Accurate Mass Differences of High Molecular Weight Unknown Compounds from Resolved Isotopic Distributions:...

Determination of the most likely candidate formulas of high molecular weight (HMW) unknown compounds by high-resolution mass spectrometry remains elusive. Several hundred thousand formulas are possible for HMW compounds such as oligonucleotides (e.g., 7000-8000 Da for a typical 20-mer antisense oligonucleotide). Implementation of the concept of the mass difference (ΔM) from a known reference compound has been greatly successful in the reduction of the total number of possible formulas by more than 3 orders of magnitude. However, determination of the accurate ΔM between isotopic peak distributions of HMW compounds is not trivial. This is because there are significant dilemmas in the use of the most abundant mass (MAbM) and the monoisotopic mass in the two distributions to accurately determine ΔM. Reliable selection of the MAbM peak is not possible due to uncertainties associated with the experimental ion signal fluctuation of the measurement. Also, reliable selection of the correct monoisotopic peak is not possible due to potential adjacent interfering peaks from other compounds. In this work, the challenges associated with the determination of accurate mass differences between isotopic distributions have been addressed by the development of two separate and equivalent approaches. An approximate ΔM is initially determined from the peak tops of the smoothed isotopic distributions as input to the two accurate methods. The first method uses the mass centroids of the isotope pairs in the two distributions separated by an empirical mass interval. The second is based on the combinatorial use of the mass centroids of all isotopes in the two distributions. The two methods produced equivalent mass accuracy, but the second has the advantages of simplicity and universality and no need for an empirical mass interval.