Abstract
Proteomics has exposed a plethora of posttranslational modifications, but demonstrating functional relevance requires new approaches. Top-down proteomics of intact proteins has the potential to fully characterize protein modifications in terms of amount, site(s), and the order in which they are deposited on the protein; information that so far has been elusive to extract by shotgun proteomics. Data acquisition and analysis of intact multimodified proteins have however been a major challenge, in particular for positional isomers that carry the same number of modifications at different sites. Solutions were previously proposed to extract this information from fragmentation spectra, but these have so far mainly been limited to peptides and have entailed a large degree of manual interpretation. Here, we apply high-resolution Orbitrap fusion top-down analyses in combination with bioinformatics approaches to attempt to characterize multiple modified proteins and quantify positional isomers. Automated covalent fragment ion type definition, detection of mass precision and accuracy, and extensive use of replicate spectra increase sequence coverage and drive down false fragment assignments from 10% to 1.5%. Such improved performance in fragment assignment is key to localize and quantify modifications from fragment spectra. The method is tested by investigating positional isomers of Ubiquitin mixed in known concentrations, which results in quantification of high ratios at very low standard errors of the mean (<5%), as well as with synthetic phosphorylated peptides. Application to multiphosphorylated Bora provides an estimation of the so far unknown stoichiometry of the known set of phosphosites and uncovers new sites from hyperphosphorylated Bora.
Highlights
electron transfer dissociation (ETD) fragmentation reveals the presence of positional isomers. For proteins up to 40 kDa these positional isomers can accurately be quantified. For in-vitro phosphorylated BoraNT a wide array of positional isomers is revealed. Use of Fragment ion false discovery rate (FDR) levels improve the quality of extracted stoichiometries
Excellent work has already been done on developing algorithms capable of extracting the sequence identity of proteins from fragmentation spectra from full LCMS runs, with well-described approaches to control the false discovery rates (FDRs) at the spectrum, protein, isoform, and proteoform level, and additional algorithms are proposed to facilitate the identification of many different Posttranslational modifications (PTMs)
False positives cannot be readily excluded and, to our knowledge, FDR calculation at the fragment ion level was not addressed in standard top-down MS software
Summary
In Brief Here we determine the quantities of the same protein modified differentially by PTMs, creating so-called positional isomers (i.e., double phosphorylation, one isomer with positions Y64 and T98, and one isomer with positions Y64 and Y120). Even though the multiplexed spectra are hard to interpret, they provide an opportunity to quantify the different positional isomers provided the set of positional isomers can be mass selected without interferences from other proteoforms, and the used fragmentation method does not induce structural biases in the fragment ion intensity depending on the position of the modification This requires an ideal fragmentation technique that does not incur modification losses that potentially lead to unspecific effects in the detected intensity levels of the fragment peaks, lowering the precision of the calculations or potentially even obfuscating the presence of the positional isomers. Such knowledge can help to direct and limit mutation studies to relatively few positions to help determine the biological relevant combination of sites
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