip. In the experiments without AlbuVoid™ enrichment, 1 mg of serum protein from the pooled breast cancer patient or unaffected individual was used directly following the same sample preparation as described above. All the enriched and unenriched serum proteins were separated in the same IEF run. The proteins were separated using the following setting: 0–7000 linear gradient voltage for 4 h hold at 7000 voltages overnight until running termination at room temperature. After IEF, the IPG strips were taken off the running unit, mineral oil from the IPG strip was absorbed with a paper towel and the IPG strip was transferred to a 12-lane refolding tray (Bio-Rad #1654025). 4 ml refolding solution was added to each lane with the IPG strip and incubated for 10 min., which allows the urea to diffuse out of the IPG strip and permits the refolding of the protein., This was followed by incubation in electrophoresis transfer buffer (Tris-glycine with 0.1% SDS), which allows for the further diffusion of urea from the IPG strip and the binding of SDS to the protein so that all the proteins were negatively charged. For protein refolding, a proprietary protein refolding solution was used; the solution contains multiple metal elements to replace the possible loss of metal ions as enzyme cofactors. A redox system to mimic the cell cytoplasm was used to assist the protein refolding process. After protein refolding, the IPG strip was laid down to a precast 2-D gel (Bio-Rad 10–20% Criterion Gel #3450107) with the acidic end of the IPG on the left side of the 2-D gel when facing the gel apparatus. The gel was operated at 80 V for 15 min. followed by running at 120 voltages until the dye front of the gel was 0.5 cm from the bottom edge of the gel.
Electroelution and protein recovery from the Protein Elution Plate (PEP)
After second dimension gel electrophoresis, the gel was taken out from the cassette, and laid on top of the PEP plate which was filled with elution solution. The proteins were transferred from the gel to the PEP plate for 60 min. at 20 V using a Semi-Blot apparatus from Bio-Rad (#1703940.). After protein transfer, the gel was carefully lifted from the PEP plate, and a multi-channel pipette transferred the eluted proteins from the PEP plate to a master plate which contained 50 μl PBS in each well. About 40–45 μl of solution could be transferred from the PEP plate to the Master Plate for a total volume of 90–95 μl in each well. In this analysis, 25 μl solutions was taken from each well in the Master Plate and transferred to an enzyme assay plate to perform the enzyme assay.
Hexokinase activity assay
Hexokinase activity can be monitored by a cascade reaction as follows:
$$ \begin{array}{l}\mathrm{Substrates}\ \mathrm{added}\left\{\mathrm{D}\hbox{-} \mathrm{Glucose} + \mathrm{ATP}\right\}\overset{\mathrm{Hexokinase}}{\to}\mathrm{Products}\left\{\mathrm{D}\hbox{-} \mathrm{Glucose}\ 6\hbox{-} \mathrm{Phosphate} + \mathrm{ADP}\right\}\\ {}\mathrm{D}\hbox{-} \mathrm{Glucose}\ 6\hbox{-} \mathrm{Phosphate} + \mathrm{\ss}\hbox{-} \mathrm{NADP}\overset{\mathrm{G}\hbox{-} 6\hbox{-} \mathrm{PDH}}{\to }6\hbox{-} \mathrm{Phospho}\hbox{-} \mathrm{D}\hbox{-} \mathrm{Gluconate} + \mathrm{\ss}\hbox{-} \mathrm{NADP}\mathrm{H}\end{array} $$
In the final assay solution, glucose was at 216 mM; MgCl2 at 7.8 mM, ATP at 0.74 mM and NADP at 1.1 mM. 25 μl of this enzyme assay solution was mixed with 25 μl of sample from the Master Plate (described above) and the enzyme activity was monitored by the 340 nm absorbance from the reduction of NADP to NADPH. The readings at 0, 1 h., 2 h. was recorded for both the normal serum and breast cancer patient serum sample. However, in lieu of purified G-6-PDH, 0.25 mg/ml beef liver protein was used as the source of Glucose-6-Phosphate Dehydrogenase (G-6-PDH). The assay thus reports the additive contributions of the endogenous hexokinase activity present in the beef liver extract, and any exogenous activity from the presence of test sera protein in the PEP plate, which may influence the reduction of NAD or NADP (the reporting signal). In light of the ambiguities that may arise from such a reporting system, the primary goal of this investigation was to generate sufficient signal intensities and activity features which could monitored and compared between the two samples types within an ‘omics’ context. Therefore, this broader spectrum assay was chosen that could potentially detect the activities of downstream glycolytic and other cross-regulating proteins from the test sera.
Protease activity
FITC-labeled casein was used as general protease substrate at 0.5 mg/mL final concentration.
25 μl each of the PEP plate sample and substrate were incubated at room temperature overnight in the dark, after protease digestion, the casein was precipitated with 10% TCA (trichloroacetic acid) and the supernatant was neutralized with Tris base and used for the fluorescence measurement.
Proteases Assay:
$$ \mathrm{Casein}\ \left(\mathrm{substrate}\right)\hbox{-} \mathrm{FITC}\overset{\mathrm{Proteases}}{\to}\mathrm{Hydrolyzed}\ \mathrm{Casein} + \mathrm{FITC} $$
Enzyme activity display
Two Microsoft Excel formats were used to display the enzyme activities. One is to use the 3-D column display and the other is to use the heat map.