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

Abstract While recent developments in mass spectrometry enable direct evaluation of monoisotopic masses ( M mi ) of smaller compounds, protein M mi is mostly determined based on its relationship to average mass ( M av ). Here, we propose an alternative approach to determining protein M mi based on its correlation with the most abundant mass ( M ma ) measurable using high-resolution mass spectrometry. To test this supposition, we first empirically calculated M mi and M ma 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 M ma of protein samples in its ion cluster with the highest signal in the mass spectrum. Generally, our method produces a short series of likely M mi in 1-Da steps, and the probability of each likely M mi is assigned statistically. It is remarkable that the mass error of this M mi is as miniscule as a few parts per million, indicating that our method is capable of determining protein M mi 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.