Abstract
The emergence of efficient fragmentation methods such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) provides the opportunity for detailed structural characterization of heavily covalently modified large peptides and small proteins such as intact histones. Even with effective gas phase ion isolation so that a single molecular precursor ion is selected, the MSMS spectrum of a heavily modified peptide may reveal the presence of a mixture of peptides with the same amino acid sequence and the same total number of posttranslational modification (PTM) moieties (same PTM composition) but with different PTM configurations or site-specific occupancy isoforms. Currently available data analysis methods depend on a deisotoping procedure, which becomes less effective when spectra (fragmentation patterns) contain many overlapping isotopic distributions. Peptide database search engines can only identify the most abundant PTM configuration (PTM arrangement on different residues) in such mixtures. To identify all the PTM configurations present in these mixtures and to estimate their relative abundances, we extended our fragment assignment by visual assistance program to search for ions representing all possible configurations, subjected to the total PTM composition constraint. This resulted in the identification of PTM configurations supported by unique fragment ions, and their relative abundances were estimated by use of a non-negative least squares procedure.
Highlights
The emergence of efficient fragmentation methods such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) provides the opportunity for detailed structural characterization of heavily covalently modified large peptides and small proteins such as intact histones
Characterization of large peptides and intact proteins by MSMS has initially been driven by the need for determination of complex patterns of PTMs1 on histones
The relative abundance ratio of configurations 0310 and 0220 is about 3:16, which agrees with the intensity ratio of c13(3Me) and c13(2Me) triply charged ions
Summary
Core histones were extracted from human 293 cells by use of a standard isolation protocol [17]. Raw ETD spectra acquired in the orbitrap were converted to monoisotopic peak lists (M ϩ H format) with the Xtract program (Thermo Fisher Scientific, Bremen, Germany). The raw data of ETD spectra are converted to monoisotopic peak lists by use of Xtract (part of the Xcalibur software from Thermo Fisher Scientific, San Jose, CA). The database search results (peptide sequence and type and number of covalent modifications) allow for generation of all possible PTM candidate configurations, and FAVA code is used to extract non-redundant ions from the original raw ETD data. RPLC fractionation of a Glu-C digest of histone H3 from human HEK-293 cells produced a fraction containing a set of molecular ions whose molecular masses match a peptide of sequence “LATKAARKSAPATGGVKKPHRYRPGTVALRE” with an additional zero to five “14-Da moiety” groups (Fig. 2)
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