Sequencing of oligonucleotides by mass spectrometry

A method of determining a 3′ terminal base sequence of a parent nucleic acid, the method comprising:

• obtaining a first tandem mass spectrum of said parent nucleic acid using a first collision energy;
• determining an ion abundance of each product ion of interest from said first mass spectrum above a predetermined ion abundance;
• obtaining a second tandem mass spectrum using a second collision energy that is different from said first collision energy;
• determining an ion abundance of each product ion of interest from said second mass spectrum above said predetermined ion abundance;
• determining an ion ratio defined by the formula:
• ion ratio=(ion abundance of said product ion of interest in a mass spectrum having a higher collision energy)/(ion abundance of said product ion of interest in a mass spectrum having a lower collision energy);
• determining a mass-to-charge ratio of an ion of interest having a high ion ratio;
• and
• comparing the obtained mass-to-charge ratio to a predetermined mass-to-charge ratio value associated with a known 3′ terminal base sequence, wherein the mass-to-charge ratio of the ion of interest being substantially equal to the predetermined mass-to-charge ratio value associated with a known 3′ terminal base sequence provides an indication of the 3′ terminal base sequence.

A method of sequencing a parent nucleic acid having a known mass, the method comprising:

• obtaining a first tandem mass spectrum of the parent nucleic acid using a first collision energy;
• determining a mass-to-charge ratio of a smallest [w] ion from said mass spectrum so as to provide a known partial 3′ terminal base sequence;
• adding a first hypothetical base to said smallest [w] ion to provide a hypothetical second smallest [w] ion;
• determining a hypothetical mass-to-charge ratio of said hypothetical second smallest ion;
• and
• comparing the hypothetical mass-to-charge ratio of said hypothetical second smallest ion to the actual mass-to-charge ratios obtained from said first tandem mass spectrum, wherein when said first hypothetical base causes the hypothetical mass-to-charge ratio of the hypothetical second smallest [w] ion to be substantially equal to an actual mass-to-charge ratio in said spectrum, said parent nucleic acid has a sequence that comprises the first hypothetical base plus the known partial 3′ terminal base sequence.

A method for sequencing a nucleic acid comprising:

• obtaining a mass spectrum of the nucleic acid using a first collision energy;
• determining a first partial 3′ terminal base sequence of said nucleic acid from a first [w] ion;
• combining a first internal ion from said mass spectrum to said first [w] ion to provide a first hypothetical [w] ion;
• determining a first hypothetical mass-to-charge ratio of said first hypothetical [w] ion, wherein the charge of the first hypothetical [w] ion is one;
• comparing the first hypothetical mass-to-charge ratio of said first hypothetical [w] ion to actual mass-to-charge ratios obtained from said mass spectrum, wherein when said internal ion causes said first hypothetical mass-to-charge ratio of said first hypothetical [w] ion to be substantially equal to an actual mass-to-charge ratio in said spectrum, said nucleic acid has a sequence that comprises the first hypothetical internal ion plus the first partial 3′ terminal base sequence.

A method for sequencing a nucleic acid comprising:

• obtaining a first tandem mass spectrum of the nucleic acid using a first collision energy;
• determining a first partial 5′ terminal base sequence of said nucleic acid;
• combining a first internal ion from said mass spectrum with said 5′ terminal base sequence to form a first hypothetical [a-Base] ion;
• determining a first hypothetical mass-to-charge ratio of said first hypothetical [a-Base] ion;
• and
• comparing the first hypothetical mass-to-charge ratio of said first hypothetical [a-Base] ion to actual mass-to-charge ratios obtained from said mass spectrum, wherein when said first internal ion causes said first hypothetical mass-to-charge ratio of said first hypothetical [a-Base] ion to be substantially equal to an actual mass-to-charge ratio in said spectrum, said nucleic acid includes a sequence that comprises the first hypothetical internal ion plus the 5′ terminal base sequence.