Electrospray ionization fourier transform ion cyclotron resonance analysis of large polymerase chain reaction products

Abstract

In previous work, we demonstrated the use of electrospray ionization to analyze small differences in size or sequence of relatively small polymerase chain reaction (PCR) products of 114 base pairs or less. The sequence information required to answer a biological question may be only a single nucleotide substitution or deletion. In many cases, the regions where these sequence variations can occur are several hundred base pairs in length, and the analysis of large PCR products is therefore desirable. Therefore, we have attempted to expand the size range of PCR products that can be analyzed by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Previous work has shown that the difficulties associated with PCR product analysis increase with product size. A revised cleanup scheme was employed to target the removal of detergents with ethanol wash or precipitation steps, followed by additional desalting. Additionally, an in-trap cleanup to collisionally induce dissociation of noncovalent salt adducts was employed. This approach was extended to a 223 base pair PCR product yielding mass measurement accuracy within 26 ppM. The mass measurement accuracy obtained illustrates that a single base substitution could be identified at this size of PCR product with a 7 tesla ESI-FTICR.