Developing Free Radical Initiated Peptide Sequencing towards the Analysis of Complex Mixtures
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Authors
Osho, Kemi Eunice
Issue Date
2025
Type
Dissertation
Language
en_US
Keywords
Alternative Title
Abstract
Proteomics characterization of complex samples is often employed to determine protein conformation and functions. Tandem mass spectrometry (MS/MS) is one of the predominate techniques for providing information on the amino acid sequence and the site of protein post-translational modification, where free radical initiated peptide sequencing (FRIPS) is an example. However, with unwanted neutral losses, complex mixture analysis can be challenging because ion abundance can be shuttled away from sequence-informative fragment ions, leading to decreased ion abundance and reduced sequence coverage. To be employed in more complex systems and proteomics workflows, the efficiency of sequence ion generation must be improved for multiply charged ions, which are frequently generated in proteomics workflows. This dissertation presents the development of radical-driven MS/MS (FRIPS) for improved peptide characterization towards the analysis of complex mixture. There have been different forms of radical-driven tandem mass spectrometry (MS/MS) techniques ranging from UV photodissociation of photolabile radical precursor (UVPD), UV photoexcitation of peptide cations (UVPE), among others. However, the radical-based method employed for this dissertation often displays distinctive benefits such as low cost (requires no extra instrumentation), unique thermal stability, and ability to examine singly and even negatively charged peptides when compared with electron-based techniques. By considering peptide radical ion fragmentation of specific PTMs, FRIPS MS/MS has often been seen as one of the most efficient techniques, able to couple to a proteomic setup without difficulty. Although several studies deal with the analysis of FRIPS-MS, few research reports the application of the para-FRIPS tag for peptide characterization. Importantly, no research has reported using negative mode collision induced dissociation-trapped ion mobility spectrometry (CIDtims) coupled with FRIPS-MS for peptides and phosphorylated peptide analysis. The para-TEMPO-FRIPS reagent allows for electron delocalization and steric protection during the reaction. The work carried out in this dissertation is expected to open new research horizons, particularly in the analysis of complex mixtures, including post-translational modification, protein pharmaceutical development, biological, and physiological processes. In addition, through this peptide backbone dissociation technique, the gas-phase digest of large PTMs-containing peptides can be examined.
In chapter 2, we demonstrate that free radical initiated peptide sequencing (FRIPS) as a radical-driven technique improves the performance of positive-ion mode peptide characterization. This is done through the comparison of the dissociation behavior of the ortho- and para-FRIPS tags. These tags are chemical reagents that when conjugated to a peptide/protein, can facilely degrades into radicals upon activation. Results from this chapter reveal that compared to the commonly utilized ortho-FRIPS tag, the para counterpart overcomes high abundance of neutral losses, increase sequence coverage, and can be a promising tool in FRIPS-MS workflow.
In FRIPS-MS, all product ions can be generated, so the complexity of FRIPS fragmentation requires the use of high-resolution mass spectrometers which are often slow. Our hypothesis is that ion mobility spectrometry (IMS) could be an alternative method for deconvoluting these spectra on a high-throughput mass spectrometer. In chapter 3, we investigate the utility of ion mobility spectrometry-mass spectrometry (IMS-MS) to assist in para-TEMPO-Bz FRIPS-based fragmentation by increasing the Δ6 direct current (DC) voltage and controlling the pressure within a trapped ion mobility spectrometry (TIMS) device. We demonstrate that a recently developed method from our group, collision induced dissociation-trapped ion mobility spectrometry “CIDtims” can initiate the homolytic cleavage of the FRIPS precursor. We then examine if the resultant ion mobility separation results in additional assignments of product ions and improved sequence coverage. We demonstrate that activation within the TIMS device promotes robust radical initiation and fragmentation of peptide cations. The generated product ions are mobility separated, enabling facile assignment and increased sequence coverage.
PTMS can be labile in positive mode analyses and efficient localization of them may require negative-mode analyses although our technique “CIDtims” has not been tested in negative mode. In chapter 4, we demonstrate that activation within the TIMS device can be further extended in the negative ion mode. Here, IMS activation was employed prior to the collisional activation step, enabling the radical initiation in doubly protonated and deprotonated peptides and in phosphorylated peptide anions within a trapped-ion mobility spectrometry device. This process does indeed promote robust radical initiation of deprotonated peptide. Generated radical species are subjected to isolation by the quadrupole and further activated in the collision cell. In negative mode, this two-step activation enables increased sequence coverage of examined peptides. Promisingly, the ion-mobility-assisted fragmentation technique can be developed as a new pseudo-MS3 workflow for applying FRIPS-based PTMs proteomics in negative mode.
From the studies described in this dissertation, in the future, we will examine the ability of this TOF-based FRIPS workflow to annotate the location of labile post-translational modifications. We will also investigate whether the “CIDtims” can be coupled with liquid chromatographic separations and utilize this FRIPS-LC workflow to annotate post-translational modification in complex mixtures.