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Download tech note (PDF)
Site-localization of malonylated peptides using the SCIEX ZenoTOF 7600 system
Joanna Bons1, Jason Cason2, Birgit Schilling1, Christie Hunter3
1Buck Institute, USA, 2SCIEX, Canada, 3SCIEX, USA
Determining the identity and the precise location of a post-translational modification on a protein is important to fully characterize function. Here, the utility of electron activated dissociation on the SCIEX ZenoTOF 7600 system was investigated for the characterization of labile post translational protein modifications. Leveraging the tunable kinetic energy, the malonyl modification on a synthetic peptide was preserved such that definitive localization was possible. In addition, the use of Zeno trapping provided significant sensitivity gains to ensure complete sequence characterization was obtained.
Post-translational modifications (PTMs) are important players in a diverse group of functions that include protein conformation and signaling. Lysine acylation, such as malonylation, is one such PTM, and is regulated in part by lysine deacylases, which are members of the sirtuin (SIRT) protein family. In a previous study investigating SIRT5-regulated lysine malonylome, it was shown that 183 malonyllysine sites (from 120 proteins) out of the 1,137 identified sites (from 430 proteins) were significantly increased in Sirt5-/- KO versus wild-type mice.1 Specifically, it revealed that malonylation regulated GADPH activity. Malonylated peptides are however traditionally difficult to characterize using mass spectrometry and CID because the modification is extremely labile.
In this work, that involves using EAD technology with tunable kinetic energy, the effects of kinetic energy ramping on the preservation of highly labile PTMs (malonylation, for example) was studied, focusing on one previously identified malonyllysine site from GADPH (K-192). Two orthogonal fragmentation modes were compared (EAD vs. CID), to investigate the utility of each for PTM site localization. In addition, samples were measured using MRMHR mode to investigate the use of EAD for quantitative PTM characterization of labile modifications.2 MS/MS data were acquired with the Zeno trap activated, which provides significant sensitivity increases and enhances the quality of EAD MS/MS spectra.
Figure 1. Sequence of the investigated malonylated peptide. Peptide at position 185-195 in mouse glyceraldehyde-3-phosphate dehydrogenase (P16858) and carrying a malonyl group on lysine K-192 was investigated. PTM site-specific ions are highlighted in blue.
Sample preparation: Malonylated synthetic peptides were obtained from Thermo Fisher Scientific and diluted in simple matrix for analysis.
Chromatography: A NanoLC 425 system plumbed for microflow chromatography (5 µL/min) was used and operated in direct inject mode. The analytical column used was a 0.3 mm x 150 mm (2.6 µm particle size) Phenomenex Omega Polar column. Column temperature was controlled at 30°C. Short gradients of 8 minutes were used.
Mass spectrometry: All data were acquired using a SCIEX ZenoTOF 7600 system and an OptiFlow Turbo V ion source4 equipped with the microflow probe and 25 µm electrodes. The system is equipped with the electron activated dissociation (EAD) cell which enabled electron-based fragmentation to be performed in a targeted manner on the malonyl peptides. MRMHR Acquisition data were collected using a TOF MS scan of 250 msec and MS/MS accumulation times of 150 msec both in CID and EAD mode. Kinetic energies were ramped and optimized per analyte. Electron current was also ramped to optimize and a final value of 5000 nA was used throughout study.
Data processing: The MRMHR data was processed using SCIEX OS software 2.1 using both Explorer and Analytics modules.
Malonylated peptide TVDGPSGKmaLWR from mouse glyceraldehyde-3-phosphate dehydrogenase (P16858) was synthetized with a malonylated lysine at position K-192 (Figure 1). MRMHR analysis using CID fragmentation mode caused significant neutral loss of CO2 (-44 m/z) from the labile malonyl group for the y-ion series (Figure 2A). However, EAD fragmentation generated intact z+1-ion and c-ion series, that were easily detectable in the MS/MS spectra (Figure 2B).5 Specifically, fragment ions were observed, ranging from z4+1 to z11+1, or also c9 and c10 that contained the labile PTM, that did not undergo a significant neutral loss, enabling confident PTM site localization.
Using the Peptide fragment pane in Bio Tool Kit, the fragment ion coverage is easily visualized between the two dissociation techniques (Figure 2C).
Figure 2. MS/MS spectra of TVDGPSGKmaLWR peptide analyzed in CID and EAD modes. The malonylated peptide (m/z 656.3320) was analyzed in (A) CID mode and (B) EAD mode (KE = 5 eV). CID fragmentation resulted in neutral loss of -CO2 (-44), and no “intact” PTM-specific differentiating ions were detectable. Comprehensive EAD fragmentation generated high-intensity fragment ions that provided evidence for straightforward PTM site localization. (C) Near complete sequence characterization was achieved with the EAD dissociation.
EAD fragmentation was performed across a range of kinetic energies, taking advantage of the tunable nature of EAD on the ZenoTOF 7600 system. This allowed determination of the optimal fragmentation parameters to preserve labile modifications on PTM modified peptides, while achieving best sensitivity. Kinetic energies were ramped from 0 eV to 11 eV. In this study, it was observed that a KE value of 5 eV generated high intensity, intact PTM site-specific fragment ions with very little background noise. When increasing KE to 8 eV or higher, MS/MS spectra became more complex, and neutral losses from fragment ions containing the PTM modification emerged (Figure 3).
Interestingly, different dissociation patterns were observed for fragment ions across the different kinetic energies during EAD MS/MS fragmentation (Figure 4). The peak area for most fragment ions containing the modified residue and the labile malonyl modification was highest at a KE value of 5 eV. With higher KEs some neutral PTM losses were observed, but a KE value of 5 eV maintained the integrity of the post-translational modification and maximized peak area intensity for site specific fragment ions.
Figure 3. Impact of the kinetic energy (KE) value in EAD on fragmentation pattern. MS/MS spectra of malonylated peptide TVDGPSGKmaLWR (m/z 656.3320) analyzed in EAD mode with a kinetic energy value of (A) 5 eV and (B) 8 eV.
Figure 4. Kinetic energy ramping for EAD MS/MS. Malonylated peptide TVDGPSGKmaLWR was analyzed in EAD mode with kinetic energy (KE) values varying from 0 eV to 11 eV. Chromatographic peaks were extracted for 16 ions. For the intact ions (two first lines), peak area values were normalized on the highest area. For the ions with a neutral loss (third line), area values were normalized to their respective intact ion.
The impact on MS/MS spectral quality and sensitivity when using the Zeno trap was also investigated. The Zeno trap is located in the back half of the collision cell and is used to reduce ion losses between the collision cell and the accelerator. Ions are captured in the Zeno trap and then ejected in order of high m/z to low m/z, such that each ion reaches the center of the TOF accelerator simultaneously. This increases the duty cycle to greater than 90% through this region and greatly increases the sensitivity for MS/MS acquisition.3
EAD MS/MS spectra, collected with and without the Zeno trap activated, illustrate the clear gain in sensitivity provided by this feature (Figure 5). The ratios of extracted fragment ion peak areas, obtained with and without the Zeno trap activated, were determined for PTM site-specific ions at two different amounts loaded (Table 1). The sensitivity gain ranged between 3.5 and 9.6-fold for the various fragment ions, with an average value of 6.4. This highlights the added value of the Zeno trap for investigating very low-abundance ions.
Figure 5. Increased sensitivity when the Zeno trap is activated. MS/MS spectra of TVDGPSGKmaLWR peptide (m/z 656.3320) analyzed in EAD mode (KE = 5 eV) (A) without and (B) with Zeno trap activated. Significant improvements in signal intensity and thus spectral quality was observed while using the Zeno trap in combination with EAD, enabling more confident PTM localization.
Table 1. Gain of MS/MS sensitivity using the Zeno trap. TVDGPSGKmaLWR peptide was analyzed in EAD mode (kinetic energy = 5 eV) with and without using the Zeno trap at various amounts (16 and 80 fmol) on column. Chromatographic peak areas were extracted and sensitivity changes between having the Zeno trap on and off were determined.
To gain first insights into the quantitative performances of EAD MS/MS, initial dilution curves were generated by performing triplicate injections of 4 different peptide concentrations (16, 80, 400 and 2000 fmol on column) (Figure 6).
Good linearity performances were achieved for 9 investigated fragment ions (R2 ≥ 0.99) in these initial assessments.
Figure 6. Linear response of fragment ions during EAD MRMHR. Peptide TVDGPSGKmaLWR was analyzed in EAD mode (KE = 5 eV), in triplicate, loading various amounts (16, 80, 400 and 2000 fmol). 9 fragment ions are displayed. The R2 coefficient for determination of the linear regression is displayed for each ion.
In this work, the utility of electron activated dissociation on the SCIEX ZenoTOF 7600 system was investigated for the characterization and quantification of labile post translational protein modifications.
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