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Differentiation of Norvaline and Valine in Peptides by Hot Electron Capture Dissociation Xiang Yu, and Wendy Zhong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00823 • Publication Date (Web): 06 May 2016 Downloaded from http://pubs.acs.org on May 13, 2016

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Analytical Chemistry

Differentiation of Norvaline and Valine in Peptides by Hot Electron Capture Dissociation

Xiang Yu1 and Wendy Zhong2,*

Merck Research Laboratories 1, Department of Pharmacokinetics, Pharmacodynamics, & Drug Metabolism (PPDM), 770 Sumneytown Pike, West Point, PA, 19486 2, Process/Analytical Chemistry, 126 E. Lincoln Ave. Rahway, NJ, 07065

* Corresponding e-mail: [email protected]

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Abstract During the production of recombinant proteins, misincorporation of Nva (norvaline) is common and causes heterogeneity or even toxicity. To characterize Nva and differentiate it from Val (Valine), a systematic study was conducted using hot electron capture dissociation (HECD) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The thorough investigation of the fragmentation behaviors of a set of model peptides led us to reveal the characteristic/diagnostic fragment ions, w ions, which can be used to differentiate Val and Nva. However, when both Nva and Val were present in one peptide, the observation of interfering ions may mislead the interpretation. Interestingly, HECD also produced v ions, which have the same nominal mass as the M+1 isotope of the w ion and can only be determined by MS with ultra-high mass resolution and high mass accuracy. The energy dependent study of the v ion provided an unambiguous identification of Nva and Val since the v ion was generated only when Val was present, not Nva within the electron energy range we studied. In addition, an electronenergy dependent curve provided an overall picture on how w ions, v ions, as well as interfering ions behaved as the electron energy increased from 1.5 eV to 14 eV. The results suggest that careful selection of electron energy during a HECD experiment is crucial for the unambiguous differentiation of Val and Nva.


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Introduction

With the great clinical and commercial success of therapeutic recombinant proteins, the pharmaceutical industry has become increasingly focused on this emerging market1. Despite extensive research efforts, it is challenging to produce highly homogenous and purified recombinant proteins due to prevalent protein modifications. In additional to post-translational modification, protein modification can also arise from misincorporation of non-cognate amino acids. Such mistranslation of the genetic code can lead to protein misfolding or even toxicity1,2. Norvaline (Nva) is a nonproteinogenic amino acid that derives from pyruvate chain elongation over α-ketobutyrate and α-ketovalerate3,4. In the early stage of biochemical evolution, Nva was also considered to be a component in primitive proteins5. As Nva and leucine (Leu) differ only by one methyl group, E. coli leucyl-tRNA synthetase (EcLeuRS) lacks specificity and treats Nva as a potential substrate in the synthetic reactions2,6. Although EcLeuRS corrects the incorporation of Nva through post-transfer editing7, it mischarges tRNALeu with Nva under conditions that favor Nva production. For example, the concentration of Nva can accumulate up to 1 mM in microaerobic conditions8. With high Nva/Leu ratio in the cell culture, E. coli replaces Leu with Nva and incorporates Nva into recombinant human hemoglobin9. In large-scale production of recombinant proteins, the Nva concentration will increase with cell density and local oxygen limitation10. Mutation of the repair mechanism for Nva- tRNALeu caused toxicity and decrease in cell number11. Hence, it is important to be able to characterize Nva incorporation into proteins and peptides. Nva is isomeric to valine (Val), which means that they have the same molecular weight and cannot be differentiated by collision-induced dissociation (CID), thereby creating significant analytical challenges for protein sequencing using established MS methods.

The characterization of Nva usually relies on conventional analytical techniques such as Edman degradation9 and high performance liquid chromatography (HPLC)12,13, which are tedious and slow.



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Recent advances in MS/MS (tandem mass spectrometry) have shown great promise in distinguishing isomeric amino acids in peptides and proteins. For instance, ECD (electron capture dissociation) MS/MS method was developed to differentiate isoAsp and Asp, where the observation of c+57 and z-57 ion pair suggested the isoAsp was present in the sequence instead of Asp14. Furthermore, multistage MS/MS (MSn) and online-offline LC-MS platforms were developed to expand the differentiation of Asp and isoAsp from peptide level to intact protein level15,16. Using hot ECD (HECD)17, Kjeldsen et al. demonstrated that characteristic side-chain losses can be produced for Leu (43.0584 u) and Ile (29.0391 u)18,19 which was used to differentiate isomeric amino acid Leu and Ile. The technique was later applied for the detection of a new hemoglobin variant20. Multiple stage MS/MS using ETD combined with HCD were also recently reported as an alternative method to differentiate Ile and Leu21. However, no MS/MS method has been reported to differentiate Val and Nva.

In this work, we have designed a systematic study to differentiate isomeric peptides containing Nva and Val using a set of model peptides with Val and Nva at different positions. HECD MS/MS of these peptides were performed at various electron energies in an ultra-high mass resolution and high mass accuracy FT-ICR instrument. Nva and Val diagnostic ions and HECD fragmentation behaviors of these peptides under different electron energy conditions were carefully investigated.


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Materials and Methods

Synthetic peptides that contain isomeric branched side-chain amino acids were obtained from Anaspec (Fremont, CA) including:

1) RGVGG(norV)GAFR (H-Arg-Gly-Val-Gly-Gly-Nva-Gly-Ala-Phe-Arg-OH); 2) RG(norV)GGVGAFR (H-Arg-Gly-Nva-Gly-Gly-Val-Gly-Ala-Phe-Arg-OH); 3) RGVGGGGAFR (H-Arg-Gly-Val-Gly-Gly-Gly-Gly-Ala-Phe-Arg-OH); 4) RG(norV)GGGGAFR (H-Arg-Gly-Nva-Gly-Gly-Gly-Gly-Ala-Phe-Arg-OH); 5) RGLGGIGAFR (H-Arg-Gly-Leu-Gly-Gly-Ile-Gly-Ala-Phe-Arg-OH); 6) RGIGGLGAFR (H-Arg-Gly-Ile-Gly-Gly-Leu-Gly-Ala-Phe-Arg-OH).

The above peptides were directly infused into mass spectrometer by TriVersa NanoMate robot (Advion, Inc., Ithaca, NY) at a concentration of 5 pmol/µL in the spray solution consisted of 50% acetonitrile, 50% water and 0.5% formic acid.

All electron activated dissociation (ExD) experiments were carried out using a 9.4-T solariX qQq-Fourier transform ion cyclotron resonance (ICR) mass spectrometer (Bruker Daltonics, Billerica, MA)15,17. Each spectrum contains 2M data points and was generated by summing over 40-100 individual spectrum depending on the signal quality. The transient length was 1.12 sec, and the estimated resolving power at m/z 400 was about 200,000. Peaks with S/N (signal over noise ratio) less than 4 were ignored. All spectra were internally calibrated using c1-c9 and z1-z9 ion series to obtain mass accuracy of less than 1 ppm, and were analyzed using the DataAnalysis software (Bruker Daltonics, Billerica, MA).



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Results and Discussion

HECD Produced Diagnostic w Ions and Interfering Ions

HECD experiment was conducted on a synthetic peptide RGVGG(norV)GAFR containing both Val and Nva in the sequence. Figure 1 shows the HECD spectrum of the RGVGG(norV)GAFR at 12 eV electron energy. A fragment ion at m/z 504.2563 was observed, corresponding to w5 ion (theoretical m/z 504.2565, Figure 1a). w5 ion was generated via the loss of the part of Nva side chain (- •CH2CH3) from z5• ion. The process is most likely initiated by radical migration from z5• ions when the excessive energy is available during HECD process as illustrated in Scheme 1a. Similarly, a fragment ion at m/z 731.3834 was also observed, corresponding to w8 ion (theoretical m/z 731.3835, Figure 1b). The w8 ion was produced via the loss of the part of Val side chain (- •CH3) from z8• ion based on the similar mechanism (Scheme 1b). Since HECD generated different side chain losses for Val 3 and Nva 6, w5 and w8 can be used as the diagnostic ions for Nva 6 (Figure 1a) and Val 3 (Figure 1b), respectively. These results demonstrate that HECD can differentiate Val and Nva by generating corresponding diagnostic w ions.

Upon closer examination of Figure 1b, an unexpected ion at m/z 717.3678 was also observed, corresponding to z8 - •CH2CH3, which could only happen when Nva is at the 3rd position instead of Val. This observation created an ambiguity that could potentially confuse the interpretation of the Val 3/Nva 3 pair. On the other hand, we did not observe z5 - •CH3 for Nva 6. We were curious to understand the origin of m/z 717.3678, and whether this originated from the side chain loss of Val or something else. To further understand this phenomenon, peptide RG(norV)GGVGAFR, was synthesized, in which the locations of Val and Nva residues were switched. Figure 2 shows the HECD spectrum obtained under the same condition. Two diagnostic fragment ions were observed: w5 ion generated from z5 - •CH3, which was diagnostic to Val 6 (theoretical m/z 518.2722, Figure 2a), and w8 ion generated from z8 - •CH2CH3, which was diagnostic to Nva 3 (theoretical m/z 717.3679, Figure 2b). Similarly, the fragment ion at m/z 6 ACS Paragon Plus Environment

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731.3840 was also present in the HECD spectrum, corresponding to z8 - •CH3 (theoretical m/z 731.3835), which could cause ambiguity in the assignment of this isomeric Nva/Val pair. Again, z5 - •CH2CH3 was not observed. The data showed a trend that the interfering ions were only observed when a second Val/Nva is present at the C-terminal of this amino acid residue. Since radical migration is common in ECD22,23, this phenomenon may be the result of the migration of the radical on the N-terminus of z• ion to the α-carbon on the C-terminal Val/Nva, which then induces side-chain loss on the second Val/Nva when excessive energy was provided during HECD activation process (Scheme 2). A similar observation was reported for the Leu/Ile isomer pair21.

To verify the origin of the interfering ions, we synthesized another set of peptides, RGVGGGGAFR and RG(norV)GGGGAFR, in which the C-terminal Nva and Val were replaced by glycine (Gly). As shown in Figure S1, the interfering ions found in the HECD spectra of RGVGG(norV)GAFR and RG(norV)GGVGAFR were absent in the HECD spectra of RGVGGGGAFR (Figure S1a, inset) and RG(norV)GGGGAFR (Figure S1b, inset). These results further confirmed that the interfering ions originated from radical migration to the Cα of the C-terminal Val or Nva, followed by side-chain losses. The presence of these interfering ions made it difficult to differentiate the N-terminal Val or Nva using HECD when both amino acids were present in one product ion. A better method is needed to differentiate these two isomers.

Observation of Diagnostic v Ion for Valine

The v ion is typically observed in high-energy CID, which are generated via the loss of an entire side chain from the y ion, according to Johnson’s high-energy CID investigations24. It is unusual to observe v ions in low-energy CID spectra, however, upon closer examination, we noticed that v8 ions were present in the HECD spectrum of RGVGG(norV)GAFR for Val 3 (theoretical m/z 718.3631, Figure 1b inset) while v5 was absent for the Nva 6 (theoretical m/z 505.2518, Figure 1a inset). Similarly, v5 ion was 7 ACS Paragon Plus Environment


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observed in the HECD spectrum of peptide RG(norV)GGVGAFR for Val 6 (theoretical m/z 505.2518, Figure 2a inset) and v8 ion was absent for Nva 3 (theoretical m/z 718.3631, Figure 2b inset). These results suggest that the v ion was only observed when a Val residue was present, but not for a Nva residue, at HECD condition (Scheme 3); therefore the presence of v ions can be used as a diagnostic ion for Val. It is worth noting that the vn ion and the second isotope of zn - •CH2CH3 differ only by 7.6 mDa, which requires resolving power of above 190,000 for baseline separation. With the ultra-high mass resolving power and high mass accuracy offered by FT-ICR, we were able to determine the v8 ion with high confidence. The data from low resolving power or less accurate instrument may mis-assign this peak as the second isotope of z8 - •CH2CH3.

Our hypothesis is that since Val has a secondary β-carbon, it can generate a more stable leaving group, while Nva has primary β-carbons, therefore, the leaving group is not quite as stable. Therefore, v ion was only observed for Val, and not for Nva. In order to confirm our hypothesis on the formation of v ions, another pair of peptides, RGLGGIGAFR and RGIGGLGAFR, were synthesized and then studied, where Val and Nva were replaced by Leu and Ile. The HECD spectrum of RGLGGIGAFR showed that v5 ion was observed for Ile 6 while v8 ion was absent for Leu 3 (Figure S2). Similarly, in HECD spectrum of peptide RGIGGLGAFR, v8 ion was observed for Ile 3 while v5 ion was absent for Leu 6 (Figure S3). These results showed that v ion was only observed for Ile residue (not Leu) in HECD. The data support our hypothesis that the radical stability on the β-carbon played the key role in the v ion formation during HECD activation process.

Electron Energy Dependence of Ion Formation

The data shows that both w and v ions can be generated via HECD experiments. We would like to know more about how electron energy affects the formation of these diagnostic ions. A systematic study on the relative intensities of these ions versus electron energy was conducted in the electron energy range from 8 ACS Paragon Plus Environment

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1.5 eV to 14 eV.

Figure 3 shows the ion intensities of w5 and w8 ions verses electron energies. For the peptide RGVGG(norV)GAFR, the intensities of both w5 (Figure 3a) and w8 (Figure 3b) ions increased with the electron energy. Similar results were found in the HECD spectrum of RG(norV)GGVGAFR (Figure 3c and 3d). These results suggested that the high-energy electron promotes the formation of w ions. Figure 3b and 3d shows that the ion intensities of the interfering ions, z8 - •CH2CH3 and z8 - •CH3, also increased with the electron energy. However, the intensity of the w diagnostic ions were always higher than that of the interfering ions at any given electron energy tested (1.5 – 14 eV); moreover, the intensities of the w diagnostic ions increased more dramatically with the electron energy compared to that of the interfering ions. As shown in the Figure 3, at low electron energy, the intensity difference between the w diagnostic ion and the interfering ion was small at low electron energy and this difference became more apparent at high electron energy, which increased the confidence in the diagnostic ion determination. Based on this data, we can safely conclude that the ion with higher intensity is the w ion while, the lower intensity ion is the interfering ion. This conclusion was further supported by a similar observation in the differentiation of Leu and Ile by HECD19, 25.

The relative ion intensities of v5 and v8 ions verses the electron energy were plotted in Figure 3. For RGVGG(norV)GAFR, the v5 ion was not observed across the electron energy range studied (1.5 – 14 eV, Figure 3a), whereas the v8 ion intensity increased drastically from 6 eV (Figure 3b). Similarly, for RG(norV)GGVGAFR, the v8 ion was not observed across the electron energy range tested (1.5 – 14 eV, Figure 3d), whereas the v5 ions intensity increased drastically from 6 eV (Figure 3c). The experimental data suggest that v ions can be used as a diagnostic ion for Val since it was only observed in Val, not Nva. Higher electron energy (up to 14 eV) also promotes the formation of v ion. In summary, the energy dependent curve provides a bigger picture for understanding how the diagnostic ions changed with the electron energy, rather than making conclusions based on one particular electron energy. 9 ACS Paragon Plus Environment


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Conclusions In this study, the fragmentation behaviors of Nva and Val in a series of custom designed peptides were investigated using HECD and high-resolution FT-ICR MS. Val and Nva isomers were successfully identified and distinguished by their diagnostic w ions respectively (•CH3 side-chain loss and •CH2CH3 side-chain loss). Although the observation of interfering ions may mislead the interpretation when both Nva and Val were present in one peptide, the observation of v diagnostic ion in HECD for Val exclusively provided additional confidence in assignment using high resolution FT-ICR MS. Systematic energy-dependent studies revealed that the intensities of w ions were higher in abundance and increased more drastically with electron energy than that of the interfering ions. Moreover, we also investigated Ile and Leu at similar experimental conditions. Similar to Val and Nva, v ions were only observed in Ile, which provided an additional means to unambiguously differentiate Ile and Leu. These findings helped to improve understanding of the fragmentation behaviors of Val/Nva, and will greatly help the characterization of these isomeric amino acids in recombinant proteins.

Supporting Information Additional figures.

Acknowledgements We are grateful to Christopher J. Welch for providing helpful comments on the manuscript. We also thank the support from Merck Future Talent Program.

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Schemes and Figures

Scheme 1, Proposed mechanisms for the w ion formation in HECD. For the peptide RGVGG(norV)GAFR, HECD produced a) w5 ion that was diagnostic to the Nva 6 residue, and b) w8 ion that was diagnostic to Val 3 residue.


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Scheme 2, Proposed mechanisms for the formation of interfering ions in HECD. During HECD, the excess energy on z8• ion facilitates the N-terminal radical migrating to the α-carbons on the C-terminus. The radical then induces corresponding side-chain loss on the C-terminus. a) For the peptide RGVGG(norV)GAFR, the loss of •CH2CH3 from the Nva 6 interferes with the determination of the w8 ion as the diagnostic ion to Val 3; b) For the peptide RG(norV)GGVGAFR, the loss of •CH3 from the Val 6 interferes with the determination of the w8 ion as the diagnostic ion to Nva 3. 13 ACS Paragon Plus Environment


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Scheme 3, Structures of a) v8 ions produced from the peptide RGVGG(norV)GAFR; b) v5 ions produced from the peptide RG(norV)GGVGAFR.


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Figure 1, HECD spectrum of the peptide RGVGG(norV)GAFR at 12 eV electron energy. a) HECD produced w5 ion which was diagnostic to Nva 6. b) HECD produced w8 and v8 ions which were diagnostic to Val 3. The z8 - •CH2CH3 ion had the same mass as the diagnostic w ion for Nva 3 and interfered with the identification of Val 3.



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Figure 2, HECD spectrum of the peptide RG(norV)GGVGAFR at 12 eV electron energy. a) HECD produced w5 and v5 ions which were diagnostic to Val 6. b) HECD produced w8 ions which was diagnostic to Nva 3. z8 - •CH3 ion had the same mass as the diagnostic ion for Val 3 and interfered with the identification of Nva 3.


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Figure 3, The energy dependent curves for the formation of w5, w8, v5, v8 and interfering ions during HECD experiments. The intensities of w5 and v5 ions were normalized to the “z5 sum”. The “z5 sum” was calculated using the intensity of all ions that were originated from z5 ion, including w5 ion (m/z 504.2565 for RGVGG(norV)GAFR, and m/z 518.2722 for RG(norV)GGVGAFR), z5 ion with loss of Phenylalanine side-chain (m/z 456.2565), z5 - •H ion (m/z 532.2878), z5 ion (m/z 533.2956), and z5 + •H ion (m/z 534.3034). The intensities of w8, v8, and interfering ions were normalized to the “z8 sum”. The “z8 sum” was calculated using the intensity of all ions that were originated from z8 ion, including w8 ion (m/z 731.3835 for RGVGG(norV)GAFR, and m/z 717.3679 for RG(norV)GGVGAFR), interfering ion (m/z 17 ACS Paragon Plus Environment


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717.3679 for RGVGG(norV)GAFR, and m/z 731.3835 for RG(norV)GGVGAFR), z8 ion with loss of Phenylalanine side-chain (m/z 655.3522), z8 - •H ion (m/z 745.3992), z8 ion (m/z 746.4070), and z8 + •H ion (m/z 747.4148).


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80x44mm (300 x 300 DPI)

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