Methods

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. MRM^HR 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 MRM^HR data was processed using SCIEX OS software 2.1 using both Explorer and Analytics modules. 

EAD preserves labile PTMs preventing neutral losses

Malonylated peptide TVDGPSGKmaLWR from mouse glyceraldehyde-3-phosphate dehydrogenase (P16858) was synthetized with a malonylated lysine at position K-192 (Figure 1). MRM^HR 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).

Kinetic energy ramping for EAD MS/MS

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.

Zeno trap boosts MS/MS sensitivity for fragment ions

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.

Preliminary assessment of quantitative response using targeted MRM^HR workflow

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. 

Conclusions

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.

• Kinetic energy for EAD can be adjusted and exactly tailored to the different analyte necessities
• For this extremely labile PTM, malonyl, it appeared that a KE value of 5 eV was a good kinetic energy impact to preserve the PTM and to achieve optimal sensitivity
• The ability to optimize the kinetic energy for a specific PTM should enable accurate site-localization of labile PTM-containing peptides and maximize/improve quantification accuracy
• In addition, use of the Zeno trap provides significant gains in sensitivity, further improving the utility of this approach for characterizing PTMs
• Preliminary results using MRM^HR with EAD fragmentation suggests this approach should provide robust quantification of labile PTMs, but work is planned to explore this further.

References

1. Nishida Y, Rardin MJ et al. (2015) SIRT5 regulates both cytosolic and mitochondrial protein malonylation with glycolysis as a major target. Mol Cell, 59:321-32.
2. Schilling B, Hunter CL et al. (2015) Multiplexed, scheduled, high-resolution parallel reaction monitoring on a full scan QqTOF instrument with integrated data-dependent and targeted mass spectrometric workflows. Anal Chem, 87: 10222-9.
3. Qualitative flexibility combined with quantitative power - Using the SCIEX ZenoTOF 7600 LC-MS/MS system, powered by SCIEX OS software. SCIEX technical note RUO-MKT-02-13053-A.
4. Single source solution for low flow chromatography. SCIEX technical note, RUO-MKT-02-9701-A.
5. PTM site localization and isomer differentiation of phosphorylated peptides - Tunable electron activated dissociation (EAD) MS/MS using the ZenoTOF 7600 system. SCIEX technical note RUO-MKT-02-13174-A.