2D-PAGE with MS principle
Proteins can be separated by two-dimensional gel electrophoresis. The proteins are identified on 2-D electrophoresis, and then the spots are eluted from the gel and identified sequences using Liquid Chromatography Mass Spectrometry (LC/MS) or western blotting. In the first dimension, proteins are resolved in according to their isoelectric points (pI) using IEF. In the second dimension, proteins are separated according to their approximate molecular weight using sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE). The 2-D PAGE consisted of isoelectric focusing in the first dimension and SDS-PAGE in the second dimension under denaturing conditions.
Fig. The schematic diagram of peptide analysis by PAGE-MS.
- For 2D-PAGE, samples are prepared for isoelectric focusing (IEF) first. The samples are resuspended in a solution containing 7 M urea and 2 M thiourea at pH 8 and dialyzed against the same buffer overnight at room temperature.
- For equilibration, the buffer is adjusted to contain a final concentration of 7 M urea, 2 M thiourea, 100 mM DTT, 4% CHAPS and 2% IPG.
- IEF is performed with immobilized pH gradients (from 3-10), 7 cm or 18 cm lengths. The gels are rehydrated overnight with the samples in equilibration buffer and run according to a standard protocol.
- After the IEF run, the IPG gel strips are incubated twice for 15 min with 50 mM Tris-HCl at pH 8 containing 6 M urea, 30% glycerol and 2%SDS. For the first incubation, 125 Mm DTT is added and the second incubation, 125 mM iodoacetamide is added. Then gels are washed with ddH2O.
- In the second dimension, SDS-PAGE is completed on a 14% acrylamide, 7cm´cm gel following usually used SDS-PAGE method.
- The gels are either silver stained and processed for mass spectrometry, or the resolved proteins are transferred onto PVDF membrane for western blotting.
- To determine the identity and function of the proteins, the spots observed on the 2-D PAGE are eluted, and treated with DTT to break disulphide linkages, alkylated with iodoacetamide and then digested with trypsin.
- Protein samples are destained and underwent a 14 h tryptic digest at 37ºC.
- The resultant peptides are extracted in washes of ammonium bicarbonate solution, ACN and 10% formic acid.
- Extraction solvent is removed under vacuum and the peptides are resuspended in 30 µl of 5% MeOH, 0.5% formic acid.
- Capillary RP HPLC separation of protein digests (desalted with a PepMap C18 cartridge) is performed using Ultimate Capillary HPLC System (LC Packings, San Francisco, CA). Samples (3 µl) are injected directly onto a PepMap reversed phase C18 column (0.075 x 150 mm) supplied by LC Packing (Dionex). The flow rate after splitting was 320 nl / min. Tandem mass spectrometric analysis is performed online using a hybrid quadrupole time-of-flight instrument (QSTAR XL hybrid LC/MS/MS) equipped with a nanoelectrospray source (Applied Biosystems, Foster City, CA).
- Tandem mass spectra are acquired using the information Dependent Acquisition mode.