Step One: Choosing an Array
ProteinChip arrays are available with different chromatographic properties, including hydrophobic, hydrophilic, anion exchange, cation exchange, and metal affinity surfaces. Arrays with higher specificity may be designed using preactivated arrays for covalently coupling proteins or peptides.
Step Two: Sample Application
Complex biological samples such as serum, cell and tissue lysates, urine, cerebrospinal fluid, or other protein homogenates (including those with high salt or detergent concentrations) can be applied directly to ProteinChip arrays. The ProteinChip cassette–compatible bioprocessor, capable of holding up to 12 arrays, is formatted to a standard 96-well microplate footprint. Sample application can be performed by manual pipetting or by liquid-handling robotics. Capture of a subset of proteins by the array occurs through simple chemical or protein-protein interactions.
Step Three: Removal of Unbound Components and Application of Energy Absorbing Molecules (EAMs)
After a short incubation period, unbound proteins and other contaminants are washed off the surface of the ProteinChip array using buffers and other wash solutions as required by the array chemistry. Only proteins interacting with the chemistry of the array surface are retained for analysis. These selective washes create on-chip protein (retentate) maps. After the wash step, a solution containing EAMs in an organic solvent is applied. EAMs are essential for ionization of the sample. Once the protein dissolves into solution with the EAMs, the solution dries, forming crystals composed of EAMs and protein on the array surface. The array is then placed into the ProteinChip SELDI reader for mass spectrometric analysis. Unlike most mass spectrometry methods, surface-enhanced laser desorption/ionization (SELDI) yields uniform crystallization of molecules and matrix, resulting in the reproducible peak intensities across a spot that are essential for differential expression analysis.
Step Four: Analysis in the ProteinChip SELDI Reader
Analysis of ProteinChip arrays involves laser desorption and ionization of proteins from the array surface and detection by time-of-flight mass spectrometry (TOF-MS). Inside the ProteinChip SELDI reader, a nitrogen laser illuminates the sample and initiates the process of ionization and desorption. This process results from a poorly understood interplay between the laser energy, the EAM, and the analyte. In short, the laser energy induces both protein ionization and a change of state from the solid, crystalline phase into the gas phase. Once in the gas phase, the protein ion can move rapidly, or fly, upon application of a voltage differential. Proteins with a positive charge are induced to fly away from the metal array, which also has a positive charge. The voltage differential applies the same energy to all of the analytes in the sample, resulting in mass-dependent flight times. The ProteinChip SELDI reader records the TOF of the analyte, and from this measurement a highly accurate and precise mass is derived.
