Abstract
Epigenetic regulation of chromatin is dependent on both the histone protein isoforms and state of their post-translational modifications. The assignment of all post-translational modification sites for each individual intact protein isoform remains an experimental challenge. We present an on-line reversed phase LC tandem mass spectrometry approach for the separation of intact, unfractionated histones and a high resolution mass analyzer, the Orbitrap, with electron transfer dissociation capabilities to detect and record accurate mass values for the molecular and fragment ions observed. From a single LC-electron transfer dissociation run, this strategy permits the identification of the most abundant intact proteins, determination of the isoforms present, and the localization of post-translational modifications.
Highlights
Over the last two decades, the integration of the unanticipated discovery of new modes of internal energy deposition and rapid technological progress in mass spectrometrybased instrument platforms has revolutionized our experimental capabilities to effectively study the inherent complexity of human and other mammalian proteomes. This includes the rapid identification of proteins, the detection and unambiguous assignment of protein isoforms, and the detection and localization of post-translational modifications (PTMs)
Considerable use for many purposes, this common strategy has a variety of limitations especially from the view of acquiring the information needed to establish and understand biological function. These limitations include the loss of information regarding the nature of protein isoforms and a loss of knowledge of the global presence of multiple PTMs that can co-occur at differing sites on the same protein molecule
With the electron transfer dissociation (ETD) fragmentation implemented on an Orbitrap mass analyzer [28], we were able to record high resolution data for both the molecular ions and the fragment ions that are necessary for the experiments carried out at the intact level
Summary
Over the last two decades, the integration of the unanticipated discovery of new modes of internal energy deposition and rapid technological progress in mass spectrometrybased instrument platforms has revolutionized our experimental capabilities to effectively study the inherent complexity of human and other mammalian proteomes This includes the rapid identification of proteins, the detection and unambiguous assignment of protein isoforms, and the detection and localization of post-translational modifications (PTMs).. Considerable use for many purposes, this common strategy has a variety of limitations especially from the view of acquiring the information needed to establish and understand biological function These limitations include the loss of information regarding the nature of protein isoforms and a loss of knowledge of the global presence of multiple PTMs that can co-occur at differing sites on the same protein molecule. The sequence identification for fragment ion spectra representing peptides with modifications that are unanticipated can be difficult to assign using common search algorithms
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