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The sites of protein-nucleic acid interaction can be determined by mass spectrometric analysis of covalent crosslinks induced between them through exposure to UV light. The method has great potential but has been limited by limited primary yields and the losses during purification. Four methods of crosslink purification were investigated: immobilized metal affinity chromatography, anion exchange, reverse phase and gel filtration; each is promising but requires further refinement, most promisingly in the form of miniaturization.
Even given large quantities of analyte, mass spectrometry's ability to determine exactly which residue entered into a crosslink with nucleic acid is dependent on where the resulting nucleopeptide fragments. Moreover, the resulting spectra obtained can be so complicated as to reveal little, or can simply fail to contain fragmentation data in a manner which unambiguously assigns the point of covalent bonding. An N-terminal sulfonic acid derivatization has the ability to direct peptide fragmentation, which deconvolutes it and generates the icons needed to assign an exact point of crosslinking. This derivatization was attempted with a post-translationally modified phosphopeptide and found to first successfully prevent fragmentation of the modification and second simplify spectral interpretation. Derivatization’s effect remains to be determined for larger modifications such as a dinucleotide photolabel. Even if it is unable to fully prevent modification fragmentation, these results indicate it will significantly deconvolute the peptide’s fragmentation, thus facilitating interpretation of any dinucleotide fragmentation.