SEPARATION AND CHARACTERIZATION OF BOVINE HISTONE H1 SUBTYPES BY COMBINED ION-EXCHANGE AND REVERSED-PHASE CHROMATOGRAPHY AND MASS-SPECTROMETRY

In order to separate and identify histone H1 subtypes from calf thymus we used both electrospray mass spectrometry (ES-MS) and matrix-assist ed laser desorption ionisation time-of-flight mass spectrometry (MALDI -TOF-MS) after a three-step chromatographic procedure consisting of re versed-phase high-performance liquid chromatography (RP-HPLC), size-ex clusion chromatography (SEC) and ion-exchange chromatography (IEC). Un der the RP-HPLC conditions described, we obtained two baseline-separat ed I-Ii-fractions which were characterised by MALDI-TOF-MS. The determ ined masses ranged from 22 850 to 22 590 for the first fraction and fr om 22 070 to 21 250 for the second fraction. Further, it was shown tha t the first fraction contained at least four and the second one at lea st five subtypes of the histone class H1. Four homogeneous pure H1 sub types were obtained by a combination of IEC followed by SEC and RP-HPL C. The molecular masses of these four subtypes determined by ES-MS wer e 22 606, 22 761, 21 347 and 21 263. We obtained six additional molecu lar masses of histone H1 subtypes from three heterogeneous fractions, namely 22 066, 21 802, 20 586 and 19 817 by ES-MS and 22 800 and 22 67 5 by MALDI-TOF-MS. The retention times of these fractions and the mole cular masses were in agreement with the data obtained from RP-HPLC fra ctions by MALDI-TOF-MS.