Abstract
Here we present a novel methodology for the identification of the targeted post-translational modifications present in highly modified proteins using mixed integer linear optimization and electron transfer dissociation (ETD) tandem mass spectrometry. For a given ETD tandem mass spectrum, the rigorous set of modified forms that satisfy the mass of the precursor ion, within some tolerance error, are enumerated by solving a feasibility problem via mixed integer linear optimization. The enumeration of the entire superset of modified forms enables the method to normalize the relative contributions of the individual modification sites. Given the entire set of modified forms, a superposition problem is then formulated using mixed integer linear optimization to determine the relative fractions of the modified forms that are present in the multiplexed ETD tandem mass spectrum. Chromatographic information in the mass and time dimension is utilized to assess the likelihood of the assigned modification states, to average several tandem mass spectra for confident identification of lower level forms, and to infer modification states of partially assigned spectra. The utility of the proposed computational framework is demonstrated on an entire LC-MS/MS ETD experiment corresponding to a mixture of highly modified histone peptides. This new computational method will facilitate the unprecedented LC-MS/MS ETD analysis of many hypermodified proteins and offer novel biological insight into these previously understudied systems.
Highlights
We present a novel methodology for the identification of the targeted post-translational modifications present in highly modified proteins using mixed integer linear optimization and electron transfer dissociation (ETD) tandem mass spectrometry
An important aspect of the proposed framework is that chromatographic information is used to correlate the modification states as a function of modification position, mass, and time
The proposed computational framework is applied to an entire LC-MS/MS ETD experiment corresponding to a mixture of highly modified histone peptides to demonstrate its utility
Summary
We present a novel methodology for the identification of the targeted post-translational modifications present in highly modified proteins using mixed integer linear optimization and electron transfer dissociation (ETD) tandem mass spectrometry. The separation must be conducted off line because the mobile phase additives used are non-volatile components, and subsequent fractionation is necessary for mass spectrometric analysis This protocol has made it possible to analyze the first 50 amino acids of the N-terminal tail of histone H3 and provided important insight regarding connectivity information between the modification sites. Unprecedented separation of the modified histone forms is achieved within a single LC-MS/MS ETD experiment, thereby introducing important chromatographic information that can be utilized in the subsequent identification and quantification of these post-translational modifications. Computational methodologies that utilize the extensive and complementary information contained within these LCMS/MS data sets are nonexistent as the technology has only recently been developed
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