Sequence and Conformational Analysis of PeptideâPolymer

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Sequence and Conformational Analysis of Peptide-Polymer Bioconjugates by Multidimensional Mass Spectrometry Sahar Sallam, Ivan Dolog, Bradford A Paik, Xinqiao Jia, Kristi L. Kiick, and Chrys Wesdemiotis Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01694 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

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Biomacromolecules 12 March 2018

Sequence and Conformational Analysis of PeptidePolymer Bioconjugates by Multidimensional Mass Spectrometry

Sahar Sallam,† § Ivan Dolog,‡ Bradford A. Paik,# Xinqiao Jia,# Kristi L. Kiick,# and Chrys Wesdemiotis†,‡,* †

Department of Chemistry, The University of Akron, Akron, Ohio 44325, United States

‡

Department of Polymer Science, The University of Akron, Akron, Ohio 44325, United States

#

Department of Material Science & Engineering, University of Delaware, Newark, DE 19716,

United States §

Department of Chemistry, Jazan University, Jazan, Saudi Arabia

ACS Paragon Plus Environment

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ABSTRACT

The sequence and helical content of two alanine-rich peptides (AQK18 and GpAQK18, Gp: Lpropargylglycine) and their conjugates with poly(ethylene glycol) (PEG) have been investigated by multidimensional mass spectrometry (MS), encompassing electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) interfaced with tandem mass spectrometry (MS2) fragmentation and shape-sensitive separation via ion mobility mass spectrometry (IMMS). The composition, sequence, and molecular weight distribution of the peptides and bioconjugates were identified by MS and MS2 experiments, which also confirmed the attachment of PEG at the C-terminus of the peptides. ESI coupled with IM-MS revealed the existence of random coil and α-helical conformers for the peptides in the gas phase. More importantly, the proportion of the helical conformation increased substantially after PEG attachment, suggesting that conjugation adds stability to this conformer. The conformational assemblies detected in the gas phase were largely formed in solution, as corroborated by independent circular dichroism (CD) experiments. The collision cross-sections (rotationally averaged forward moving arears) of the random coil and helical conformers of the peptides and their PEG conjugates were simulated for comparison with the experimental values deduced by IM-MS in order to confirm the identity of the observed architectures and understand the stabilizing effect of the polymer chain. Cterminal PEGylation is shown to increase the positive charge density and to solvate intramolecular positive charges at the conjugation site, thereby enhancing the stability of α-helices, preserving their conformation, and increasing helical propensity.


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INTRODUCTION Bioconjugates are hybrid materials containing biomolecules covalently linked to synthetic polymers.1 The most widely used polymer for this purpose has been poly(ethylene glycol) (PEG), a water soluble and nontoxic material that has been approved by FDA since 1990 for use in drug conjugates and biomedical devices.2,3 Covalent attachment of PEG to other molecules, known as PEGylation, is primarily applied to therapeutic peptide and protein drugs. This process improves the bioavailability as well as the physiochemical and pharmacokinetic properties of such drugs3,4 by increasing their solubility and stability, decreasing their aggregation proclivity, and generally minimizing adverse immune system responses against them.2,5-9 PEGylated therapeutic drugs are employed for the treatment of several chronic diseases, including cancer, hepatitis C, kidney disease, and Crohn’s disease.5 PEGylated drugs that have been approved by FDA include PEG-aspargase for acute lymphocytic leukemia treatment, methoxy polyethylene glycol-epoetin beta for kidney disease treatment, and Certolizumab pegol for rheumatoid arthritis and Crohn’s disease treatment. 2,3,7 Such compounds and many similar bioconjugates cannot generally be prepared in crystalline or highly purified form for molecular structure characterization by spectroscopic methods that probe average structures, like NMR and X-ray diffraction spectroscopy.10-12 This limitation can be overcome by mass spectrometry based methods, where the desired product can usually be separated from impurities, residual reactants, and/or byproducts by its unique mass, so that its primary structure can be examined by tandem mass spectrometry (MS2) fragmentation.13,14 Further separation efficiency as well as shape/size selectivity is gained with ion mobility mass spectrometry (IM-MS), which interfaces dispersion according to mass-to-charge ratio (MS dimension) with dispersion by collision cross-section and


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charge (IM dimension).15,16 Combining a soft ionization method, such as matrix-assisted laser desorption ionization (MALDI) or electrospray ionization (ESI), with IM-MS and tandem mass spectrometry fragmentation (MS2 dimension) creates a multidimensional technique that can provide insights into the composition, structure, and architecture of bioconjugates and other complex biomacromolecules.14,17 Since the complete analysis takes place in the mass spectrometer without the need for offline degradation, derivatization, or fractionation, this approach qualifies as a top-down multidimensional methodology.18,19 Its applicability has been demonstrated with the comprehensive characterization of the primary structure and architecture of an acrylate-based branched glycopolymer,20 a poly(acrylic acid)-peptide biomaterial,21 and

Figure 1. Amino acid sequence of the peptides and PEGylated peptides investigated.


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most recently a polyether dendron conjugated with two different bioactive peptides.22 A pertinent question that has remained unanswered in these studies is whether conjugation affects biomolecular conformation and folding, causing denaturation, which could have a negative effect on the efficacy of bioconjugate drugs. This issue is explored here with an investigation of the alanine-rich peptides AQK18 and GpAQK18 (where Gp represents propargylglycine) and their PEGylated forms AQK18-PEG and GpAQK18-PEG in which the polymer was attached at the Cterminus through an amide bond (Figure 1). 9,23 The choice of peptides was based on previous studies showing that alanine-rich sequences endow association and aggregation capabilities, which can be modulated by PEGylation for various biomedical applications.6,24 The present study illustrates the utility of the MS2 and IM-MS / MS2 techniques for the determination of the sequence, derivatization site, and conformation of bioconjugates which play an increasingly important role in polymer-based biopharmaceutics.25

MATERIALS AND METHODS Materials. The materials and solvents used for the synthesis and mass spectrometry characterization of the peptides and their conjugates were purchased from Fisher Scientific (Pittsburgh, PA), Sigma-Aldrich (St. Louis, MO), and ChemPep (Wellington, FL); all were used as received. Peptides with the sequences Ac-KAAAQAAAQAAAQAAAQK-NH2 (AQK18) and AcGpKAAAQAAAQAAAQAAAQK-NH2 (GpAQK18), where Gp denotes L-propargylglycine, and their conjugates with PEG (Figure 1), were prepared by standard Fmoc-based solid-phase synthesis protocols using a PS3 peptide synthesizer (Protein Technologies, Tucson, AZ). Details


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of the synthesis and characterization of AQK18 have been reported elsewhere.9 These methods were also used for the synthesis and purification of GpAQK18. The expected molecular weight of GpAQK18 (1774.9 Da) was verified by electrospray ionization (ESI-MS) using a Thermo Finnigan LCQ Advantage mass spectrometer (Thermo Finnigan, San Jose, CA). TentaGel PAP Resin (Rapp Polymere GmbH, Tubingen, Germany) was used for solidphase synthesis of the peptides directly onto a PEG chain with an average molecular weight (Mn) of 3 kDa. The conjugate was cleaved from resin in 95% trifluoracetic acid (TFA), 5% thioanisole for 12 hours. TFA was removed via evaporation, and the conjugates were precipitated twice into cold ethyl ether. Samples were redissolved in water, frozen in liquid nitrogen, and lyophilized. Dried samples were then dissolved in water and dialyzed against water using a 1000 MWCO regenerated cellulose dialysis membrane (Spectrum Labs, Rancho Domingo, CA); water was exchanged twice daily for three days. Samples were again frozen in liquid nitrogen and lyophilized. The successful synthesis of the conjugates was verified by 1H nuclear magnetic response (NMR) spectroscopy, using a Bruker AV600 (600 MHz) with samples dissolved in D2O (cf. Figure S14 and S15). The synthetic procedures employed for the peptides and the peptide-PEG conjugates yield peptides that were acetylated at the N-terminus; the free peptides were amidated at the C-terminus. All peptides and conjugates contain four blocks of three alanine and one glutamine residue, i.e. (A3Q)4, which are equipped with lysine residues (K) at the C-terminus and either K or GpK residues at the N-terminus, cf. Figure 1. All peptides and conjugates were stored as dried solids at 4 °C. MALDI-MS and MS2 experiments. MALDI-MS and MALDI-MS2 spectra were acquired on a Bruker UltraFlex III MALDI tandem time-of-flight (ToF/ToF) mass spectrometer


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(Bruker Daltonics, Billerica, MA), as described in the Supporting Information (SI) section, where these data are also presented and discussed. ESI-MS, IM-MS, and MS2 experiments. ESI-MS and ESI-MS2 experiments were carried on a Waters Synapt HDMS quadrupole/time-of-flight (Q/ToF) mass spectrometer (Waters, Beverly, MA), equipped with an ESI source and traveling-wave ion mobility mass spectrometry (IM-MS).26 The interface region between the Q and ToF mass analyzers comprises a Triwave region containing three confined cells in the following order: trap cell (closest to Q), ion mobility (IM) cell (intermediate), and transfer cell (closest to ToF). Stock solutions of the peptides and conjugates were prepared in H2O at 10 mg mL-1. These solutions were diluted with MeOH / H2O (1:1, v/v) to 0.01 mg mL-1 (peptides) or 0.10 mg mL-1 (conjugates) before being introduced into the ESI source by direct infusion at a flow rate of 10 µL min-1. The instrument was operated in positive ion mode with a capillary voltage of 3.15 kV, cone voltage of 35 V, sampling cone voltage of 3.2 V, desolvation gas flow rate of 550 L h-1 (N2), trap cell collision energy (CE) of 6.0 eV, transfer cell CE of 4.0 eV, trap/transfer gas flow of 1.5 mL min-1 (Ar), source temperature of 80 °C, and desolvation temperature of 150 °C. IM-MS experiments were carried out by applying a traveling-wave velocity of 350 m s-1, a traveling-wave height of 7.5 V, and a bath gas (N2) flow rate of 22.7 mL min-1 to the IM cell; all other parameters were set as summarized above. Experimental collision cross-section (CCS) values were derived from measured drift times through the IM cell after calibration of the drift time scale with polyalanine standards.21,22,27 All CCS values determined in this study were measured in nitrogen, while the calibrant CCS data were measured in helium bath gas (cf. Table S1 and Figure S16). ESI-MS2 and ESI-IM-MS2 experiments were carried out via collisionally


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activated dissociation (CAD) in the trap cell or (for mobility-separated ions) transfer cell, respectively, with Ar as collision gas and a CE of 40-80 eV. Molecular modeling. Molecular dynamics simulations were performed using the leapfrog algorithm to solve Newton’s equation of motion with the GROMACS 5.0 software28-33 and CHARMM27 force field34 in 1-fs steps. The VMD program35 was used for the analysis and images. Structures were simulated in vacuum. Center-of-mass translation and rotation around the center-of-mass were removed to avoid artifacts. Before starting an annealing simulation, the system was energy-minimized, using the steepest descent algorithm in vacuum, in order to avoid unrealistic interactions. The collision cross-sections of the computationally optimized structures were calculated (in helium) using the trajectory method in the MOBCAL suite of programs.36

RESULTS AND DISCUSSION ESI-MS (MS2) and ESI-IM-MS analysis of peptides AQK18 and GpAQK18. AQK18 is an alanine-rich peptide with 18 amino acid residues, including four blocks of three alanine units (Ala or A = C3H5NO, 71.037 Da) plus one glutamine unit (Gln or Q = C5H8N2O2, 128.059 Da). The peptide is terminated with lysine residues (Lys or K = C6H12N2O, 128.095) at both termini. GpAQK18 has the same sequence as AQK18, with the addition of a single propargylglycine residue (Gp = C5H5NO, 95.037 Da) at the N-terminus. The addition of the propargylglycine residue enables the future production of higher-number block polymers of the conjugates, and thus baseline characterization of the chemically reactive GpAQK18 and GpAQK18-PEG is of interest. Furthermore, propargyl glycine is widely used in click chemistry to change the functionality or property of peptides. Addition of markers via click chemistry could lead to


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simplification or tracing of chemicals in complex biological environments.4,23 Both peptides were acetylated at the N-terminus and amidated at the C-terminus, giving rise to elemental compositions of C70H121N25O23 (1679.907 Da) for AQK18 and C75H126N26O24 (1774.944 Da) for GpAQK18.

Figure 2. (a) ESI-MS spectrum of AQK18 peptide. Peaks at m/z values labeled with a superscripted # arise from incomplete sequences (missing either one Lys or one Ala residue). Peaks without m/z labels are fragments from the doubly or triply charged peptide. (b) ESI-MS2 spectrum of doubly protonated AQK18 (m/z 840.952), acquired at a collision energy of 40 eV. The bn and yn fragment series observed corroborate the sequence Ac-KAAAQAAAQAAAQAAAQK-NH2 (see Figure 1).

The ESI mass spectra of AQK18 (Figure 2) and GpAQK18 (Figure S1) mainly show triply and doubly charged ions of the intact peptides. Low-intensity signals from incomplete sequences and fragment ions from in-source fragmentation are also present in both spectra.


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Mass-selection and collisionally activated dissociation (CAD) of the doubly protonated peptides ([M + 2H]2+) gives rise to ESI-MS2 spectra with contiguous series of singly charged N-terminal bn and C-terminal yn fragments that affirm the corresponding peptide sequences (cf. Figures 2 and S1). Very similar conclusions are drawn by MALDI-MS and MALDI-MS2 characterization of AQK18 (Figure S2) and GpAQK18 (Figure S3).

Figure 3. (a) 2D ESI-IM-MS plot (m/z vs. drift time) of AQK18 peptide and (b,c) mass spectra extracted from the mobility regions of (b) doubly and (c) triply charged AQK18 ions. Color brightness correlates with ion abundances; warmer colors indicate higher ion abundances and colder colors indicate lower ion abundances.

A more precise molecular characterization is achieved by interfacing ESI-MS with ion mobility (IM) separation. With the IM cell turned on, the ions travel through a bath gas under the influence of an electric field and are separated in this process by their sizes, shapes, and charge states before mass analysis.14-17 In the overall IM-MS experiment, 2D dispersion occurs, according to the drift time of the ions in the IM cell (IM dimension) and their m/z value in the


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mass analyzer (MS dimension). This procedure separates chemical noise and fragments from the intact peptides in various charge states, as illustrated by the 2D IM-MS plot of AQK18 in Figure 3a. The most intense regions in this plot contain intact peptide with 2+ or 3+ charges (Figures 3b and 3c, respectively). 2D dispersion enables the observation of minor products, such as peptide dimer in 3+ charge state and intact peptide with 4+ charges, which cannot be detected conclusively without the IM dimension (cf. Figure 3a vs. 2a). Similar compositional insight is gained for GpAQK18 by IM-MS analysis (cf. Figure S4).

Figure 4. IM-MS drift time distributions (mobilograms) of (a) doubly protonated vs. protonated-sodiated AQK18 and (b) quadruply protonated vs. triply protonated-sodiated AKQ18-PEG70. Drift times are marked next to the peaks; see Table 1 for the corresponding Ω values.

Mass-selected ions can also be sent through the IM region to examine their isomeric or conformational purity and detect any overlap of isobaric charge states based on the ensuing drift time distributions (“mobilograms”).14,15,37,38 The mobilogram of [AQK18 + 2H]2+ shows two peaks centered at 5.42 ms and 6.77 ms, while only a single peak centered at 6.77 ms is observed


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for [AQK18 + H + Na]2+ (Figure 4a). These data provide evidence for the existence of two distinct groups of conformations for doubly charged AQK18, which are assigned to a set of conformers with compact random coil structure (drifting faster) and a set of more elongated αhelical conformers (drifting more slowly).39 The same conformational distribution is observed for [GpAQK18 + 2H]2+ vs. [GpAQK18 + H + Na]2+, cf. Figure S5a. The drift time of an ion through the IM cell is proportional to its collision cross-section (CCS or Ω) which is the rotationally averaged forward-moving area of the ion as it drifts within the IM bath gas. CCS values derived from drift times measured by IM-MS provide information about the architecture and conformation of the macromolecular ion under study, analogous to that revealed for the neutral macromolecule by hydrodynamic volumes derived from retention times in size exclusion chromatography experiments. The CCS values of the random coil and αhelical components of doubly protonated and protonated-sodiated AQK18 and GpAQK18 are listed in Table 1 and fall within the ranges found for other random coils and α-helices of alaninerich peptides.39 Table 1. Experimental collision cross-sections (Ω) Ion

m/z

random coil [AQK18 + 2H]2+

840.965

[AQK18 + H + Na] [GpAQK18 + 2H]

2+

888.478

[GpAQK18 + H + Na]2+

[AQK18-PEG70 + 3H + Na]

4+

4+

[GpAQK18-PEG70 + 3H + Na]4+ a

5.42

6.77

random coil

α-helix

335

380

6.77 5.78

7.40

899.478

[AQK18-PEG70 + 4H]4+ [GpAQK18-PEG70 + 4H]

α-helix

851.957

2+

Ω (Å2) a

drift time (ms)

380 347

7.40

400 400

1191.434

9.21

11.50

900

1020

1196.938

9.30

11.50

905

1020

1204.191

9.48

12.00

915

1045

1209.699

9.57

12.00

919

1045

±4%.


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The random coil and helical conformers collapse to partially unfolded structures with a single drift time distribution when a third charge is added (cf. Figure S6). This is most likely caused by increased repulsive forces between the like charges. Structure and collision cross-secton simulations for AQK18. In order to corroborate the peak assignments made for the IM-MS drift time distributions (mobilograms) in Figure 4, molecular mechanics/dynamics simulations were performed on the AQK18 peptide. Two initial conformations were used for doubly protonated AQK18: an extended conformer and a right hand α-helix. The amine groups on the N- and C-terminal lysines and the amide groups of the glutamines were considered as protonation sites. The simulation results show that adding protons to both lysines (K1 and K18, see numbering in Table 2) results consistently in a random coil conformation (Figure 5a) with a CCS of 343 Å2. Coil structures persist if one proton is retained on the N-terminal lysine (K1) while the other is moved to one of the glutamines (Q5, Q9, Q13, or Q17), cf. Figure S7; their larger average CCS of 365 Å2 (Table 2) suggests a less dense hydrogen bonding network than in the random coil generated by protonation of the two lysines. On the other hand, retaining one proton on the C-terminal lysine (K18) and placing the other on one of the glutamines gives rise to helical conformations (Figures 5b and S7), having an average CCS of 388 Å2 (Table 2). Our IM-MS mobilogram of [AQK18 + 2H]2+ (Figure 4a) and experimental CCS values (Table 1) are best reconciled by the presence of a major random coil conformer protonated at the two lysines and a minor helical conformer protonated at the Cterminal lysine and one of the glutamine residues. The alanine residues are unlikely protonation sites because of their much lower proton affinity (902 kJ mol-1)41 relative to that of glutamine (964 kJ mol-1)42 or lysine (996 kJ mol-1).41 Meanwhile, the higher proton affinity of lysine, compared to glutamine, justifies the predominance of the random coil structure.


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Table 2. Simulated structures and collision cross-sections (Ω)

Ion [AQK18 + 2H]2+

Conformer

Charge sites

Ω (Å2) a

Random coil

K1 (H), K18 (H)

343 (3)

Random coil

K1 (H), Q5 (H)

365 (9)

or K1 (H), Q9 (H) or K1 (H), Q13 (H) or K1 (H), Q17 (H) α-helix

Q5 (H), K18 (H)

388 (17)

or Q9 (H), K18 (H) or Q13 (H), K18 (H) or Q17 (H), K18 (H) [AQK18 + H + Na]2+

α-helix

Q5 (Na), K18 (H)

407 (8)

or Q9 (Na), K18 (H) or Q13 (Na), K18 (H) or Q17 (Na), K18 (H) partial or bent

K1 (H), Q5 (Na)

helix

or K1 (H), Q9 (Na)

371 (12)

or K1 (H), Q13 (Na) or K1 (H), Q17 (Na) [AQK18-PEG70 + 4H]4+

Random coil

K1 (H), K18 (H), PEG (2H)

968 (17)

α-helix

Q17 (H), K18 (H), PEG (2H)

1040 (98)

or K18 (H), PEG (3H) or K1 (H), PEG (3H) [AQK18-PEG70 + 3H + Na]4+

Random coil

K1 (H), K18 (H), PEG (H + Na)

902 (52)

α-helix

Q17 (H), K18 (H), PEG (H + Na)

1038 (15)

or Q17 (Na), K18 (H), PEG (2H) a

Average value for the listed charge sites. The number in parenthesis is the standard deviation of the

individual CCSs used to calculate the average value.


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Figure 5. [AQK18 + 2H]2+ conformers; (a) random coil structure protonated at lysines K1 and K18 and (b) α-helical structure protonated at glutamine Q17 and lysine K18. See Figure S7 for other random coil and α-helical tautomers.

For protonated-sodiated AQK18, the proton was attached to one of the lysines because of their high proton affinity (vide supra), and the sodium ion to one of the glutamines because of the high sodium ion affinity of this residue (222 kJ mol-1)43 vs. alanine or lysine (167 or ~212 kJ mol-1, respectively).44,45 Protonation at K18 predominantly yields an α-helix, independent of which Q residue binds Na+ (Table 2 and Figure S8); the average CCS predicted for these conformers is 407 Å2. Interestingly, moving the proton to K1 does not produce random coil structures but partial or bent helices with smaller CCS (372 Å2). The measured CCS of 380 Å2 strongly suggests that all these helical structures are sampled in the IM-MS experiment. With either type of charge, compact structures (drifting faster) are associated with a pool of random coil conformations, whereas more extended components of the sample (drifting more slowly) are associated with α-helical conformations. ESI-IM-MS (MS2) analysis of bioconjugates AQK18-PEG and GpAQK18-PEG. The ESI mass spectra of the PEGylated peptides are complex due to superimposed charge and molecular weight distributions. Use of the IM dimension is imperative to overcome this problem


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Figure 6. (a) 2D ESI-IM-MS plots (m/z vs. drift time in IM cell) and (b) extracted mass spectra of charge state 4+ of bioconjugates AQK18-PEG (top) and GpAQK18-PEG (bottom). Warmer colors indicate higher ion abundances and colder colors indicate lower ion abundances.

and separate the ion mixture by charge, so that interpretable mass spectra can be extracted. The 2D IM-MS plots of the bioconjugates (Figure 6a) contain five well separated regions for charge states 3+ to 7+. The least convoluted mass spectra are extracted from the group of ions with 4+ charges (Figure 6b), which are generated by the addition of mainly 4 protons and, to a lesser extent, 3 protons plus a sodium cation. More combinations of H+/Na+ charges and/or poorer signal-to-noise ratio are observed in the spectra of the other charge states. Measured m/z ratios are in excellent agreement with those calculated for the bioconjugates, which contain a (C2H4O)n chain (44.02621n Da), capped with either the AQK18 peptide (C70H121N25O23; 1679.907 Da) or


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the GpAQK18 peptide (C75H126N26O24; 1774.944 Da). For example, the [M+ 4H]4+ ions of AQK18-PEG70 and GpAQK18-PEG70 are observed at m/z 1191.434 and 1215.207, respectively, which match within 7 ppm the corresponding calculated m/z values of 1196.443 and 1215.203, respectively.

Figure 7. (a) ESI-IM-MS mobilogram of quadruply protonated AQK18-PEG71 (m/z 1202.45). (b) 2D ESI-IM-MS2 plot of [M + 4H]4+, acquired by IM separation followed by CAD at a collision energy of 80 eV. Two IM bands are observed for m/z 1202.45, corresponding to a compact (random coil) and an extended (α-helix) conformation of AQK18-PEG71 (see text). Barely any fragments are formed from the α-helical conformer, consistent with a higher stability and dissociation after collapse to the random coil structure. (c) ESI-IM-MS2 spectrum extracted from the ions drifting at 9.30 ms (random coil conformer).

Under ESI-IM-MS2 conditions, [M + 4H]4+ ions from the bioconjugates dissociate to form N-terminal (an and bn) and C-terminal (yn) fragments, as demonstrated for the 71-mer of AQK18-PEG in Figure 7. None of the N-terminal fragments but all of the C-terminal fragments contain the PEG chain, validating that the polymer is conjugated at the C-terminus. MALDI-MS


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and MS2 experiments corroborate the successful synthesis of the bioconjugates (Figure S9) and the C-terminal location of the polymer (Figure S10). The conformational composition of the PEGylated peptides was examined by drift time analysis of the 4+ ions. Generally, native solution conformations are retained in lower charge states, where charge repulsion in the gas-phase ions formed by ESI is minimized (vide supra). The lowest charge state with adequate intensity to permit the acquisition of drift time distributions was 4+ (cf. Figure 6a); those resulting from mass-selected [AQK18-PEG70 + 4H]4+ (m/z 1191.434) and [AQK18-PEG70 + 3H + Na]4+ (m/z 1196.938) are included in Figure 4b, whereas the mobilograms of the corresponding congeners from GpAQK18-PEG70 are depicted in Figure S5b. Interestingly, the bioconjugates show a bimodal drift time distribution both when quadruply protonated or when charged by 3H+ + Na+. In both cases, the faster drifting compact conformers are more abundant than the more slowly drifting extended conformers; and the proportion of the extended structures slightly increases in the presence of the Na+ cation. Different conformations can be adopted by linear PEG chains depending on their length and charge.46-49 For PEG chains with

Sequence and Conformational Analysis of PeptideâPolymer

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