Relative quantitation of oligonucleotides and impurities is commonly measured by LC-UV trace peak integration. Oftentimes multiple oligonucleotides elute within the same integrated UV peak, complicating UV quantitation. An example is shown below, with multiple coeluting oligonucleotide impurities.
The Byos UV-MS Report Tab uses the Deconvoluted MS Intensity ratio to approximately quantitate multiple oligonucleotides observed in the same UV peak. This can be observed in the UV-MS column in the Report tab. An example UV-MS Report Tab is shown below, calculating relative abundance of a 40-mer full length product and several observed impurities for two replicate samples.
The UV-MS Report Tab rows are divided into three levels. First by datafile (Sample name column 1), second by UV integrated peak for each sample (Peak # column 2), and third by deconvoluted assigned masses in each UV Peak (Oligonucleotide name, Oligo Candidate, and Delta name columns 8, 9 and 10). Only assigned deconvoluted masses are included in the UV-MS Report Tab and MS Ratio% calculation (discussed in detail below).
Four columns are included for quantitation: Normed area %, Intensity, MS Ratio%, and UV-MS.
- Normed area % is the percent Area of a UV peak relative to the measured total area of all other peaks in the sample’s UV trace. The measured total area is limited by the Global trace peak filters Show peaks within range retention time limit.
- Intensity is the height of a deconvoluted mass peak.
- MS Ratio% is an assigned mass peak Intensity divided by the sum of all assigned masses' Intensity in the same UV peak.
- Finally, UV-MS is the product of Normed area % of a UV peak and the MS Ratio% of an assigned mass observed in the UV peak.
UV Peak # 1 in the 40-mer Rep 1 sample is used as an example to explain how UV-MS is calculated.
First, MS Ratio% is calculated for each assigned mass in UV Peak # 1. The formula below shows how MS Ratio% is calculated for the Agilent 40-mer [24-40] impurity mass assignment:
Second, UV-MS is calculated for each mass assigned to UV Peak # 1. The formula below shows how UV-MS is calculated for the Agilent 40-mer [24-40] impurity mass assignment:
Adding the UV-MS Report Tab:
As of Byos v5.4, the UV-MS Report Tab is not yet included as a preset. Please reach out to support@proteinmetrics.com for access. Direct access from Byos and further enhancements to the report template will be available in future releases.