Peptide Sequence Influence on the Differentiation of Valine and

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Peptide Sequence Influence on the Differentiation of Valine and Norvaline by Hot Electron Capture Dissociation Zhidan Liang, Xiang Yu, and Wendy Zhong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04808 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Analytical Chemistry

Peptide Sequence Influence on the Differentiation of Valine and Norvaline by Hot Electron Capture Dissociation Zhidan Liang 1, Xiang Yu2, Wendy Zhong1* 1. 2.

Analytical Research & Development, MRL, Merck & Co., Inc., Rahway, NJ 07065, USA Department of Pharmacokinetics, Pharmacodynamics, & Drug Metabolism (PPDM), MRL, Merck & Co., Inc.,West Point, PA 19486, USA

Abstract Isomeric amino acid residues such as valine (Val) and norvaline (Nva) are common in recombinant proteins. The mis-incorporation of Nva for leucine (Leu) causes heterogeneity and in some cases even toxicity. Previous studies have shown that hot electron capture dissociation (HECD) is able to differentiate Val from Nva by producing diagnostic w ions on custom designed synthetic model peptides. To broaden the utilization of HECD in proteomic studies and to define the critical structural features, a thorough investigation was performed on representative peptides including specifically designed synthetic peptides as well as biological peptides bearing tryptic digest-like features and peptides with posttranslational modifications. Experimental evidence confirmed that the formation of a w ion is directly dependent upon the presence of the corresponding z ion. The results suggested that a charge carrier residue at the C-terminus is required for the detection of diagnostic w ions for Nva. Thus, peptides resulting from trypsin digestion, with arginine (Arg) or lysine (Lys) at the C-terminus, can be analyzed using the HECD method. Post-translational modification (PTM) such as phosphorylation did not prevent the generation of the requisite side chain fragmentation w ions. These results suggest the general applicability of HECD for unambiguous identification of Val and Nva especially in structure characterization of therapeutic proteins.

Introduction

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Characterization of the primary amino acid sequence of peptides and proteins is one of the most important fundamentals of proteomics studies. To a large extent, the primary structure defines the threedimensional structure through protein folding, which protein function directly depends on1. The two important features of the primary structure include the amino acid sequence and any post-translational modification(s) (PTM) that are readily reflected by the molecular weight of the peptide or protein. Tandem mass spectrometry (MS/MS) has evolved into an essential technique for the structural characterization of proteins and peptides 2. Ions are selected based on their mass-to-charge (m/z) ratio and varied peptide sequence ions are produced by different fragmentation methods. The most widely used fragmentation method in MS/MS experiment is collision-induced dissociation (CID), which breaks amide bonds and forms b-/y- ions3. Electron-capture dissociation (ECD) has been developed as an alternative activation technique, wherein precursor ions react with low energy electrons to produce c-/z- ions upon cleavage of the N-Cα bond 4. Sequence definition is subsequently accomplished by identifying a consecutive series of fragment ions, corresponding to b-, y-type or c-, z-type ion series, or a combination of both. Constitutional isomers such as aspartic (Asp)/isoaspartic acid (isoAsp), leucine (Leu)/isoleucine (Ile), and valine (Val)/norvaline (Nva)5-6 are widely present in peptides and proteins. For example, Leu and Ile count for 16% of all amino acids in proteins 7. Nva differs from Leu by only one methyl group, and mis-incorporation of Nva is common during the production of recombinant proteins 8. However, routinely employed CID fragmentation of peptide precursor ions that contain isomeric amino acid residues generates product ions of the same m/z value and consequently the unequivocal assignment of constitutionally different isobaric species is impossible. Such uncertainty poses a great challenge to establishing correct peptide and proteins sequences.


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Various strategies have been developed to address this problem, and majority of them are focused on generating diagnostic ions that are specific to each amino acid residue. The differentiation of Asp and isoAsp can be achieved by generating c+57 and z-57 diagnostic ions via cleavage of Cα-Cβ bond on the peptide backbone using either ECD 9, electron transfer dissociation (ETD) 10, or in-source decay11 approach. Biemann and co-workers first reported the distinction of Leu and Ile using high energy CID in a sector mass spectrometer 12 to produce amino acid side chain secondary fragmentation. The diagnostic w ions were generated via the characteristic loss of .CH2CH3 (29.0391 Da) from Ile or .CH(CH3)2 (43.0548 Da) from Leu, respectively. However, this method did not have broad application since sector instruments cannot be readily coupled with LC systems and was discontinued shortly after. With the introduction of ECD technique by McLafferty and coworkers 4, the differentiation of Leu and Ile was then achieved on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, where hot electron capture dissociation (HECD) can also produce diagnostic w ions, and thus allow the differentiation of the two residues in synthetic peptides 13, bovine milk PP3 protein 14 and a hemoglobin variant 15. Recently, using a combined HCD-ETD MS3 approach 16-19 the differentiation of Leu and Ile had also been demonstrated on Orbitrap Fusion mass spectrometer. Analogs to HECD, while ETD breaks N-Cα bond, subsequently, HCD provides excessive energy for radical migration during ETD, induces the secondary side-chain fragmentation, and generates diagnostic w ions 18. HECD was also employed to differentiate another pair of amino acid isomers, Val and Nva, in synthetic peptides by Yu, et al. 20 using a FT-ICR mass spectrometer. The synthetic peptide sequences employed in the study were specifically designed to minimize potential interference from other amino acid residues with only glycine (Gly) inserted between Val and Nva in the peptides tested. The identification and differentiation of Val and Nva isomers were achieved by their diagnostic w ions, with the loss of .CH3 and .CH2CH3, respectively. Differentiating Nva from Val has thus far only been accomplished using specifically designed model peptides that does not reflect the complexity of naturally occurring peptides in various biological 3 ACS Paragon Plus Environment


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systems. As mis-incorporation of Nva has been well documented 8, 21-24, the confident characterization of Nva and Val isomers is crucial for a high-quality recombinant protein production. The goal of this study is to identify key structure features that promote secondary fragmentation in HECD experiments, which provides guidance in the unambiguous assignment of Val/Nva in broader field such as in characterization of recombinant proteins in biotherapeutics and proteomic studies. To examine the impact of different side chains, charge carriers, and post-translational modifications, we performed HECD experiments on a set of specifically designed peptides. In a shotgun proteomics study, large proteins are normally digested prior to mass spectrometric analysis by trypsin, which affords peptide fragments with a C-terminal Arg or Lys. Thus we chose a set of Leu- and Val-containing biological peptides that have C-terminal Arg or Lys residues and then replaced Leu with Nva to evaluate the generation of the corresponding w and v ions necessary for differentiation using HECD. Post-translational modification is common in biological proteins and peptides. However, the modified groups are normally unstable during tandem mass fragmentation and are cleaved off. We used phosphorylated peptides to study the influence of a labile modification on amino acid side chain fragmentation during HECD fragmentation. Through this study, we examined the utility of this approach to differentiate Val/Nva in representative peptide models and to provide guidance for the potential application of the method in recombinant protein analysis. Experimental Materials Peptides employed in this study included the following peptide standards: human fibrino peptide A (ADSGEGDFLAEGGGVR); syntide-2 (PLARTLSVAGLPGKK); human big endothelin-1 fragment (22-38) (VNTPEHVVPYGLGSPRS); phospho-cholecystokinin (IKNLQ-pS-LDPSH), which were purchased from Bachem (Bachem Americas, Inc., Torrance, CA, USA). Synthetic peptides employed in this study were synthesized by AnaSpec (AnaSpec, Fremont, CA, USA) included RGVGDG-Nva-GAFR,


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RGVGKG-Nva-GAFR, RGVGFG-Nva-GAFR, RG-Nva-GDGVGAFR, RG-Nva-GKGVGAFR, RGNva-GFGVGAFR; Nva-replaced peptide standards: modified human fibrinopeptide A (ADSGEGDFNva-AEGGGVR and ADSGEGDF-Nva-AEGGGVA); modified syntide-2 (PLARTLSVAG-Nva-PGKK and PLART-Nva-SVAGLPGKK); modified human big endothelin-1 fragment (22-38) (VNTPEHVVPYG-Nva-GSPRS); modified phospho-cholecystokinin, (IKNLQ-pS-Nva-DPSH, IKNNva-Q-pS-LDPSR, and IKNLQ-pS-Nva-DPSR). All solvents used were Optima LCMS grade from Fisher Chemical. MS/MS Analysis HECD experiments were performed on a 9.4T solariX qQq-FTICR mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Each sample was directly infused into the mass spectrometer by a TriVersa NanoMate robot (Advion, Inc., Ithaca, NY, USA) at a concentration of 5 pmol/µL in a spray solution of 50:50 acetonitrile:water with 0.1% formic acid. Mass spectra were collected in the positive ion mode, with 2 M data points. Electron energies used were ranged from 1.7 to 21 eV. ECD and HECD MS/MS spectra were summed over as many as 100 scans to ensure an acceptable signal-to-noise (S/N) ratio. The transient length was 1.12 s, and the estimated resolving power was ~200,000 at m/z 400. The S/N threshold was set to 3; signals below that threshold were ignored. Data were analyzed using DataAnalysis 4.4, and a mass accuracy of less than 5 parts per million (ppm) was used. Results and Discussion HECD of Synthetic Peptides A study by Savitzki and co-workers9 suggested that side chain fragmentation in HECD conditions was due to excess energy in the molecule or fragment. As noted in that study, side chain losses in HECD occurred widely and the majority of the 20 naturally occurring amino acids had cleavable side chains. These side chain losses can be used as additional structural information to confirm amino acid assignments. Since isomeric amino acid residues such as Leu and Ile generate different w ions from side 5 ACS Paragon Plus Environment


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chain fragmentation, these isomeric amino acid residues can be differentiated using diagnostic w ions. The formation of w ions is believed to involve radical migration to the neighboring residues from the corresponding z ions 25. As an initial effort to investigate the formation of diagnostic w ions from Val/Nva side chain fragmentation, peptide sequences were specifically designed to minimize possible side chain fragmentation interferences from other residues in our previous work 20 by placing two Gly between Val and Nva. In this study we inserted an additional amino acid residue between the two Gly residues in the synthetic peptide sequences, including an aromatic phenylalanine (Phe) residue, an acidic aspartic acid (Asp), or a basic lysine (Lys), respectively, to explore the impact of different types of amino acid side chain structures on secondary fragmentation of Val and Nva during HECD processes. Our HECD results are shown in Figure 1 for the three peptides, RGVGFG-Nva-GAFR, RGVGDG-Nva-GAFR, and RGVGKG-Nva-GAFR, and these results demonstrated that the corresponding diagnostic w5 ion for Nva was successfully produced in each case. A fragment ion at m/z 504.256 was observed, corresponding to the w5 ion (theoretical m/z 504.2566) that was generated via the loss of Nva side chain (•CH2CH3) from z5 ion. Similarly, diagnostic ions for Val including a w9 ion (loss of .CH3 from z9) and a v9 ion were observed as shown in Figure S1. These results confirm the generation of diagnostic w/v ions for Val and Nva under HECD conditions. Moreover, this observation also establishes that the presence of other amino acid residues with bulky side chains, such as Asp, Lys, and Phe, will not prevent the secondary fragmentation of the Val and Nva side chains. Doubly charged fragment ions at m/z 516.28404, 500.26321, and 506.79740 were most likely generated via the side chain loss (.C7H7) of Phe residue at the 10th position for all three peptides, respectively. Close examination of the HECD spectra revealed the presence of fragment ions that had a m/z of z9-•CH2CH3, which could be mistaken as diagnostic ions for Nva. The corresponding residue to the z9 ion should be Val, and as a result z9-•CH2CH3 was referred to as an interference ion in our previous work 20. We observed that interference ions were only produced when there was a second Val/Nva present near the C-terminus. The proposed mechanism for the generation of the interference ions is the migration of 6 ACS Paragon Plus Environment

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radicals to Cα of the C-terminal Val or Nva with subsequent side chain fragmentation. To further examine the impact of the addition of amino acid residues with cleavable side chains on the generation of interference ions, the corresponding set of peptides with the positions of Val and Nva switched, RG-NvaGFGVGAFR, RG-Nva-GDGVGAFR, and RG-Nva-GKGVGAFR were studied. In an analogous fashion, if the hypothesis advanced above is valid, interference ions (z9-•CH3) for Nva should also be observed. As shown in Figure S2, the interference ions (z9-•CH3) for Nva were indeed observed. The results of this study were in agreement with previous work, where the interference ion was only found to be present when both Val and Nva were in the sequence. It is also worth noting that the intensity of the interference ion was always lower than the diagnostic w ion as predicted based on our previous work 20. These results gave us the confidence to conclude that despite the addition of amino acid residues with a bulky side chain, w and v ions can still be used as diagnostic ions for the identification of Val and Nva in a peptide sequence.

HECD of Tryptic Digest-like Peptides Biological peptides have more complicated and unpredictable sequence compositions, and the applicability of HECD differentiation of isomeric Val and Nva has not been well demonstrated. To address this limitation, we next applied the HECD method to a set of representative biological peptide standards that are commercially available. The Leu residue in the peptide sequence was then replaced by Nva to mimic the possible mis-incorporation that could occur during recombinant protein synthesis 21-22. The fragmentation behaviors of peptide standards and Nva-modified peptides under HECD conditions were carefully studied. We chose human fibrinopeptide A and syntide-2 to represent peptides that are generated from trypsin digestion of proteins with arginine (Arg) and lysine (Lys) as typical cleavage sites. These peptides will be referred to as “tryptic digest-like peptides” in this study.



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For fibrinopeptide A, and the Nva-substituted fibrinopeptide A, the diagnostic w ions for Leu/Ile and Val/Nva were produced under HECD conditions as shown in Figure 2a and 2b. It is also worth noting that the w8 ion of fibrinopeptide A and Nva-substituted fibrinopeptide A share the same m/z. The only structural difference between Leu and Nva is one methyl group, and during the HECD fragmentation process, •CH(CH3)2 and •CH2CH3 cleaved off of corresponding z ions of Leu and Nva, respectively, resulting in identical remaining fragments. In addition, the generation of diagnostic Nva w ions is more difficult than that of a Leu diagnostic w ion since isopropyl is a better leaving group than an ethyl group. Two additional representative peptides, syntide-2 peptide PLARTLSVAGLPGKK, where Leu 5, was replaced with Nva and human big endothelin-1 fragment VNTPEHVVPYGLGSPRS, where Leu 6 was replaced with Nva, were also subjected to HECD. In the syntide-2 peptide sequence we observed w5 and w10 ions for corresponding Leu fragmentations under HECD conditions (Figure S3a and S3b), with w5 exhibiting almost a10-fold higher signal intensity than was observed for the w10 ion. A similar observation was made for the z5 and z10 ions, which supports the proposed mechanism that the formation of the w ion is directly related to its corresponding z ion. When the intensity of the z ion is too low, it will hinder the production of the corresponding w ion. When Leu at the 5th position (C-terminal) was replaced with Nva, the corresponding w5 ions for Nva were also generated under HECD conditions (Figure S3c). A human big endothelin-1 fragment, with an Arg at the 2nd position, was also examined. In a similar manner, the w6 ion from Leu in the big endothelin-1 fragment and w6 of the ion from the Nva-substituted sequence were both produced under HECD conditions, with electron energies above 16 eV (Figure S4). This data suggests that HECD is capable of generating diagnostic w ions to differentiate Nva from Val for biological tryptic digest-like peptides. The successful application of HECD on a set of tryptic digest-like peptides led us to further investigate the dependence of the secondary fragmentation upon overall peptide structures. The tryptic digest-like peptides used above have a C-terminal charge carrier residue, either Arg or Lys. Based on our observation that the formation of a z ion dictated the formation of corresponding w and v ions, we moved 8 ACS Paragon Plus Environment

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on to investigate the impact of the charge carrier residue on the formation of z ions and the subsequent formation of the diagnostically useful w and v ions. We then chose peptides that did not have C-terminal Arg or Lys such as autocamtide-2-related inhibitory peptide KKALRRQEAVDAL. We were able to detect w10 (m/z 1111.28306) at an ECD current of 1.6 A, and 8 eV of electron energy for Leu. However, when Leu was replaced with Nva, w10 for Nva was not detected despite our effort using a wide range of electron energies from 8 eV to 20 eV. One of the key differences in the peptide sequences was the lack of C-terminal charge carrier residue. To verify that the C-terminal charge carrier was essential to the formation of w ions for Nva, we next purposely replaced C-terminal Arg with Ala in a Nva-substituted fibrinopeptide A ADSGEGDF-Nva-AEGGGVA. As shown in Figure 2c, the w8 ion for Nva was not observed, while z8 and z8+1 ions were present in relatively low abundance. In addition, we compared the peptide backbone cleavage of two peptides ADSGEGDF-Nva-AEGGGVA and ADSGEGDF-NvaAEGGGVR in HECD experiments as shown in Figure S5. The data indicates that major fragments are z ions when a charge carrier was present at C-terminus and c ions are the most dominant fragment ions when a charge carrier is absent at C-terminus. As a result, the formation of the corresponding w ion is hindered when a z ion is either missing or formed at a very low abundance. These findings support the importance of C-terminal charger carrier for the differentiation of Nva using diagnostic side chain fragment ions. Using various tryptic digest-like peptide standards and purposely replacing Leu in the sequences with Nva, we examined the production of w ions under HECD conditions. It has been proposed that the formation of w ion is from the corresponding z ion in the previous study20. These results provide solid experimental evidence to support the dependence of w ion on z ions during HECD as well as ETD-HCD experiments . Our findings suggest that differentiation of isomeric amino acid residues such as Leu/Ile and Val/Nva via diagnostic w ions is possible when C-terminal charge carrier residues are present. In proteomic studies, tryptic digested peptides have Arg or Lys at the C-terminus, and thus this approach can be readily applied. These results greatly expand the applicability of HECD in the unambiguous



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identification of isomeric amino acid residues, especially for Val/Nva, which was only demonstrated on specific synthetic model peptides in the previous study. Peptides with Post-translational Modification Post-translational modification (PTM) is widely observed in proteomic studies. ECD-based methods have been extensively applied to characterize protein and peptide PTMs, since they have the capability of inducing peptide backbone fragmentation while preserving labile modification groups 13. HECD, in contrast, has primarily been applied to induce secondary fragmentation to improve peptide identification. However, a determination of whether the presence of labile modification group(s) will interfere with the secondary fragmentation during HECD has not been systematically investigated. Herein, we employed a phosphorylated model peptide, phospho-cholecystokinin, to investigate the potential impact of modifying groups on secondary fragmentation under HECD conditions. The HECD results suggest that the phosphate group did not prevent the generation of secondary fragments. In phospho-cholecystokinin there are two Leu residues in the sequence that were examined for corresponding secondary fragments. Corresponding diagnostic w ions were observed for all three residues, w5 (m/z 509.19839) and w8 (m/z 917.33687) as shown in Figure S6a and S6c. We also detected a diagnostic v8 for Leu8. To evaluate the impact of a phospho-group on peptide side chain fragmentation, we next examined the mass spectrum to determine if the phospho-group remained intact during the HECD process as shown in Figure S6e. The experimental results demonstrate that de-phosphorylated fragments corresponding to w8 and v8 of Leu were generated simultaneously. These data suggest that the cleavage of labile phosphoryl moiety during HECD process did not prevent peptide side chain fragmentation. As a result, isomeric amino acid differentiation in peptides with PTM can still be achieved by tracking diagnostic w/v ions.. Next, we replaced Leu at two separate positions (Leu5 or Leu8) with Nva in phosphocholecystokinin, but we were not able to obtain diagnostic w fragment ions for Nva at either position as shown in Figure S6b and S6d. The results were consistent with our earlier finding that a C-terminal 10 ACS Paragon Plus Environment

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charge carrier residue is essential to facilitate the generation of secondary fragmentation ions for Nva. To further confirm our hypothesis, we then replaced the C-terminal histidine (His) group with Arg in the Nva-substituted peptide sequences, yielding peptide sequences of IKNLQ-pS-Nva-DPSR and IKN-NvaQ-pS-LDPSR, respectively. HECD fragmentation of these two peptides was shown in Figure 3 and Figure 4. With Arg at the C-terminus, diagnostic w5 or w8 ions for Nva at different positions were detected at m/z of 528.24011 and 936.38011. Our study of HECD on peptides with PTMs demonstrates that cleavage of the PTM group occurs to a lower degree compared to side chain fragmentation. Hence when a modification group is present in a given peptide sequence, diagnostic w and v ions generated during HECD can still be used as a complementary means to identify and differentiate isomeric amino acid groups such as Leu and Ile, Val and Nva. Peptide structure features such as C-terminal charge carrier residue will greatly impact the detection of side chain fragmentation ions. Conclusions Side-chain fragmentation under HECD conditions can be used to differentiate isomeric amino acid residues such as Leu/Ile and Val/Nva. The generation of diagnostic w ions was found to be dependent on several factors during the HECD activation processes. We demonstrated the impact of the peptide structure on the fragmentation behavior using a series of specifically designed as well as biologically relevant peptides. Inserting amino acid residues with bulky side chains such as Phe, Lys and Asp did not affect Val/Nva secondary fragmentation. The findings in our previous study 20, namely that the w ion is generated from the corresponding z ion, and that interfering ions are not major secondary fragments and thus do not interfere the identification of Nva/Val isomers using diagnostic w ions, still applies here. Charge carrier residues such as Arg at C-terminus proved to be critical to the secondary fragmentation process and the corresponding detection of diagnostic w ions for Nva/Val. Modification groups such as phosphorylation can also be cleaved off during HECD fragmentation, however, diagnostic w ions for Nva/Val were more substantial during HECD. Therefore, the findings in tryptic digest-like 11 ACS Paragon Plus Environment


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peptides also hold true in peptides with modification groups when using diagnostic w ions for Nva/Val differentiation. Ambiguity is still present in peptide identification using mass spectrometry, and often a combination of different techniques will need to be employed for accurate and confident peptide identification. Secondary fragmentation occurs during HECD, making the resultant mass spectra more complicated than conventional ECD spectra. A better understanding of HECD fragmentation behavior is important to further advance its application in peptide identification. Acknowledgements: We would like to thank Dr. Gary Martin for critical reading of the manuscript and helpful discussions. The authors also gratefully acknowledge the support from the MRL Postdoc Research Fellow Program.

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11. Yu, X.; Sargaeva, N. P.; Thompson, C. J.; Costello, C. E.; Lin, C., In-Source Decay Characterization of Isoaspartate and beta-Peptides. Int J Mass Spectrom 2015, 390, 101-109. 12. Johnson, R. S.; Martin, S. A.; Biemann, K.; Stults, J. T.; Watson, J. T., Novel fragmentation process of peptides by collision-induced decomposition in a tandem mass spectrometer: differentiation of leucine and isoleucine. Anal Chem 1987, 59 (21), 2621-5. 13. Kjeldsen, F.; Haselmann, K. F.; Budnik, B. A.; Jensen, F.; Zubarev, R. A., Dissociative capture of hot (3-13 eV) electrons by polypeptide polycations: an efficient process accompanied by secondary fragmentation. Chem Phys Lett 2002, 356 (3-4), 201-206. 14. Kjeldsen, F.; Haselmann, K. F.; Sorensen, E. S.; Zubarev, R. A., Distinguishing of Ile/Leu amino acid residues in the PP3 protein by (hot) electron capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry. Anal Chem 2003, 75 (6), 1267-74. 15. Williams, J. P.; Creese, A. J.; Roper, D. R.; Green, B. N.; Cooper, H. J., Hot electron capture dissociation distinguishes leucine from isoleucine in a novel hemoglobin variant, Hb Askew, beta54(D5)Val-->Ile. J Am Soc Mass Spectrom 2009, 20 (9), 1707-13. 16. Xiao, Y.; Vecchi, M. M.; Wen, D., Distinguishing between Leucine and Isoleucine by Integrated LC-MS Analysis Using an Orbitrap Fusion Mass Spectrometer. Anal Chem 2016, 88 (21), 10757-10766. 17. Bagal, D.; Kast, E.; Cao, P., Rapid Distinction of Leucine and Isoleucine in Monoclonal Antibodies Using Nanoflow LCMS(n). Anal Chem 2017, 89 (1), 720-727. 18. Zhokhov, S. S.; Kovalyov, S. V.; Samgina, T. Y.; Lebedev, A. T., An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides. J Am Soc Mass Spectrom 2017, 28 (8), 1600-1611. 19. Lebedev, A. T.; Damoc, E.; Makarov, A. A.; Samgina, T. Y., Discrimination of leucine and isoleucine in peptides sequencing with Orbitrap Fusion mass spectrometer. Anal Chem 2014, 86 (14), 7017-22. 20. Yu, X.; Zhong, W., Differentiation of Norvaline and Valine in Peptides by Hot Electron Capture Dissociation. Anal Chem 2016, 88 (11), 5914-9. 21. Moghal, A.; Mohler, K.; Ibba, M., Mistranslation of the genetic code. FEBS Lett 2014, 588 (23), 4305-10. 22. Cvetesic, N.; Palencia, A.; Halasz, I.; Cusack, S.; Gruic-Sovulj, I., The physiological target for LeuRS translational quality control is norvaline. EMBO J 2014, 33 (15), 1639-53. 23. Apostol, I.; Levine, J.; Lippincott, J.; Leach, J.; Hess, E.; Glascock, C. B.; Weickert, M. J.; Blackmore, R., Incorporation of norvaline at leucine positions in recombinant human hemoglobin expressed in Escherichia coli. J Biol Chem 1997, 272 (46), 28980-8. 24. Lin, T. J.; Beal, K. M.; Brown, P. W.; DeGruttola, H. S.; Ly, M.; Wang, W.; Chu, C. H.; Dufield, R. L.; Casperson, G. F.; Carroll, J. A.; Friese, O. V.; Figueroa, B., Jr.; Marzilli, L. A.; Anderson, K.; Rouse, J. C., Evolution of a comprehensive, orthogonal approach to sequence variant analysis for biotherapeutics. MAbs 2019, 11 (1), 1-12. 25. Kjeldsen, F.; Zubarev, R., Secondary losses via gamma-lactam formation in hot electron capture dissociation: a missing link to complete de novo sequencing of proteins? J Am Chem Soc 2003, 125 (22), 6628-9.



Page 14 of 22

Figures

Figure 1.

HECD spectra of synthetic peptides RGVGFG-Nva-GAFR, RGVGDG-Nva-GAFR, and RGVGKG-Nva-GAFR at 16 eV; HECD produced diagnostic w5 ions for Nva. 2+ 516.28404

RGVGFG Nva-GAFR z5 533.2956 w5 504.2565

w5 504.25591

z5 533.29509

RGVGDG Nva-GAFR c5

w5

z5 533.2956 w5 504.2565

515.28035

504.25571

515.34070 z5 533.2956 w5 504.2565

w5 504.25604 505

533.29496

RGVGKG Nva-GAFR

c5

2+ 506.79740

z5

510

515

520

525

z5

533.29539 530

m/z


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Figure 2.

HECD spectra of fibrinopeptide A and Nva-substituted fibrinopeptide A at 16 eV; 2a). HECD produced w8 ion for Leu; 2b). HECD produced w8 ion for Nva in Nva-substituted fibrinopeptide A; 2c). HECD did not produce a diagnostic w ion for Nva when Arg at Cterminal was replaced by Ala.

w8

2a

ADSGEGDF LAEGGGVR

699.34223

z8 742.3968 w8 699.3420

z8

723.32288

742.39698

z8

2b

728.37949

ADSGEGDF Nva-AEGGGVR z8 728.3812 w8 699.3420

w8

739.34166

699.34041 700

705

710

715

720

725

730

735

740

m/z

2c ADSGEGDF Nva-AEGGGVA z8 643.3172 w8 614.2781

[z8+1]-•CH 629.33488

3

z8+1

644.34566 580

590

600

610

620

630

640

650

660

m/z



Figure 3.

Page 16 of 22

HECD spectra of modified phospho- cholecystokinin: IKNLQ-pS-Nva-DPSH and IKNLQpS-Nva-DPSR at 16 eV; 3a) HECD produced a z5 ion when substituting Leu with Nva, but no w5 ion for Nva was generated; 3b) Substituted C-terminal His residue with Arg, HECD produced z5 and w5 ions for Nva.

IKNLQ-pS- Nva-DPSH

512.35511

z5 538.2382 w5 509.1991

549.97009 554.25651 507.29236 500

3a

538.23783

510

530

520

540

550

m/z

549.97009

z5

554.25651

538.23783 558.28702

IKNLQ-pS- Nva-DPSR

3b

z5+1

z5 557.2804 w5 528.2413

z5 w5

565.66044

528.24011 525

530

535

540

545

550

555

560

565


m/z

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Figure 4.

HECD spectra of modified phospho- cholecystokinin: IKN-Nva-Q-pSLDPSH and IKN-NvaQ-pSL DPSR at 16 eV; 4a) no z8 or w8 was produced under HECD when the C-terminal residue was His; 4b) w8 and z8 ions for Nva were generated when His was replaced by Arg at the C-terminal.

4a IKN Nva-Q-pS-LDPSH z8 960.3948 w8 917.3401

951.47933 946.37976 966.42570

IKN- Nva-Q-pS-LDPSR

4b

z8+1

z8 965.4214 w8 936.3697

z8

938.39512

w8 936.38011 920

930

940

950

960

970


m/z


141x109mm (300 x 300 DPI)

ACS Paragon Plus Environment

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177x168mm (300 x 300 DPI)



192x118mm (300 x 300 DPI)


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177x113mm (300 x 300 DPI)



71x59mm (300 x 300 DPI)


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Peptide Sequence Influence on the Differentiation of Valine and

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