Ion Mobility-Mass Spectrometry to Augment Middle-Down Fragmentation Coverage
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Authors
Satish Reddy, Rachitha
Issue Date
2025
Type
Thesis
Language
en_US
Keywords
CIDtims , Ion Mobility Spectrometry , LCMS/MS , Middle-down , Proteomics , Trapped Ion Mobility Spectrometry
Alternative Title
Abstract
Proteomics aims to characterize all proteins in a sample including their identification, quantification and any chemical modifications. Proteins are the primary functional molecules in all living organisms carrying out many processes necessary for life. To respond to external stimuli and accomplish these tasks, proteins are strictly regulated and a single gene will give rise to multiple proteoforms each possessing variability due to alternative splicing, sequence variations, and/or post-translational modifications (PTMs). Understanding this protein diversity helps us uncover how cells function, the mechanism of disease, and aid in the development of drug design.Mass spectrometry is a powerful tool and can rapidly identify and characterize proteins with high sensitivity and provide detailed information on the PTM status of proteins. There are three main mass-spectrometry proteomics approaches, and they are bottom-up, middle-down and top-down methods. Bottom-up is a well-developed and high-throughput technique involving the digestion of proteins into peptides and separation of the peptides by liquid chromatography and analysis by mass spectrometry. While bottom-up proteomics is highly mature and sensitive, there are frequent gaps in the sequence coverage due to the digestion process generating very short peptides that go undetected. Additionally, reliance on digestion can result in the inability to assign concurrent PTM modifications making it an unreliable method for technique for the analysis of proteoforms.
In middle-down analysis the protein is partially digested to generate larger peptides and subunits. Middle-down approach is able to preserve proteoform information better than bottom-up and assign concurrent PTM’s. This makes it an excellent option to study both mid-size proteins and big proteins like antibodies. Although the middle-down approach sounds promising, the larger peptides result in highly complex tandem mass spectra, overlapping product ions, decreased signal to noise ratios, and reduced fragmentation coverage when compared to bottom-up.
Ion mobility spectrometry (IMS) techniques have been successful in deconvoluting overlapping product ions, enabling their facile annotation. Trapped ion mobility spectrometry is a type of IMS which offers great resolution. The benefits of pairing collision-induced dissociation and this IMS technique to generate and separate product ions derived from protein ions has been extensively studied but its application to middle-down workflows has yet to be investigated. In this work, we utilize CIDtims to sequence middle-down peptides and subunits. We assess five proteins ranging in mass from 8.6 kDa ubiquitin to a 150 kDa immunoglobulin G. While the benefits for small proteins like cytochrome c and ubiquitin were minimal, increases in sequence coverage of 43.6%, 24.5%, and 39.3% were measured for myoglobin, carbonic anhydrase, and NIST antibody, respectively. We can conclude that CIDtims is an effective method to improve fragmentation coverage by middle-down approach.