Ion trap tandem mass spectrometry applied to small multiply charged oligonucleotides with a modified base

Two isomeric oligodeoxynucleotide hexamers, 5′-d(N-6meATGCAT)-3′ and 5′-d(ATGSmeCAT)-3′, were subjected to analysis by electrospray and ion trap mass spectrometry. In the case of the isomer with a modified adenine, location of the modified base in the sequence was straightforward and a triple mass spectrometry experiment provided information on the identity of the modification. In contrast, the isomer with the methylated cytosine did not yield definitive information on the location or identity of the modification. Tandem mass spectrometry data in this case could indicate that the modification was present on either the third or fourth nucleoside. The two isomers represent extremes in the facility with which modified bases can be identified and located in a small oligonucleotide via multiple mass spectrometry of multiply charged anions. A preference for loss of particular bases strongly influences which structurally diagnostic ions are formed upon collisional activation. The likelihood for locating and identifying a modified base is dependent, therefore, upon the likelihood that the base is lost directly from the parention.