Nano-LC-MALDI is a relatively wide used method in proteomics and has gained more importance in recent years. The major aim of this master work was to establish a new nano-HPLC system at VetCore Facility.
In a first part of the study, standard proteins such as bovine serum albumin and cytochrome c were used to show system suitability. The entire peptides of the proteins were separated using the nano-HPLC system and the fractions were collected with a robot system (ProbotR), which spotted directly on a MALDI target plate. Several parameters of both systems had to be considered and optimized. In the robot system the sample can be mixed with MALDI matrix directly. Thus the amount of matrix has to be defined. Furthermore, the time interval and the spotting direction as well as the alignment of the sample carrier have to be optimized. Separated peptides were analyzed with MALDI-TOF/TOF.
After establishing the nano-HPLC coupled to fraction collector, the method was applied to identify some fungal proteins. After performing SDS PAGE and silver staining some bands were cut out and destained. Reduction and alkylation were followed by in-gel digestion with trypsin. After extraction of peptides and ZipTip purification, a part of the sample was spotted directly on the MALDI target and the remaining part was analyzed using the newly established nano-LC-MALDI method to directly compare the two strategies. Processed spectra were searched via Mascot against the NCBI database. The results were analyzed closer with regard to the number of obtained MSMS-spectra, the protein identification, the score and the matching peptides.
Concluding, it can be stated, that the nano-LC-MALDI method is superior to simple MALDI analysis.Much more MSMS-spectra could be obtained. Due to fractionation with nano-LC prior to MALDI the complexity of the sample is reduced which should reduce ion suppression. Thus more peptides can be analyzed, thereby increasing the number of obtained MSMS-spectra as well as of the matching peptides. This leads to a higher score since the probability that the match is a random event is decreased. Alltogether, more proteins can be identified with nano-LC-MALDI due to a higher number of MSMS-spectra.