Determination of accurate protein monoisotopic mass with the most abundant mass measurable using high-resolution mass spectrometry

While recent developments in mass spectrometry enable direct evaluation of monoisotopic masses (Mmi) of smaller compounds, protein Mmi is mostly determined based on its relationship to average mass (Mav). Here, we propose an alternative approach to determining protein Mmi based on its correlation with the most abundant mass (Mma) measurable using high-resolution mass spectrometry. To test this supposition, we first empirically calculated Mmi and Mma of 6158 Escherichia coli proteins, which helped serendipitously uncover a linear correlation between these two protein masses. With the relationship characterized, liquid chromatography–mass spectrometry was employed to measure Mma of protein samples in its ion cluster with the highest signal in the mass spectrum. Generally, our method produces a short series of likely Mmi in 1-Da steps, and the probability of each likely Mmi is assigned statistically. It is remarkable that the mass error of this Mmi is as miniscule as a few parts per million, indicating that our method is capable of determining protein Mmi with high accuracy. Benefitting from the outstanding performance of modern mass spectrometry, our approach is a significant improvement over others and should be of great utility in the rapid assessment of protein primary structures.