Over the course of the semester, our trainees are reviewing webinars in their given fields and preparing abstracts to help colleagues outside their discipline make an informed choice about watching them. As our program bridges diverse disciplines, these abstracts are beneficial for our own group in helping one another gain key knowledge in each other’s fields. We are happy to share these here for anyone else who may find them helpful.
Dr. John Yates III. Scripps Research Institute ; Dr. Kosuke Ogata. University, Kyoto
March 10, 2021
The high complexity of the human proteome compared to the genome can be explained by post-translational modifications (PTMs) of signaling peptides and proteins. These PTMs include covalent addition of molecules, partial degradation, enzymatic cleavages, rearrangements, etc. These modifications result in functional diversity of translational products, protein folding, etc. — hence, they are very important in health and disease pathology. Identification of these modifications and their respective functional roles is essential in understanding cellular events.
In this webinar, Dr. John Yates, and Dr. Kosuke Ogata discussed advances in mass spectrometry approaches used in identifying post-translational modifications. Dr. Yates explained how post-translational modification can affect the fate of a cell using the cancer paradigm (protein misfolding in cancerous cells). He also elaborated on how genomic plasticity occurring in coding sequence can lead to gain or loss of protein function. He asked, “Can a single point mutation transform a normal cell to a malignant cell?”
In answering the question, they employed a covalent protein painting method, where different isotope of dimethyl tags was used to label intact protein (light labeling) and tryptic digest (heavy labeling). This procedure allows the identification of protein-accessible residues since the light labeling of the intact protein only tags surface-accessible lysine residues. A single mutation introduced into normal breast epithelial cells leads to the generation of a tumorigenic cell line (Hart et al).
After the alteration, changes in the genome, as well as the proteome, were monitored using mRNA profiling techniques and the covalent painting methods described above. Cytoskeletal proteins such as actin filament and microtubules were seen to be disrupted which may be because of misfolding. These observations probably explain why cell remolding occurs after point mutation. Although their approach was successful in measuring protein misfolding, peptide sequence coverage for protein identification was low.
A Bruker TIMS TOF Pro equipped with the 4th dimension of separation (trapped ion mobility) and a PaSEF technology was used to improve the labeled protein coverage. Here, a machine learning algorithm was developed to predict the collisional cross-section (CCS) of the peptides. The addition of CCS values (ion mobility) in peptide identification significantly increased the number of peptides, peptides coverage for proteins, and the overall confidence in PTM identification. Specifically, the latter approach increased peptide coverage by approximately 24% for unmodified peptides, 15% for PTM-modified peptides, and 104% for phosphate PTMs.
Dr. Kosuke focused his talk on how peptide ion mobility separation can improve phosphate PTM measurements. First, he evaluated the effectiveness of ion mobility separation in discriminating between the contaminant and endogenous phosphopeptides. Using tandem mass tag (TMT) quantitative assay, phosphorylated E. coli peptides were easily distinguished from human HeLa cell phosphopeptides after incorporating trapped ion mobility in analysis. Additionally, the same peptides with phosphorylation at different amino acid residues were successfully resolved by the addition of ion mobility separation.
The effect of peptide phosphorylation on its CCS was also evaluated. Here, a linear regression model was developed to predict the CCS of phosphopeptides. In theory, an increase in the mass of peptides after phosphorylation should result in an increase in CCS. However, one-third of the phosphopeptides showed an opposite trend. Further analysis revealed that the relative position of the phosphorylation can impact the CCS, thus phosphorylation near the C or N terminal sites increases compaction through intramolecular interaction. Also, the number of basics groups and the distance between the basic and phosphate group affect peptides’ size. The degree of compactness of phosphopeptides increases with increasing distance between basic and phosphate as well the number of basic sites.
In summary, this webinar demonstrates the improved capabilities of mass spectrometry in PTM identification when combined with ion mobility separations. (E.g., TIMSTOF Pro). This new technology can be employed in neuroproteomics and peptidomics to effectively unravel post-translational modifications and their effects on neuronal function.