Protein Expression Analysis | Identification of Proteins by LC-MALDI System
Peptide Mass Fingerprinting (PMF) is a method for identifying proteins using a mass spectrometer.
PMF is an effective method of identifying proteins. However, the identification of trace-level proteins, low-molecular-weight proteins, and post-translational modified proteins is difficult due to the following reasons: it is difficult to attribute them in the database because the number of digest fragments is insufficient; sufficient number of fragments cannot be obtained for trace-level proteins; and the target proteins are post-translationally modified.
MS/MS ion searches are used when identification by PMF is difficult. However, the identification accuracy for difficult-to-identify proteins can be further improved using automated MS/MS ion searching with an LC-MALDI system incorporating nano-LC.
Enzyme digests separated and purified by nano-LC are spotted onto individual positions on the sample plate to reduce ion suppression in MALDI and permit highly sensitive automated MS/MS ion searching of peptides.
2D electrophoresis was conducted on a sample of 300 µg protein extracted from HeLa cells.
After staining with SYPRO®Ruby, multiple spots were excised and subjected to PMF. MS/MS ion searching with an LC-MALDI system was performed on spots that could not be identified by PMF. This method identified the proteins in all four spots.
The LC-MALDI system comprising a Prominence nano Nanoflow High-Performance Liquid Chromatograph, AccuSpot MALDI Plate Spotter, and AXIMA Performance MALDI-TOF MS performs automatic identification by MS/MS of samples separated by nanoflow LC from complex mixtures. It dramatically increases the analysis efficiency of proteome analysis.