Capillary Electrophoresis-Electrospray Ionisation-Mass Spectrometry (CE-ESI-MS) for PTM Analysis of H1 Histones: Strengths and Limitations

摘要

The use of M7C4I as a less positively charged coating than the PEI coating and the reduction of separation voltage from 25 kV to 12kV yielded both an increased separation window from 3 min to 13 min and an increased number of modified + unmodified peptides. Using these improved separation conditions the analysis of 300 fmol H1 histones peptides with CE-ESI-MS showed roughly the same number of peptides as a 30 pmol sample applied to LC-ESI-MS. Using IMAC, a substantial increase in the number of identified phosphopeptides could be observed. In total 44 modification sites could be identified on different histone H1 subtypes, 25 by both methods, 8 solely by CE- and 10 solely by LC-ESI-MS. Therefore, CE interfaced to MS via the sheathless high sensitivity porous sprayer can be considered as a complementary technique to nanoLC-ESI-MS for analysis of posttranslational modifications of linker histones.