Free Solution Oligonucleotide Separation by CE-MS in Acidic Buffers and Positive ESI Ionization

Area of Science:

• Analytical Chemistry
• Biochemistry
• Molecular Biology

Background:

• Oligonucleotide (ON) separation is crucial for molecular biology applications.
• Capillary electrophoresis-mass spectrometry (CE-MS) offers high-resolution analysis.
• Optimizing CE-MS parameters is key for accurate ON characterization.

Purpose of the Study:

• To develop and optimize a CE-MS method for separating synthetic oligonucleotides (ONs) in free solution.
• To investigate the impact of acidic background electrolytes (BGEs) on ON separation selectivity.
• To determine optimal conditions for ionization and detection of ONs with varying lengths.

Main Methods:

• Capillary electrophoresis-mass spectrometry (CE-MS) was employed for ON separation.
• Acidic background electrolytes (BGEs) including formic acid (FA) were utilized.
• Nanospray ionization (NSI) in positive mode and time-of-flight mass spectrometry (TOF-MS) were applied.

Main Results:

• Optimal ON separation and peak shape were achieved at acidic pH 2-2.5.
• Nucleobase acidity constants (pKa) were experimentally determined, guiding pH selection.
• Specific sheath liquid compositions were identified for efficient ionization of short and long ONs.

Conclusions:

• Acidic BGEs significantly enhance separation selectivity for oligonucleotides based on differential protonation.
• CE-MS in positive mode with optimized NSI parameters provides effective separation and detection of diverse ON lengths.
• This optimized method is valuable for analyzing synthetic oligonucleotides in various research applications.