Electron Capture Dissociation in Combination With High Performance Cyclic Ion Mobility Separation for Detailed Protein Studies

Electron capture dissociation (ECD) is an alternative fragmentation technique of increasing use in the study of biomolecules.¹ Its advantages include comprehensive fragmentation of large peptides and proteins, distinction of isomeric amino acid residues, and retention of labile post-translational modifications. ECD is available as an option on the SELECT SERIES™ Cyclic™ IMS Mass Spectrometer, a unique, cyclic ion mobility-enhanced quadrupole time-of-flight mass spectrometer with high mass and mobility resolving power.

In this application brief we demonstrate the power of ECD in combination with cyclic ion mobility. Firstly, we show the separation of phosphopeptide isomers at a mobility resolving power >320 CCS/∆CCS and their subsequent ECD-based sequencing. Secondly, we present the utility of pre-mobility ECD for topdown protein sequencing, exploiting ion mobility separation of fragment ions to increase sequence coverage. Together, these data reveal the power of ECD and cyclic ion mobility for increasing the information content for protein studies, of particular relevance for fundamental protein research and for characterizing biotherapeutics in the biopharmaceutical industry.

Benefits

• ECD provides increased information content in peptide and protein sequencing experiments, boosting confidence in protein characterization results
• Unambiguously identify peptide isomers utilizing high resolution ion mobility separations in combination with ECD
• Increase confidence in top-down protein sequencing results by boosting the number of fragment ions detected

Studies of proteins in the life science sector rely heavily on mass spectrometry for qualitative and quantitative assays. Most frequently, tandem mass spectrometry is used to identify and characterize proteins by virtue of amino acid sequence-informative ions, however, challenges exist that preclude detection of these ions in all cases. Firstly, post-translational modifications such as phosphorylations, acetylations, and glycosylations, must be retained in tandem MS experiments to determine their amino acid site. In many cases collision-induced dissociation provides complete coverage and retention of PTMs, but labile modifications such as phosphorylations and glycosylations may not.² Furthermore, for large peptides and for topdown protein sequencing, high coverage can be prevented due to incomplete fragmentation and spectrum complexity.

The SELECT SERIES Cyclic IMS Mass Spectrometer is an ion mobility-enabled instrument with a flexible geometry (Figure 1). Collision-induced dissociation is implemented in two collision cells, one before and one after the ion mobility device, offering a wide range of experimental options. The SELECT SERIES Cyclic IMS instrument is also available with optional ECD capability which can be implemented either pre- or post-IMS, offering the same flexibility in fragmentation as for CID.³ We have previously demonstrated the use of ECD on this instrument for the sequencing of O-glycopeptides in biotherapeutics.⁵ In this application brief we demonstrate the flexibility of the SELECT SERIES Cyclic IMS Mass Spectrometer with key examples which demand the high performance of cyclic ion mobility and superior Tof mass spectrometry. We show that high ion mobility resolving power in excess of 320 CCS/∆CCS enables the separation of phosphopeptide isomers and that post-mobility ECD enables their sequencing and unequivocal identification. Furthermore, we show that pre-mobility ECD offers advantages for topdown protein sequencing. By utilizing ion mobility to separate ECD product ions we significantly increase the coverage of the model protein ubiquitin and double the number of ions detected, increasing redundancy and therefore confidence in the assignments.

Separation and Sequencing of Isomeric Phosphopeptides

Phosphorylations are a common post-translational modification found on proteins and are highly important in cell signalling processes. The modification itself, however, is prone to dissociation under traditional collision-based conditions. ECD is an attractive technique to employ to locate phosphosites as the process can retain phosphorylations and yield high quality peptide sequencing data.

The isomeric phosphopeptides RSpYpSRSR and RYpSpSRSR (from Sigma Phosphomix 1 and 2) were chosen for this analysis as they are structural isomers with the same positional phosphosites, the same mass, but slightly different amino acid sequences. In this experiment we subjected the [M+H]²⁺ ion of the 1:1 mixture to 25 passes of mobility separation in the cyclic device, equating to a resolving power of approximately 325 (CCS/∆CCS). Two distributions were observed (Figure 2, Top); species A at arrival time 208 and B at 210 ms. As the phosphopeptides exited the cell according to their mobilities they passed into the ECD cell for fragmentation. The position of the ECD cell post-mobility means that the product ions generated are mobility-aligned with their precursors giving non-chimeric spectra for each peptide. Full coverage of both peptides was obtained, allowing both location of the phosphosites and confirmation of the amino acid sequences. From the data it was clear that species A was peptide RSpYpSRSR and species B was peptide RYpSpSRSR, a distinction made possible only through the combination of high resolution ion mobility and ECD fragmentation.

Enhancing Sequence Coverage With Mobility Separation of ECD Product Ions

Top-down protein product ion spectra are highly complex and congested due to the wide range of product ions and charge states generated during the dissociation process. Also, in ECD there is the high abundance precursor ion that can overlap in m/z with key fragments. To demonstrate the ability of ion mobility to disentangle these ions, pre-IMS ECD was performed on the 10+ charge state of bovine ubiquitin.

Figure 3 shows the top-down ECD ion mobility mass spectrum of the 10+ charge state of bovine ubiquitin. The product ions are separated according to their mass, shape and charge in the drift vs m/z space. This increase in peak capacity makes the product ions easier to assign due to a reduced overlap in the m/z dimension. A primary driver of the mobility separation is charge, with the singly-charged product ions separated out into a distinct series (Figure 3). The rest of the product ions inhabit a narrower drift vs m/z region due to the square root relationship to charge, with these ions being multiply-charged, larger and more conformationally diverse.

By exploiting the ion mobility dimension, clearer product ion spectra are obtained (Figure 4) allowing more confident assignment and less demanding deconvolution. Indeed, Figure 5 shows the results of the top-down sequencing of ubiquitin. In the case where ion mobility is utilized, sequence coverage is boosted from 89% to 97%. Furthermore, the number of product ions detected is doubled, which increases the redundancy in product ion assignments and therefore the confidence in the sequencing results.

In this application brief we have demonstrated the power of ECD when implemented on the SELECT SERIES Cyclic IMS Mass Spectrometer. The ability to place the ECD pre- or post-IMS provides the ultimately flexibility for protein and peptide characterization experiments. The high resolving power mobility separation enables the separation of isomeric phosphopeptides and when coupled to ECD the peptides can be fully sequenced and identified. Furthermore, when implementing the ECD cell pre-IMS, the mobility separation can be utilized to increase the number of ions detected in top-down characterization experiments. Together with the high resolving power time-of-flight mass analyzer, these capabilities offer an attractive platform for protein researchers in the fundamental academic field and the pharmaceutical industry.

1. Zubarev RA, Kelleher NL, McLafferty FW. Electron Capture Dissociation of Multiply Charged Protein Cations. A Nonergodic Process. J Am Chem Soc. 1998 Apr 1;120(13):3265–6.
2. Bakhtiar R, Guan Z. Electron Capture Dissociation Mass Spectrometry in Characterization of Peptides and Proteins. Biotechnol Lett. 2006 Jul 1;28(14):1047–59.
3. Shaw JB, Cooper-Shepherd DA, Hewitt D, Wildgoose JL, Beckman JS, Langridge JI, et al. Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry. Anal Chem. 2022 Mar 8;94(9):3888–96.
4. Ippoliti S, Cooper-Shepherd DA, Yu Y-Q, Langridge, JI. Confident O-glycosylation Site Identification for ENBREL (etanercept) Using the ECD Functionality of the SELECT SERIES Cyclic IMS System. Waters Application Brief. 720007458. 2021.
5. Fellers RT, Greer JB, Early BP, Yu X, LeDuc RD, Kelleher NL, Thomas PM. ProSight Lite: Graphical Software to Analyze Top-Down Mass Spectrometry Data. Proteomics. 2014 Dec 12;(15):1235–1238.