Mindy I. Davis Ph.D. National Institute of Health

New Method for Optimization of Fragmentation Parameters Could Be Applied to any Protein <30 kDa

Kinases are involved in a whole host of biological processes, so there is an interest in accurately determining the sites these enzymes phosphorylate. One method that has been used to study these post-translational modifications is digestion of the protein substrate into peptides followed by mass spectrometry analysis. While this method can identify phosphorylation sites, it is difficult to be able to ascertain when multiple sites are present and temporally phosphorylated, which sites may be present at the same time on a given substrate. The method of top-down analysis described herein allows an intact protein to be used and fragmented in the Orbitrap Fusion instrument, an instrument that combines quadrupole, linear ion trap, and Orbitrap mass analyzers. A limitation of top-down analysis can be insufficient sequence coverage due to incomplete protein fragmentation. The authors here tested multiple single and sequential methods of fragmentation to determine whether there was a benefit to a combined method. The 17.5 kDa N-terminal protein Bora, a substrate of Aurora A (AurA) and Polo-like kinase 1 (PLK1), was studied here. They found that sequential electron transfer dissociation (ETD) then high-energy collisional dissociation (HCD) in a single event, termed EThcD, provided the best coverage of Bora (∼75%) and preserved labile PTM sites. The authors tested two time points and showed that Aurora A phosphorylated Bora up to three times while extending the time to 2 h identified up to four phosphorylation events per protein. Polo-like kinase 1 at 2 h yielded four phospho states as well. The first site of phosphorylation by Aurora A was S64, but the second event was at one of three locations: T149, S48, or T20. For polo-like kinase 1 phosphorylation, T43 is the first site phosphorylated with S89 being the second site; either S37 or S64 was the third site phosphorylated. This method allowed the identification of the distinct phosphorylation sites and phosphorylation sequence by the two kinases. The authors mention that this method of optimization of parameters for fragmentation could be applied to any protein <30 kDa, but they caution that data should still be manually interpreted to be confident in the phosphosite localization.

(B) Localization of the primary and secondary Plk1-specific phosphorylation sites on Bora to residues T43 and S89, identified with ETD 8 ms ETD ion/ion reaction time. Inter-residue cleavages are indicated in purple for matched b-ions, in green for c-ions, blue for y-ions and red for z-ions. (C) Summary of the AurA- and Plk1-specific phosphorylation sites on Bora identified by top-down mass spectrometry.

Abstract from Analytical Chemistry 2015; DOI: 10.1021/acs.analchem.5b00162

Top-down analysis of intact proteins by mass spectrometry provides an ideal platform for comprehensive proteoform characterization, in particular, for the identification and localization of post-translational modifications (PTM) co-occurring on a protein. One of the main bottlenecks in top-down proteomics is insufficient protein sequence coverage caused by incomplete protein fragmentation. Based on previous work on peptides, increasing sequence coverage and PTM localization by combining sequential ETD and HCD fragmentation in a single fragmentation event, we hypothesized that protein sequence coverage and phospho-proteoform characterization could be equally improved by this new dual fragmentation method termed EThcD, recently been made available on the Orbitrap Fusion. Here, we systematically benchmark the performance of several (hybrid) fragmentation methods for intact protein analysis on an Orbitrap Fusion, using as a model system a 17.5 kDa N-terminal fragment of the mitotic regulator Bora. During cell division, Bora becomes multiply phosphorylated by a variety of cell cycle kinases, including Aurora A and Plk1, albeit at distinctive sites. Here, we monitor the phosphorylation of Bora by Aurora A and Plk1, analyzing the generated distinctive phospho-proteoforms by top-down fragmentation. We show that EThcD and ETciD on a Fusion are feasible and capable of providing richer fragmentation spectra compared to HCD or ETD alone, increasing protein sequence coverage, and thereby facilitating phosphosite localization and the determination of kinase specific phosphorylation sites in these phospho-proteoforms. Data are available via ProteomeXchange with identifier PXD001845.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Anal Chem titled "Benchmarking multiple fragmentation methods on an Orbitrap Fusion for top-down phospho-proteoform characterization." Authors of the paper are Brunner AM, Lossl P, Liu F, Huguet R, Mullen C, Yamashita M, Zabrouskov V, Makarov A, Altelaar AFM, Heck AJR.