A fast protein liquid chromatography method for purification of myoglobin from different species

The aim of this study is to describe a fast method for the purification of high-purity myoglobin for Mass Spectroscopy analyses and to use it as standard-grade material. A three-step Fast Protein Liquid Chromatography (FPLC) method was used to produce high-purity myoglobin. SEC 650 gel filtration followed by an Enrich Q anion exchange chromatography was used to produce myoglobin in acceptable purity for most research methods. A second filtration step was carried out by narrow field SEC 70 gel to prepare high-purity myoglobin at standard grade purity and capable of Mass Spectroscopy analyses. At least 90% pure myoglobin was obtained by applying two chromatography steps in the samples of three species, and over 99% pure myoglobin was obtained in standard material quality and suitable for mass spectroscopy when the additional narrow field SEC 70 chromatography step was applied. The proposed method provides higher purity compared to other methods and can be applied in a shorter time. FPLC columns significantly reduce the duration of the chromatography steps. At the same time, the use of solid extraction columns instead of dialysis reduced the long overnight dialysis process to a few minutes.