I synthesized two chemical probes with masses
(C24H36N6O2 : 440) and (C24H32N6O2 : 444) as light and heavy probes, respectively . The idea is to use these two probes to label and quantify protein cleavage sites by known enzymes. First, a known protease is used to digest a known substrate. This will generate a new N-terminus at every cleavage site. Digested protein( 2 similar tubes and same conditions) is precipitated and treated with the light and heavy probes, precipitated. Precipitated light and heavy label proteins are resuspended and combined, click reaction, pulled down, trypsin digested, and eluted, LC-MS/MS. Raw files obtained are run on maxquant software and msms files imported into skyline software to identify cleavage sites and relative quantify as heavy vrs light pairs. To run raw file, I created two separate labels for light and heavy according to their composition and masses in maxquant as shown above. These probes can label the neo-N-terminus of any residue, so I select any N-terminus, label, then select all residues in the specificity section. I select the light in the light channel, then heavy in the heavy channel. trypsin digestion as semi specific, and all other parameters as default. I run against database for protein of interest in maxquant.
Unfortunately, it runs very slow, freezes and crushes. And when I leave the specificity blank, I don’t get the heavy and light label peptide pairs I’m looking for.
Is there anything I'm doing incorrect in maxquant?
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