Core Concepts
Scientific Fundamentals Explained

Understanding Mass Spectrometry of Oligonucleotides

Oligonucleotide synthesis is a complex process that requires more than one hundred sequential chemical reactions to make a single, 25-base sequence. Contemporary synthesis chemistry is robust and modern synthesis platforms are reliable and highly automated. Still, each oligonucleotide synthesized at IDT is evaluated for quality before shipping to ensure that the correct sequence was made. The best method available to assess compound identity in a high throughput environment is mass spectrometry (MS).

IDT Oligo MALDI-TOF Analysis
 Calculated Molecular Weight: 5,167.4 Measured Molecular Weight: 5,171.9
Figure 1. Routine MALDI-TOF Mass Spectrogram of a 17mer Oligonucleotide.

What is Mass Spectrometry?

In MS analysis, a small amount of a synthesized oligonucleotide is ionized and the ions are propelled into a mass detector/analyzer where molecular weight is measured. The analysis compares the calculated molecular weight of the given sequence to the measured molecular weight. Two methods of mass spectrometry are routinely used. These are MALDI−TOF (matrix-assisted laser desorption ionization−time of flight) and ESI (electrospray ionization) MS. (Read more about these MS methods in Mass Spectrometry Analysis of Oligonucleotide Syntheses in the Technical Reports section of the TechVault).

Interpreting MS Results

MALDI-TOF (Figure 1) and ESI (Figure 2) MS results both have a main peak representing the synthesized oligonucleotide. MS analysis at IDT can detect deletions, additions, or substitutions. IDT will remake oligonucleotides with a significant amount of these products, or with significant secondary peaks. However, there can be, and often are, additional peaks present in the final traces. During MALDI-TOF analysis, depurination of the oligonucleotide can occur as a result of heating (laser ionization) in an acidic environment (the matrix). Depurination also can occur during ESI analysis because of heating in the transport region of the ESI instrument.

IDT Oligo ESI Analysis
 Calculated Molecular Weight: 5,167.4 Measured Molecular Weight: 5,171.9
Figure 2. Routine ESI Mass Spectrogram of a 20mer Oligonucleotide.

Depurination can create secondary peaks having approximately 135 (dA) or 151 (dG) mass units less than the major peak. These species are created by the process as the sample is measured, but are not present in the product itself.

In addition, synthetic oligonucleotides made using phosphoramidite chemistries employ protecting groups on the primary amines in dA, dC, and dG phosphoramidites in order to prevent branching and other undesired side reactions during chain elongation. Protecting groups are cleaved off post-synthetically during the final steps. Incomplete removal of these side groups results in additional masses that are easily detected by MS.

Finally, oligonucleotide modifications add mass to the product. Modifications commonly used in oligonucleotides are well characterized and their masses are taken into account in the final mass spectrograms produced. A list of mass contributions for the most often requested modifications of DNA and RNA oligonucleotides are listed on the IDT Modifications page. Further, the anhydrous molecular weight of both unmodified and modified oligonucleotides can be calculated using OligoAnalyzer.

MS Data Available for IDT Orders

IDT offers MS QC free of charge on all standard oligonucleotides. This QC data is available online for oligonucleotides that were ordered on the website once they have shipped.

To view the data, go to My Account > Order History and find the order for which you would like to see the data. Click on the zip file located under the QC/COA heading for the order you want to see. This file will contain all of the trace files for your order. 

Author: Jaime Sabel is a Scientific Writer at IDT

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