SubscribePhoteomix outline standard procedures for protein biomarker discovery research.
STEP 1: Pre-fractionation
- Optional: Serum can be de-albuminated using Cibacron Blue chromatography or using anti-albumin antibodies (human only).
- IEX fractionation: samples from complex mixtures are fractionated using strong anion ion exchange support into 4 - 6 sub-fractions.
- Other novel pre-fraction procedures are possible from a wide range of chromatographic media, including proprietary biomarker capture & display matrices (click here for more details).
STEP 2: MS Analysis
- Fractionated protein samples are subjected to SELDItof MS analysis using Ciphergen ProteinChip arrays.
- Alternative MS analysis by direct MALDI-tof (useful for large scale early biomarker probing).
- Several surface chemistries are available for analysis including hydrophobic, weak or strong anion/cation exchange, normal phase, IMAC (immobilised metal affinity chromatography) with Cu2+, Ni2+, etc (or Ga3+ for phosphopeptides).
- MS analysis is performed with two matrices (CHCA or SPA) to cover the peptide to large protein range (500 Da to 200 kDa).
STEP 3: Data mining
- Multiple sample profiles are analysed for similarity and differences in their expression levels, identifying differences in the protein peaks and sorting them as a function of multiple sample groups.
STEP 4: Data Analysis
- Optimal conditions are chosen from the above based on biomarker detection sensitivity and the number of candidate biomarkers found. A larger population of samples is screened and the data submitted to statistical analysis.