Charge-Reduced Oligonucleotide Mass Spectrometry for Clearer Sequencing
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Solution Overview
Problem
Existing mass spectrometry methods face challenges in sequencing and quantifying highly charged oligonucleotides due to their fragmentation into small fragments and low intensity in mass spectra, exacerbated by charge-state distribution dependence on sequence and LC-MS conditions.
Innovation Solution
A method involving ionization of oligonucleotides to generate negatively charged ions, followed by interaction with positively charged reagent ions to reduce their negative charge state, using protonated species like peptides, within a branched RF ion trap, and subsequent mass selective extraction and fragmentation for analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If highly charged oligonucleotides are analyzed using conventional CID, then sequencing information can be obtained, but the molecules break up into small fragments which renders sequencing difficult
Solution Approach 1:
The invention changes the charge state parameter of the oligonucleotide ions from highly charged (−7 to −9) to lower charged (−3 to −5) through interaction with positively charged reagent ions. This parameter change reduces the excessive fragmentation during CID while maintaining sufficient charge for detection and sequencing.
2Stability of the object's composition
If oligonucleotides with lower charge are used, then fragmentation is reduced, but the intensity in the resultant mass spectrum becomes low
Solution Approach 1:
The invention introduces positively charged reagent ions as an intermediary to transfer charge to the oligonucleotide ions. This mediator enables the conversion from highly charged (low stability) to lower charged (high stability) state, achieving both reduced fragmentation and maintained detection intensity through controlled charge transfer.
3Illumination intensity
If highly charged oligonucleotide ions are produced, then sufficient signal intensity is achieved, but the charge-state distribution depends on sequence and LC-MS conditions which renders quantitation difficult
Solution Approach 1:
The invention changes the charge state parameter from highly charged (−7 to −9) to lower charged (−3 to −5) through reagent ion interaction. This parameter change reduces the sensitivity of charge-state distribution to sequence and LC-MS conditions variations, thereby improving quantitation reliability while maintaining sufficient signal intensity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Reduces the complexity of mass spectra by lowering the negative charge state of oligonucleotides, facilitating efficient sequencing and quantitation with improved signal intensity and reduced fragmentation.
Implementation Method 1
In other embodiments, positively charged reagent ions are generated via electron impact ionization or chemical ionization
Implementation Method 2
each of the positively charged reagent ions comprises at least one protonated species that contributes one or more protons to at least one of the negatively charged oligonucleotide ions. In some embodiments, such transfer of protons can neutralize one or more phosphoric acid groups, thiophosphoric acid groups, or other suitable acid groups of the negatively charged oligonucleotide ions
Data Source
AI summary
In one aspect, a method of performing mass spectrometry is disclosed, which comprises ionizing a plurality of oligonucleotides to generate a plurality of negatively charged oligonucleotide ions, and interacting a plurality of charged reagent ions with the negatively charged oligonucleotide ions to reduce the negative charge state of the negatively charged oligonucleotide ions.