Site-Specific characterization of peptide-polymer conjugates in various stoichiometries by MALDI-Tandem mass spectrometry

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Site-Specific Characterization of Peptide-Polymer Conjugates in Various Stoichiometries by MALDI-Tandem Mass Spectrometry

¨ u¨ Ulk ¨ u¨ Writing – Original DraftInvestigationData curationValidationVisualization , Oyk Mehmet Atakay Writing – Original DraftWriting – Review & EditingConceptualizationMethodologyData curationVisu Matin Yazdani Kohneshahri InvestigationValidation , Cengiz Uzun ConceptualizationMethodology , Bekir Salih Writing – Original DraftWriting – Review & EditingConceptualizationMethodologyProject AdministrationF PII: DOI: Reference:

S0026-265X(19)32615-3 https://doi.org/10.1016/j.microc.2019.104467 MICROC 104467

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

17 September 2019 22 November 2019 23 November 2019

¨ u¨ Ulk ¨ u¨ Writing – Original DraftInvestigationData curationValidationVisualization , Please cite this article as: Oyk Mehmet Atakay Writing – Original DraftWriting – Review & EditingConceptualizationMethodologyData curationVisu Matin Yazdani Kohneshahri InvestigationValidation , Cengiz Uzun ConceptualizationMethodology , Bekir Salih Writing – Original DraftWriting – Review & EditingConceptualizationMethodologyProject AdministrationF Site-Specific Characterization of Peptide-Polymer Conjugates in Various Stoichiometries by MALDI-Tandem Mass Spectrometry, Microchemical Journal (2019), doi: https://doi.org/10.1016/j.microc.2019.104467

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Highlights

 Conjugates having various stoichiometric peptide:PEG unit ratios could be detected with MALDI-MS analysis.  Site-specific

characterization

of

peptide-PEG

conjugates

having

various

stoichiometries could be carried out with MALDI-tandem mass spectrometry analyses.  MALDI-MS2 fragmentation of conjugates confirmed that the PEG chains having acid halide end groups are primarily bonded to the free amine group of the peptide at Nterminus.  The second conjugation site via hydroxyl group of tyrosine amino acid could be detected with MALDI-MS2 analysis for di-PEGylated peptide conjugate.

Site-Specific Characterization of Peptide-Polymer Conjugates in Various Stoichiometries by MALDI-Tandem Mass Spectrometry Öykü Ülkü1, Mehmet Atakay1, Matin Yazdani Kohneshahri, Cengiz Uzun, Bekir Salih* Department of Chemistry, Hacettepe University, 06800-Ankara, TURKEY

Abstract PEGylation, covalent linking of polyethylene glycol (PEG) chains, is one of the most common ways for enhancing the drug properties of biopharmaceuticals. The linkage of PEG chains improves the pharmacokinetic properties of therapeutic biomolecules by increasing their hydrodynamic radius and also shields them against immune system. The chemical composition and structural features of biomolecule-PEG conjugates should be wellcharacterized in a fast and easy methods to project their therapeutic efficiency. For this purpose, mass spectrometry is the best analytical technique to analyze biopharmaceuticals in very low amounts with high sensitivity. Also, the stoichiometric ratio of species composing biomolecule-PEG conjugates can be easily calculated by using data obtained from mass spectrometric analysis. Moreover, tandem mass spectrometry (MS2) analysis, controlled breakdown of the isolated ions in the gas phase, also gives detailed information on chemical structures of species. The binding site of PEG chains on biomolecules can be determined by evaluating data obtained from MS2 analysis. In the study, angiotensin II peptide was PEGylated by using PEG chains with Mw=600 containing modified acid halide end-groups. MALDI-MS and MALDI-MS2 analyses were performed for obtaining data on determination of binding sites and stoichiometric ratio of angiotensin II and PEG chains for conjugates. Keywords: PEGylation; Peptide-polymer conjugates; MALDI-Tandem mass spectrometry; Site-specific analysis

* Corresponding Author. 1

These authors equally contributed to the paper.

E-mail address: [email protected] (B. Salih)

1. Introduction

PEGylation is one of the most widely used conjugation method for improving the pharmacokinetic properties and pharmacodynamics of therapeutic biomolecules [1-3]. The biocompatibility and favorable solubility of PEG chains in both water and organic solvents make them possible to bind to biological molecules easily by various end-group modification and conjugation processes [4]. The first step of the PEGylation process is to make the one or both end groups of PEG chains suitably functional by using the appropriate chemical groups. In this way, the PEG chains are activated and prepared for binding to the desired molecule via selective functional group or groups. The PEGylation process increases the complexity of the structure and makes the characterization of the products much more challenging [5-7]. In the characterization of the products obtained as a result of PEG conjugation, the number of PEG chains and their binding sites on the biomolecule are determined specifically [8]. The content of the product depends, to a large extent, on the number of possible conjugation sites on the biomolecule, the chemical properties of the end groups of activated PEG chains and the conditions of the conjugation reaction [9]. Although PEGylation is a good strategy for improving the therapeutic properties of biomolecules, it may lead to problems such as product heterogeneity, loss of bioactivity and the production of conjugates with lower yields [10, 11]. Several studies have focused on sitespecific PEGylation methods to address these problems [12]. Most of the PEG conjugation reactions are carried out by nucleophilic attack of the functional group in the amino acid side chain of a polypeptide to the appropriate electrophilic center of a synthetic polymer. Sitespecific PEGylation usually takes place at the N-terminus or lysine and histidine amino acids of a peptide chain by targeting their primary amine side chains [13, 14]. Reactive thiol side chains of free cysteine amino acids are also possible conjugation sites for PEGylation resulting formation of disulfide bonds between PEG chain and biomolecule [15, 16]. The characteristics of biomolecule-polymer conjugates depend on the elaborative selection of the polymer type and the active group to be used for end-group modification. For example, the active esters of PEG chains having carboxylic acid end groups are the most commonly used acylating species in biomolecule conjugation processes and react with the primary amines in the conjugated biomolecule structure to form stable amide bonds [17]. The average molecular weight of the polymer and the number of polymer chains bound to the biomolecule are also determinant for the physicochemical properties of total conjugate structure [18]. The consistent control of these parameters is very important in terms of

assessing the suitability of all species that are already present in the conjugation reaction mixture and final product sample. PEGylated biomolecules are highly complex species with different structural heterogeneity. Various conjugate isoforms may differ due to the number of PEG chains attached to a single peptide chain, the position of the conjugation sites and the lengths of the PEG chains [19]. Hence, the heterogeneity and polydispersity of the conjugation product make the analytical characterization of the biomolecule-PEG conjugate very difficult. Mass spectrometry is the primary analytical technique to characterize the heterogeneity of biomolecule-polymer conjugates [20]. Mass spectrometric techniques are the most favored and up-to-date analytical tools for determining the polydispersity of the conjugation product, the PEGylation sites, and the number of attached PEG chains on conjugates [6]. In case of suitable instrument capabilities, the top-down analysis approach may be preferred in mass spectrometric analysis of protein-PEG conjugates [19]. In topdown mass spectrometry analysis approach, proteins or protein conjugates are directly analyzed without any prior enzymatic digestion [21].

As well as single-step mass

spectrometric analysis of intact form of conjugates, tandem mass spectrometric (MS2) analysis methods can be performed to identify the binding sites in the conjugates [22]. MS2 analysis of protonated peptides or proteins generates sequence indicative fragment ions. The amino acid sequences of polypeptides and the binding sites of the modifications on the polypeptide chains such as PEGylation can be clearly identified by analyzing these ions generated in the gas phase [23]. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are the most commonly used ionization techniques in mass spectrometric analysis of protein-PEG conjugates. However, ESI provides more complex mass spectra due to generating highly charged signals, while MALDI is usually advantageous because single charged ions are predominant, creating less overlapping polymer distribution, giving simple mass spectra [24]. In this study, we have used the direct and fast MALDI-MS and MALDI-MS2 analysis methods for characterization of PEGylated angiotensin II molecules. Angiotensin II peptide is a vasoconstrictor

and

important

in

regulating

cardiovascular

hemodynamics

and

cardiovascular structure [25]. This peptide was conjugated in this study with PEG chains having acid halide end groups. Conjugation products having various stoichiometries were characterized individually with high sensitivity and mass resolution using MALDI mass spectrometer. For each conjugation group, conjugation sites on angiotensin II molecules

could be determined specifically by using data obtained from MALDI-MS2 analysis in the complex media without using any additional separation technique in a very short analysis time. 2. Experimental 2.1. Materials Polyethylene glycol (PEG) 600 (Mw=600), angiotensin II (human), thionyl chloride, dichloromethane (DCM), potassium bromide, 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), sodium hypochlorite, ethyl acetate, hydrochloric acid, n-hexane, sodium sulfate, sodium hydroxide, dimethyl sulfoxide (DMSO), sodium bicarbonate, 2,5-Dihiydroxybenzoic acid (DHB), acetonitrile (ACN) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water used in the study was obtained from an Expe-Ultrapure Water System (Mirae St Co., Korea). 2.2. Preparation of Poly(ethylene glycol) diacid (PEGDA) Hydroxyl end groups of PEG chains were oxidized to carboxylic acid using TEMPO radical [26]. One gram of PEG (Mw = 600 g/mol, 1.6 mmol), and KBr (70 mg, 0.6 mmol) were dissolved in 15 mL of water. The solution of TEMPO (20 mg, 0.12 mmol, dissolved in warm water) was added into the prepared PEG solution. The pH of PEG solution was adjusted to 9.0 by adding NaOH solution (1.0 M). Then sodium hypochlorite (15% chlorine) solution was added drop by drop while stirring at room temperature. During the reaction process, the pH of solution was still maintained at 9.0 using NaOH solution (1.0 M). The addition of sodium hypochlorite was continued until the pH remained constant. Then, 15 mL of ethanol was added to deactivate excess sodium hypochlorite in the medium. pH of the solution was reduced to 1.0 by the addition of concentrated HCl (%35 w/w). Poly(ethylene glycol) diacid (PEGDA) was extracted with ethyl acetate, dried over Na2SO4 and filtered off. PEGDA was finally obtained by removal of ethyl acetate under reduced pressure followed by dissolved in DCM and precipitation in n-hexane. 2.3. Preparation PEGDA-Angiotensin II Conjugate At first, previously prepared PEGDA was dried by azeotropic distillation with toluene in Dean-Stark. In the two-neck flask, 1.5 mg (2 µmol) of PEGDA was dissolved in 1.0 mL of anhydrous DCM and 1.0 µL (12 µmol) of thionyl chloride was added into the solution. The solution was stirred 2h at room temperature under N2 atmosphere. Excess of thionyl chloride was removed at 50 oC with continuous flow of N2 gas and condensed at trap. The

conjugation reaction was carried out with addition of 1.0 mg of previously dried angiotensin II to poly(ethylene glycol) diacid chloride (PEGDACl) and stirring overnight. 2.4. Mass Spectrometry Analysis MALDI-MS and MALDI-MS2 analyses were performed using a Bruker Rapiflex MALDI tandem time-of-flight (ToF/ToF) mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a smartbeam™ 3D laser in positive ion mode. DHB is used as matrix in MALDI analyses of all samples. The solutions of DHB (10 mg/mL) were prepared in H2O:ACN mixture in the ratio 1:1 (v/v). One microliter of sample solutions (1.0 mg/mL in water) were directly spotted onto the MALDI sample target and allowed to dry at room temperature. Then, 1.0 μL of matrix solution was added to the sample spot and allowed to dry at ambient conditions before spectral acquisition. The data were acquired in reflectron mode. The mass calibration was performed prior to MS and MS2 analyses using Bruker Peptide Calibration Standard II at m/z scale of interest. MS2 spectra were acquired using the LIFT technique, which involves using excessive laser energy without using any additional collision gas [27]. Precursor ions were isolated using an isolation window of m/z ±5 for MS2 analyses. All acquired MS and MS2 data were evaluated using FlexAnalysis 4.0 software. 3. Results and Discussion 3.1. MALDI-MS analyses of unmodified PEG and diacid form of PEG MALDI-MS analysis of unmodified polyethylene glycol (PEG) sample was initially performed. Fig. 1A shows positive ion MALDI-MS spectrum of unmodified PEG containing protonated, ([M+H]+), sodiated ([M+Na]+), and potassiated ([M+K]+) ion distributions of PEG oligomers. The difference between consecutive signals is about m/z 44 for each distribution, corresponding to the mass of one ethylene oxide (C2H4O) repeating unit. Signals of singly charged PEG ions with H- and -OH end groups could be observed in the MALDI-MS spectrum (Figure 1A) ranging from m/z 400 to m/z 1000. The PEG sample was then analyzed following the end-group modification process. The singly charged modified PEG ions were obtained as [M+H]+, [M+Na]+, and [M+K]+ forms in 400-1000 m/z range of the MALDI-MS spectrum (Fig. 1B). Signals of the modified PEG chains with C 2H3O2- and –C2H3O3 end groups could be observed in the MALDI-MS spectrum shown in Fig. 1B. The comparison between the data obtained from the MALDI-MS analysis of unmodified and modified PEG chains confirms the modification of each end group of PEG chains in the sample.

A * 525.78 *

* *

*

*

500

*

*

*

*

600

* 746.02 * * 790.06 * * 834.09 * * * * * ** * * 800 900 m/z

* 700

* 641.84

B

* * 729.92 685.88

* 818.00 * 862.02

*

*

* *

* 500

+

* [M + H] + * [M + Na]+ * [M + K]

* 773.96

* 597.79 553.74

*

*

*

*

481.72

*

* 657.93 * 613.88 * 701.98

*

569.83

*

*

*

*

600

*

*

* *

* *

700

* 906.05 * * 800

**

**

* 950.07

**

900

**

* m/z

Figure 1. MALDI mass spectra of (A) Polyethylene glycol (Mw= 600) (B) Diacid form of polyethylene glycol (PEGDA) (Mw= 600). The mass differences between consecutive polyethylene glycol chains are equal to 44 Da. The asterisks indicate protonated, sodiated, and potassiated polyethylene glycol ions, in green, red, and blue color, respectively. The conversion reaction of both end groups of each PEG chains to carboxylic acid units is shown in Scheme S1. The end groups of the PEG chains were then converted to unstable acid halide groups which were active for peptide conjugation (Scheme S1). These groups were not stable and could not be analyzed by MALDI-MS. Therefore, PEG chains with acid halide end groups were stored under nitrogen atmosphere prior to use in conjugation with angiotensin II peptide. 3.2. MALDI-MS analysis of conjugate mixture Carboxylic acid end groups of PEGDA chains were converted to active acid halide forms prior to the conjugation process (Scheme S1). The site-specific conjugation was carried out by mixing the PEG chains having acid halide end groups (PEGDACl) with angiotensin II in 2:1 molar ratio (PEG:angiotensin II). The PEGylation reaction itself was controlled by MALDI-MS analysis. Figure S1 illustrates the MALDI mass spectrum of sample obtained from the conjugation of angiotensin II with PEG units in 1:2 mole ratio. Singly charged protonated ions of all species could be obtained in the MALDI mass spectrum. Besides mono- and di-

PEGylated angiotensin II conjugates, signals of conjugates having two angiotensin II units on each end side of PEG chains could be obtained in the mass spectrum. The obtained product also contains non-PEGylated angiotensin II which shows signals in the mass spectrum corresponding to its singly charged monomer and dimer ion forms (Figure S1). This data shows that the conjugation of angiotensin II with PEG chains was accomplished by using the aforementioned method. Different types of conjugates having various stoichiometric angiotensin II:PEG unit ratios were formed as a result of the conjugation process. The mass spectrometric analysis of the conjugation product could differentiate ions having different m/z values. However, additional MS2 analysis could only provide detailed data to determine the conjugation sites on the peptide chain. In this study, angiotensin II was selected as a standard peptide model for use in PEGylation processes. In conjugation, it is particularly aimed to obtain a complex mixture containing conjugation products that may be formed as a result of more than one conjugation site by using PEG chains which are active at both ends. In this way, it is predicted that it is more likely to test the performance of the site-specific MALDI-MS2 analysis method. 3.3. Site-specific MALDI-MS2 analysis of angiotensin II-polyethylene glycol conjugate Angiotensin II-polyethylene glycol (11-mer) conjugate ion (m/z 1646.90) was selected as precursor and fragmented using LIFT technology by applying excessive laser energy. The MALDI-MS2 spectrum of the conjugate in 1:1 stoichiometry, shown in Figure 2, agrees well with the amino acid sequence of angiotensin II conjugated with PEG. An intense fragment signal (m/z 1602.80) corresponding to the neutral loss of CO2 from both carboxylic acid side chains of amino acids (Asp4 and Phe8) and free end group of conjugated PEG chain is observed in the MALDI-MS2 spectrum. The mass difference between the intense peak (m/z 1602.80) and the precursor ion (m/z 1646.90) seems quite close to the mass of one ethylene glycol unit. However, precursor ions were isolated using an isolation window of m/z ±5 for MALDI-MS2 analyses. The m/z range of applied isolation window does not include the ion having one less ethylene glycol unit than the precursor ion. The obtained peak at m/z 1602.80 must only appear as a fragment due to the fragmentation of the precursor ion (m/z 1646.90). Also, as it is seen from the molecular structure of the precursor, the precursor ion angiotensin II-polyethylene glycol (11-mer) conjugate cannot be fragmented by losing one of its ethylene glycol units (Scheme S2). Because the repeating units are not located at the end

of the conjugate while they are in between the angiotensin II molecule and the carboxylic acid end group. Six homologous fragment series can be formed from bond dissociations in the backbone of peptides. Series an, bn, and cn symbolize fragment ions that retain the Nterminus side of the peptide, while xm, ym, and zm symbolize fragment ions containing the Cterminus of the peptide [28]. Collisionally activated dissociation (CAD) of peptide precursor ions generally forms b and y ions by breaking the peptide bonds between C and N atoms, which form the polypeptide backbone of the proteins primarily [29]. The most abundant bn and yn fragment series are observed with a n fragments as a result of concomitant CO loss. Figure S2 shows the chemical structure of the angiotensin II peptide with the b n /yn fragment notation and its three-letter amino acid code. y7

1602.80

- CO2

931.51

b4

b5

b6

b7

1247.63

[Ang II + H]+

971.52

y6

676.35

166.05

* 200

400

y4

600

1106.56

872.44

*

716.33

513.29

800

b4 + PEG

y5

y1

a4 + PEG

y3

400.20

b2 + PEG

775.40

b1 + PEG

y2

263.13

1219.66

*

1134.55

* 1000

1384.69

1356.73

1499.76

b6 + PEG + H2O

b3

a5 + PEG

b2

1646.90

1481.74

b6 + PEG

D R V Y I H P F b1

b7 + PEG

y1

b7 + PEG + H2O

y2 y3

a6 + PEG

y4

b3 + PEG

PEG (n=11)

y5

b5 + PEG

y6

y7

1402.73

1200

1400

1600

m/z

Figure 2. MALDI-MS2 spectrum of angiotensin II-polyethylene glycol (11-mer) conjugate (m/z 1646.90). bn and yn fragments are shown on the one-letter code for amino acid sequence of the angiotensin II-polyethylene glycol (11-mer) conjugate. Signals correspond to an fragments (bn – CO) are also labeled in the spectrum. The asterisks denote neutral H2O or NH3 losses from fragment ions. A signal with low intensity corresponding to angiotensin II is obtained in the spectrum, indicating the cleavage of the bond between PEG and angiotensin II due to the applied excessive laser energy. Signals from the consecutive losses of both water and ammonia from the bn and yn fragments were also obtained and denoted with asterisks in the MALDI-MS2 spectrum of the conjugate. In the MALDI MS2 spectrum, for the conjugated angiotensin II, no

yn fragments (from y7 to y1) including a PEG chain were observed (Figure 2). However, all bn and an fragments in the MALDI MS2 spectrum contain PEG chains which are attributed to the conjugation of angiotensin II with PEG chains via primary amine group of aspartic acid at Nterminus (Scheme S2). Data obtained from MALDI-MS2 analyses of angiotensin II-PEG conjugates confirm that the PEG chains are highly likely to bind to the free amine group of the aspartic acid amino acid at the N-terminus of the peptide. Such active primary amine groups at the available end of the peptides are capable of covalent bonding with the end groups of the PEG chains and enabled the structure to be conjugated as determined by MALDI-MS2 analysis. 3.4. Site-specific MALDI-MS2 analysis of angiotensin II-polyethylene glycol-angiotensin II conjugate Angiotensin II-polyethylene glycol (11-mer)-angiotensin II conjugate ion (m/z 2674.48) was selected as precursor and fragmented by applying excessive laser energy (LIFT technology). A highly intense signal at m/z 2612.45 corresponding to the neutral loss of CO2 in conjunction with H2O loss from angiotensin II molecules is observed in the MALDI-MS2 spectrum of the conjugate (Figure 3). This type of conjugates does not include any attached PEG chains having a free carboxylic acid end group. Both end groups of a PEG chain are bound with angiotensin II molecules (Scheme S3). Therefore, the signal obtained from only the loss of CO2 from the angiotensin II-polyethylene glycol (11-mer)-angiotensin II conjugate ion (m/z 2674.48) could not be as intense as obtained in the MALDI-MS2 spectrum of angiotensin IIpolyethylene glycol (11-mer) conjugate ion (m/z 1646.90) (Figure 2). The loss of CO2 could only be arisen from the carboxylic acid groups of Asp4 and Phe8 amino acids at the angiotensin II part of the conjugate. Intact angiotensin II and angiotensin II bounded with a PEG unit fragments were also formed by the cleavage of fragile amide bonds between PEG and angiotensin II molecules presence in the conjugate. Highly intense signal of intact angiotensin II molecule (m/z 1046.50) and angiotensin II bounded with a PEG unit (m/z 1628.81) obtained in the MALDI-MS2 spectrum of the conjugate endorses the presence of two different angiotensin II units in the conjugate structure (Scheme S3). b-, y-, and a-ion series consistent with the amino acid sequence of angiotensin II are obtained in the MALDIMS2 spectrum (Figure 3). The peaks corresponding to y5 (m/z 676.30), y6 (m/z 775.39), and y7 (m/z 931.46) fragment ions were labeled in the MALDI-MS² given in Figure 3. The signals of y5 and y6 fragment ions have very low intensity when they are compared with intensities of

the other signals appeared in the spectrum. The y7 fragment ion has much more significant peak with higher intensity than the y 5 and y6 fragment ions in the spectrum. y7 fragment ion has an additional arginine amino acid residue in its structure compared to y 5 and y6 fragment ions. The presence of this basic arginine amino acid having primary amine group which can be easily protonated increases the ionization efficiency of the y 7 fragment ion in the positive ion mode. Also, the presence of hydroxyl group on tyrosine and carboxylic acid group on the C-terminus of y5 and y6 fragment ions lowers the ionization efficiencies of these fragment ions in the positive ion mode. This intensity difference between y- type ions is also appeared in the MALDI-MS² given in Figure 2. All obtained bn and an fragments are carrying a PEG unit, whereas unbound yn fragments containing free C-terminus of angiotensin II molecules are detected. These data show that a PEG chain having both available end groups could be conjugated with two different angiotensin II molecules through their free primary amine groups of aspartic acids on N-termini. Ang II 1046.50

y1

2612.45

y6

y7

y5

b7

y4

y2

y3

1628.81

y1

1743.83 1646.83

PEG (n=11)

D R V Y I H P F b1

b2

b3

b4

b5

b6

b7

y7

513.25

y2

y

3 263.12 400.17

676.30

y6

775.39

*

1071.57

a1 + PEG + a5

y4

a2 + PEG + a2

y5

y1

166.07

a1 + PEG + a2

931.46

1901.00

1959.04

500

750

1000

1250

2510.37

2412.23

2276.40

*

1290.58

250

1942.01

b7 + PEG + Ang II

b6

b5

b6 + PEG + Ang II

b4

b5 + PEG + Ang II

b3

a5 + PEG + a6

b2

- CO2 - H2O

a4 + PEG + a7

b1

PEG + Ang II

D R V Y I H P F

b1 + PEG + Ang II

y2 y3

b2 + PEG + Ang II

y4

y5

PEG + Ang II + H2O

y6

y7

1500

2674.48

* 1750

2000

2250

2500

m/z

Figure 3. MALDI-MS2 spectrum of angiotensin II-polyethylene glycol (11-mer)-angiotensin II conjugate (m/z 2674.48). bn and yn fragments are shown on the one-letter code for amino acid sequence of the angiotensin II-polyethylene glycol (11-mer)-angiotensin II conjugate. Signals correspond to an fragments (bn – CO) are also labeled in the spectrum. The asterisks denote neutral H2O or NH3 losses from fragment ions. In this study, PEG chains containing active end groups at both sides were used during the conjugation of angiotensin II molecules on purpose. Monomethoxy PEG (mPEG) chains

having a methoxy group on one of their end groups are mostly used in protein conjugation processes to prevent crosslinking of proteins [2]. However, in some of the studies, the crosslinking of proteins or separate subunits via PEG chains may be desired to form more stable molecular architectures by preserving their activity [30, 31]. After these type of miscellaneous conjugation processes, the various types of conjugates in the final product sample must be characterized in detail. Here, we attempted to increase the complexity of peptide-PEG conjugate mixture by using PEG chains having active groups at both ends, and to test the effectiveness of the direct MALDI-MS2 analysis in the determination of conjugation sites on the peptide molecule. According to the obtained data, it was concluded that PEG chains prefer both free primary amine groups on aspartic acid amino acids located at the N-terminus of angiotensin II molecules. 3.5. Site-specific MALDI-MS2 analysis of di-pegylated angiotensin II conjugate Di-pegylated angiotensin II conjugates could be detected in the MALDI-MS analysis of the sample obtained after the conjugation process (Figure S1). The presence of di-pegylated conjugates shows that PEG chains could attach angiotensin II peptides through two different amino acid sites. Polyethylene glycol (x-mer)-angiotensin II- polyethylene glycol (y-mer) conjugate having total 22 number of repeating units (x + y = 22; m/z 2247.58) was selected as precursor ion and excessive laser energy was applied to the ion in the MALDI-MS2 analysis. PEG attached bn, an fragments, and unbound yn fragments are mainly observed in the MALDI-MS2 spectrum of the di-pegylated angiotensin II precursor ion (Figure 4). Due to the difference in the number of angiotensin II units in the conjugates, the protonated molecular ion signal of angiotensin II (m/z 1046.72) could be observed in the MALDI-MS2 spectrum at lower intensity when comparing with the analogous signal obtained in the MALDI-MS2 spectrum given in Figure 3. Dissociation of bonds between PEG units and angiotensin II due to the excessive laser energy used for the fragmentation also reveals the free PEG ions (n=11-17) at m/z between 600 and 900. Intense signals of ions generated from both neutral CO2 and H2O losses from the conjugate are observed in the MALDI-MS2 spectrum individually (Figure 4). MALDI-MS2 fragmentation pattern of the di-pegylated angiotensin II conjugate also includes the bn (m/z 750-1060) and yn (m/z 1250-1700) fragments carrying PEG units at lower signal intensities. The regions of the MALDI-MS2 spectrum containing the fragments are given as expanded insets in Figure 4. These

fragments contain the PEG binding sites Asp1 (N-terminus) and Tyr4 which are the most potential binding sites for PEG units on angiotensin II.

b2 + [PEG]15

[Ang II + H]+

y7

y6

PEG

b2

b2 + [PEG]14

b2 + [PEG]13

b2 + [PEG]12

b1 + [PEG]15

[PEG]17 b2 + [PEG]11

b1 + [PEG]14

[PEG]16 b2 + [PEG]10

900

950

b4

b3

b6

b5

y1

- H2O - CO2

b7

* PEG (n=y)

1000

1050 m/z

a4 + ●

a6 + ●

1707.18

[Ang II +

a7 + ●

1958.31

b4 + ●

2056.48

1735.21

H]+

a5 + ●

2247.58

b6 + ●

1820.27

b5 + ● 1986.12

1849.16

y7

y3

263.18 400.26

400

*

600

800

1000

1200

1400

y7 + [PEG]10

200

[PEG]11 - [PEG]17

1532.04

y7 + [PEG]9

1800

2000

2200 m/z

1576.09

y7 + [PEG]13

y5 + [PEG]14

y7 + [PEG]8

y5 + [PEG]13

y5 + [PEG]11

1488.04

1276.96

1600

1444.01

1620.12

#

1408.99 1399.97

# 1515.07

#

# 1647.18

1603.12

1559.10

y7 + [PEG]14

y4

513.40

*

y7 + [PEG]12

166.07

931.74

y7 + [PEG]11

y2

y1

y5 + [PEG]12

b1 + [PEG]13

[PEG]15 b2 + [PEG]9

b1

850

y2 y3

D R V Y I H P F

(n=x)

800

y4

y5

y7

# 1691.20

1664.08

1364.99

1320.96

1250

1300

1350

1400

1450

1500

1550

1600

1650

m/z

Figure 4. MALDI-MS2 spectrum of polyethylene glycol (x-mer)-angiotensin II- polyethylene glycol (y-mer) conjugate (x + y = 22; m/z 2247.58). bn and yn fragments are shown on the one-letter code for amino acid sequence of the polyethylene glycol (x-mer)-angiotensin IIpolyethylene glycol (y-mer) conjugate (x + y = 22). Signals correspond to a n fragments (bn – CO) are also labeled in the spectrum. Purple points shown as added to the an and bn fragments symbolize the double PEG units having 22 total number of repeating units. The asterisks and number signs denote neutral H2O and NH3 losses from fragment ions, respectively. During the MALDI-MS2, only the di-pegylated angiotensin II conjugate ion having m/z 2247.58 was intended to be selected as precursor. However, during the MALDI-MS2 fragmentation, di-pegylated conjugates having exactly the same number of ethylene glycol repeating units (x + y = 22) (Scheme S4) and m/z value, but possessing different chemical structures, were also subjected to excessive laser energy as precursor ion. This has led to obtain the signal distributions of fragment ions originating from more than one species in the acquired MALDI-MS2 spectrum (Figure 4). By taking advantage of the high sensitivity and resolution advantages provided by the MALDI-tandem mass spectrometry, complex signal distributions in the obtained MALDI-MS2 spectrum could be evaluated in detail and the PEGylated sites on the conjugate structure could be accurately identified. 4. Conclusion Angiotensin II peptide was conjugated with end-group modified PEG chains in this study. End groups of PEG chains were primarily converted to their acidic forms then to have active acid halide groups at each chain end for the conjugation process. The end-group modification was controlled by MALDI-MS analysis and the obtained mass spectrometric data confirmed that the end-groups of PEG chains could be converted to desired functional groups successfully. After the end-group modification step, the PEG chains having suitable end groups for the conjugation could be attached to angiotensin II peptide by acylation reaction. As a result of the conjugation, besides the signals of mono- and di-PEGylated angiotensin II conjugates, the signals of conjugates having two angiotensin II units linked with a single PEG chain could be obtained in the MALDI mass spectrum. The formation of angiotensin IIpolyethylene glycol-angiotensin II conjugates was expected owing to the presence of PEG chains having two available acid halide end groups for the conjugation. MALDI-MS2 analyses of each conjugate group were performed to determine the conjugation sites on the peptide molecule specifically. It has been determined that the PEG chains having acid halide end

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CRediT author statement Öykü Ülkü: Writing – Original Draft, Investigation, Data curation, Validation, Visualization.: Mehmet Atakay: Writing – Original Draft, Writing – Review & Editing, Conceptualization, Methodology, Data curation, Visualization.: Matin Yazdani Kohneshahri: Investigation, Validation: Cengiz Uzun: Conceptualization, Methodology.: Bekir Salih: Writing – Original Draft, Writing – Review & Editing, Conceptualization, Methodology, Project Administration, Funding Acquisition, Supervision

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.