Abstract
It has long been an analytical challenge to accurately and efficiently resolve extremely dense overlapping isotopic envelopes (OIEs) in protein tandem mass spectra to confidently identify proteins. Here, we report a computationally efficient method, called OIE_CARE, to resolve OIEs by calculating the relative deviation between the ideal and observed experimental abundance. In the OIE_CARE method, the ideal experimental abundance of a particular overlapping isotopic peak (OIP) is first calculated for all the OIEs sharing this OIP. The relative deviation (RD) of the overall observed experimental abundance of this OIP relative to the summed ideal value is then calculated. The final individual abundance of the OIP for each OIE is the individual ideal experimental abundance multiplied by 1 + RD. Initial studies were performed using higher-energy collisional dissociation tandem mass spectra on myoglobin (with direct infusion) and the intact E. coli proteome (with liquid chromatographic separation). Comprehensive data at the protein and proteome levels, high confidence and good reproducibility were achieved. The resolving method reported here can, in principle, be extended to resolve any envelope-type overlapping data for which the corresponding theoretical reference values are available.
Highlights
We report an alternative resolving method for Overlapping isotopic envelopes (OIEs) based on exact isotopic envelopes computed from the elemental composition of the product ions’ actual amino acids
An overlapping isotopic peak (OIP) is an experimental isotopic peak with its m/z values matched by theoretical isotopic peak m/z values from two or more product ions within the m/z tolerance
An experimental isotopic envelope is considered an ideal isotopic envelope if all of its experimental isotopic peaks are observed and their m/z and relative abundance are within the tolerance of IPMD and IPAD, respectively
Summary
We report an alternative resolving method for OIEs based on exact isotopic envelopes computed from the elemental composition of the product ions’ actual amino acids. This method is in contrast to the strategies existing in published work that are used to determine the theoretical isotopic envelopes, which are composed of Averagine units. The final individual abundance of the OIP in each OIE is defined as its ideal experimental abundance multiplied by 1 + R D This method has been implemented in our automated intact protein database search engine ProteinGoggle[44] with user-friendly graphical user interfaces. The method reported here is expected to perform well with the tandem mass spectra of small peptides and better with the tandem mass spectra of large proteins in comparison with the current methods reported in the literature
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