Abstract
Oligonucleotides containing black hole quenchers are widely used as the probes for quantitative and RT-PCR applications. For reproducible and stable PCR results it is essential to use high grade probes, which makes oligonucleotide purification and QC steps important for success. Typical methods, such as HPLC and capillary electrophoresis yield information on overall purity of the probe, while mass spectrometry provides more chemical information hardly accessible by other analytical techniques, such as nature of synthetic failure products, degree of deprotection, presence of deterioration products due to apurinization etc. While MALDI-TOF mass spectrometry is a method of choice for QC of synthetic oligonucleotides it?s application toward modified oligonucleotides containing dark quenchers is limited because of complication of spectra due to photo-dissociation of quencher group during desorption-ionization step. These in-plume photo-decomposition reactions lead to appearance of spectral artifacts interferencing with the actual signals and lead to more propensity for peak broadening at elevated laser powers. Such effects are observable for both nitrogen gas (337nm) and solid Nd-YAG (355nm) lasers typically used in MALDI mass spectrometers. In this work we discussed mechanism of photo-degradation and investigated various ways to minimize it. Short oligonucleotides containing BHQ-1, BHQ-2, and BHQ-3 labels were synthesized for testing purposes. The photo-fragmentation degree was evaluated in presence of various MALDI matrices. Different approaches to quench the exited states of quencher groups during ionization were investigated. That includes collisional cooling by using neutral additives (such as sugars) in order to facilitate energy dissipation from BHQ-groups and application of various organic additives bearing red-ox functionality, which could quench in-plume exited triplet states of azido groups. The novel matrix composition has been developed, which inhibits the photo-fragmentation of BHQ-groups during desorption-ionization step, thus eliminating spectrum artifacts and allows for successful analysis of BHQ-labeled oligonucleotides by MALDI MS.