ABSTRACT
INTRODUCTIONNanoliter-LC coupled to tandem mass spectrometry (nano-LC-MS/MS) permits the rapid and sensitive determination of protein-protein interactions. By using a specific purification technique such as coimmunoprecipitation or affinity purification in conjunction with nano-LC-MS/MS, not only are proteins identified, but specific protein-protein interactions are elucidated as well.
ABSTRACT
INTRODUCTIONThis protocol provides guidance for setting the parameters for a typical data-dependent MS/MS acquisition analysis using the ThermoFinnigan LCQ system. The method consists of a continual cycle beginning with one scan of MS (scan one), which records all of the m/z values of the ions present at that moment in the gradient, followed by two rounds of MS/MS. The initial MS/MS scan is of the first most-intense ion recorded from the MS scan. The second MS/MS scan is of the second most-intense ion recorded from scan one. Dynamic exclusion is activated to improve the protein identification capacity during the analysis.
ABSTRACT
A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.
