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Bioconjugate Chem. 2009, 20, 384–389
Transglutaminase-Mediated PEGylation of Proteins: Direct Identification of the Sites of Protein Modification by Mass Spectrometry using a Novel Monodisperse PEG Anna Mero,†,§ Barbara Spolaore,†,‡ Francesco M. Veronese,*,§ and Angelo Fontana*,‡ CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy, and Department of Pharmaceutical Sciences, University of Padua, Via F. Marzolo 5, 35131 Padua, Italy. Received October 6, 2008; Revised Manuscript Received December 30, 2008
Poly(ethylene glycol) (PEG) has been widely used to prolong the residence time of proteins in blood and to decrease their immunogenicity and antigenicity. A drawback of this polymer lies in its polydispersity that makes difficult the identification of the sites of protein modification. This is a mandatory requirement if a PEGylated protein should be approved as a drug. Here, a fast and reliable method is proposed to characterize proteins conjugated at the level of glutamine (Gln) residues using microbial transglutaminase (TGase). The novelty resides in the use of a monodisperse Boc-PEG-NH2 for the derivatization that allows the direct identification of the sites of PEGylation by electrospray ionization mass spectrometry (ESI-MS). The procedure has been tested on three model proteins, namely, human granulocyte colony-stimulating factor, human growth hormone, and horse heart apomyoglobin. The Gln residues linked to the polymer chain were easily identified by ESI-MS and tandem MS analyses, demonstrating the advantage of using a monodisperse polymer in combination with mass spectrometry for an easy characterization of conjugated proteins. Interestingly, the PEGylation reaction led to the production only of mono- and bis-derivative products, indicating that the TGase-mediated PEGylation can be extremely selective and thus very useful for the derivatization of protein drugs.
INTRODUCTION Recombinant proteins are nowadays assuming a relevant role for human therapy in a variety of diseases. However, protein drugs suffer from several limitations, including susceptibility to degradation by proteases, rapid kidney clearance, and propensity to generate immunogenic reactions. To date, the covalent attachment of poly(ethylene glycol) (PEG1) on the surface of proteins represents the most promising approach to improve their therapeutic efficacy, thus making them more stable to proteolytic digestion, non-immunogenic, non-aggregating, and with a longer half-life than native proteins (1-3). The most widely used chemical methods of PEGylation of proteins involve the covalent conjugation of PEG at the level of the ε-amino group of lysine residues by using acylating PEG derivatives. A drawback of these procedures resides in multiple sites of conjugation and thus in the substantial heterogeneity of the PEGylated proteins. In order to obtain site-specific PEGylation, other chemical approaches were developed, such as the selective PEGylation at the level of the thiol group of cysteine residues or at the N-terminal amino group of a polypeptide chain (2, 4). More recently, a very promising enzymatic method has been * Address correspondence to
[email protected] and
[email protected]. † These authors contributed equally to this work. § Department of Pharmaceutical Sciences. ‡ CRIBI Biotechnology Centre. 1 Abbreviations: GCSF, human granulocyte colony stimulating factor; hGH, human growth hormone; apoMb, apomyoglobin; TGase, transglutaminase; PEG, poly(ethylene glycol); E/S, enzyme to substrate ratio; ESI-MS, electrospray-ionization mass spectrometry; MS/MS, tandem mass spectrometry; HPLC, high-performance liquid chromatography; RP, reverse-phase; TFA, trifluoroacetic acid; Tris, tris(hydroxymethyl)aminomethane; Boc, tert-butyloxycarbonyl; MALDI, matrixassisted laser-desorption ionization.
proposed that makes use of transglutaminase (TGase) for the covalent attachment of PEG moieties at the γ-carboxamide group of Gln residues of proteins (5, 6). For this purpose, a PEG derivative bearing an amino group is being used (PEGNH2). The development of PEGylated biotech drugs involves the stringent need of a very detailed chemical characterization of the PEGylated proteins, as well as of the raw PEG material used for peptide and protein conjugation. Proper characterization of PEG requires determination of the end group structure, the mass of the repeating unit, and the average molecular weight (MW), as well as the MW distribution. These analyses are made rather difficult because of the polydispersity of the PEG polymer samples. Mass spectrometry (MS) has played an important role in the characterization of polymers and of polypeptide polymer conjugates. In particular, matrix-assisted laser-desorption ionization (MALDI) MS has been often employed, since this MS technique produces ions carrying few charges and thus generates mass spectra of lower complexity (7-9). Nevertheless, the MALDI technique suffers from poor ionization efficiency when applied to large synthetic polymers or PEGylated proteins. Consequently, accurate average MW can be obtained only for relatively small polymers (10). On the other hand, electrospray ionization mass spectrometry (ESI-MS) has a strong tendency to form multiply charged ions of PEGylated proteins, thus hampering the analysis of polymers with molecular masses above a few kilodaltons (11, 12). It should be emphasized that the complete chemical characterization of PEGylated protein drugs also requires the identification of the sites of conjugation. Several analytical methods based on classical protein chemistry methodologies have so far been developed, but they are generally quite time- and protein sample-consuming. Among these methods, the peptide mapping procedure is based on the identification of peptide(s) missing in the digest of the PEGylated protein with respect to that of
10.1021/bc800427n CCC: $40.75 2009 American Chemical Society Published on Web 02/02/2009
Technical Notes
Bioconjugate Chem., Vol. 20, No. 2, 2009 385
Figure 1. Amino acid sequence of human granulocyte colony stimulating factor (GCSF) (A), human growth hormone (hGH) (B), and apomyoglobin (apoMb) (C). The Gln residues potential sites of TGase PEGylation are shown in bold.
the nonmodified protein. Missing peptides are supposed to be those carrying the site of polymer conjugation. Therefore, this method is based on negative information, but it should be noted that a missing peptide may arise also from partial enzymatic hydrolysis of the protein. Moreover, confident identification of the derivatized amino acid residue(s) can be obtained only by the Edman N-terminal sequencing analysis of the purified modified peptide, which is not easy to obtain (13). As an alternative, mass spectrometry (MS) appears to be the most suitable technique for the identification of the sites of modification in PEGylated proteins, but until now its use has been limited by the polymer polydispersity (see above). To overcome this problem, our group has previously proposed the derivatization of proteins with a BrCN-cleavable PEG derivative. In this case, the PEGylated protein is reacted with BrCN in order to remove the polymer, while a reporter group of low molecular mass remains linked to the protein chain, thus allowing the identification of the modified amino acid residue(s) by ESI-MS (14). In this paper, a method is described that exploits the sensitivity of MS analyses for the identification of the modified Gln residues in PEGylated proteins prepared using TGase. The novelty resides in the conjugation of proteins with a monodisperse Boc-PEG-NH2, whose mass can be determined with good accuracy by ESI-MS. With respect to other procedures, in the same MS analysis and with the use of a small amount of digested protein, it is possible to identify the modified peptide and to determine the derivatized amino acid residue by MS/MS analysis. This method has been validated on three model proteins, granulocyte colony stimulating factor (GCSF) and human growth hormone (hGH) (Figure 1A,B), which represent two important pharmaceutical proteins, and apomyoglobin (apoMb), which is a most used model for studies of protein structure, folding, and stability (Figure 1C). The results of this study highlight the advantages of analyzing by MS proteins that are modified with monodisperse polymers. Moreover, it is shown that the use of TGase for the PEGylation of GCSF, hGH, and apoMb yielded only mono- and bis-derivatized products, thus emphasizing
the usefulness of the TGase-mediated conjugation for the production of homogeneously modified proteins.
EXPERIMENTAL PROCEDURES Materials. TGase from Streptomyces mobaraensis was purchased from Ajinomoto Co. (Tokyo, Japan), whereas V8protease from Staphylococcus aureus and trypsin were from Sigma Aldrich (St. Louis, MO). GCSF and hGH were supplied by Bioker SpA (Cagliari, Italy). Boc-PEG-NH2 (556.36 Da) was purchased from LCC Engineering GmbH (Egerkingen, Switzerland) and PEG-NH2 (5 kDa and 20 kDa) from Shearwater Polymers Inc. (Huntsville, AL). All other chemicals where purchased from Sigma. Apomyoglobin (apoMb) was obtained from horse heart holomyoglobin by removal of heme using the acetone extraction procedure (15). TGase-Mediated PEGylation of Proteins. Boc-PEG-NH2, previously dissolved in a small amount of acetonitrile, was added at a 10-fold molar excess to solutions containing GCSF or hGH (1.5 mg/mL) in 10 mM phosphate buffer, pH 7.0. TGase was then added at an enzyme to substrate ratio (E/S) of 1:75 (by weight). The reaction mixtures were incubated at room temperature for 4 h and the product(s) purified by RP-HPLC using a Zorbax C18 column (4.6 × 250 mm; Agilent Technologies, Palo Alto, CA) with a linear gradient of 50-70% acetonitrile containing 0.05% TFA over a 25 min period, followed by an isocratic wash at 80% acetonitrile containing 0.05% TFA. The effluent from the column was monitored by measuring the absorbance at 226 nm. The PEGylated proteins of the chromatographic peaks were collected, lyophilized, and used for a peptide fingerprinting analysis by mass spectrometry. In the case of apoMb, under the experimental conditions given above, a high yield of cross-linked dimer was obtained. Better results were obtained using a protein concentration of 0.7 mg/ mL, a large excess of Boc-PEG-NH2 (100 equiv), and TGase at an E/S ratio of 1:50 (by weight). The reaction was allowed to proceed at room temperature for 3 h and then analyzed by RP-HPLC on a Jupiter C18 column (4.6 × 150 mm; Phenomenex, Torrance, CA), eluted with a gradient of acetonitrile/ 0.085% TFA versus water/0.1% TFA from 5% to 40% in 5 min and from 40% to 50% in 25 min. The effluent from the column was analyzed as above.
386 Bioconjugate Chem., Vol. 20, No. 2, 2009
Proteolytic Digestion of PEGylated Proteins. Native and PEGylated proteins (200 µg) were dissolved in 6 M Gdn-HCl, 50 mM Tris-HCl, pH 9.0, to reach a protein concentration of about 1 mg/mL. In order to reduce disulfide bridges, tris(2carboxyethyl)phosphine was added to the protein solution at a final concentration of 5 mM and the reaction mixture kept for 1 h at 37 °C. The reduced protein was then reacted with 25 mM iodoacetamide, and the S-alkylation was allowed to proceed for 30 min at 37 °C in the dark. Protein samples were purified by RP-HPLC on a Zorbax C18 column as reported above. The reduced and S-carboxamidomethylated samples of GCSF and PEG-GCSF were dissolved in 8 M urea and diluted in 50 mM phosphate buffer, pH 7.9, to reach a final protein concentration of 0.8 mg/mL and a urea concentration of 0.8 M. An aliquot from a stock solution of V8-protease in 50 mM acetic acid was then added at an E/S ratio of 1:50 (by weight), and the proteolysis was allowed to proceed at 37 °C overnight. The digestion mixtures, desalted by PepClean C-18 spin columns (Pierce, Rockford, IL), were directly analyzed by mass spectrometry. For hGH and its PEGylated samples, the same procedures were employed, but trypsin was used for protein digestion. In the case of apoMb, the protein and its PEGylated derivative were purified by RP-HPLC, lyophilized, and digested with trypsin overnight at 37 °C in 0.1 M ammonium carbonate buffer, pH 7.8, using an E/S of 1:100 (by weight). Since apoMb does not contain cysteine residues, the steps of reduction and S-alkylation were omitted. Mass Spectrometry Analyses. MALDI mass spectrometry measurements were performed on a REFLEX time-of-flight instrument (Bruker-Franzen Analytik, Bremen, Germany) equipped with a SCOUT ion source operating in positive linear mode. Ions generated by a pulsed UV laser beam (nitrogen laser, λ 337 nm) were accelerated to 25 kV. A saturated solution of sinapinic acid in acetonitrile/water (1:1, v/v) was used as matrix and mixed with the samples dissolved in 0.1% TFA aqueous solution at a v/v ratio of 1:1. Electrospray mass spectrometry (ESI-MS) and tandem MS (MS/MS) measurements were performed on a Q-TOF Micro mass spectrometer (Waters, Milford, MA) equipped with a Z-spray nanoflow electrospray ionization interface. Mass spectra of the peptide digests of proteins and PEG derivatives were acquired using the nanoelectrospray source operating at capillary, cone, and extractor voltages of 2000, 30, and 1 V, respectively (positive ion mode). PEGylated derivatives purified by RP-HPLC were lyophilized, redissolved in acetonitrile/water (1:1, v/v) containing 0.1% formic acid, and then analyzed by ESI-MS. MS/MS analyses were performed only for the peptide 124-162 of GCSF and its PEG derivative utilizing the same parameters of the MS instrument as above, using argon as the collision gas and a collision energy setting of 40 V. External calibration was performed using a solution of 0.1% (v/v) phosphoric acid in 50% aqueous acetonitrile. Nanoelectrospray ionization capillaries were prepared in-house. Instrument control, data acquisition, and processing were achieved with the Masslynx software (Waters).
RESULTS AND DISCUSSION TGase-Mediated PEGylation of Proteins. Figure 2 shows the mass spectra of a polydisperse 5 kDa PEG and a monodisperse PEG as Boc-PEG-NH2. While the polydispersity of the usually employed PEG prevents analysis by ESI mass spectrometry, the recently available monodisperse one (556.36 Da) offers characteristics that make it suitable for analyzing PEGylated proteins by ESI-MS and MS/MS. This possibility was verified using two proteins of pharmaceutical interest, namely, GCSF and hGH, and a model protein as apoMb. As given by
Mero et al.
Figure 2. Analysis by mass spectrometry of polydisperse (5 kDa) and monodisperse (556.36 Da) PEG. (A) The MALDI mass spectrum of polydisperse PEG. (B) ESI-TOF mass spectrum of monodisperse PEG. (C) Chemical structure of monodisperse PEG used in this study. The derivative contains the tert-butoxycarbonyl (Boc) moiety as N-protecting group.
their amino acid sequences (see Figure 1), these proteins contain several Gln residues as potential sites of TGase-catalyzed conjugation. RP-HPLC analysis of the reaction mixture of GCSF with monodisperse PEG (Figure 3A) shows the complete PEGylation of the protein. The reaction product separated by RP-HPLC was analyzed by ESI-TOF mass spectrometry and a mass of 19207.2 Da was measured, which corresponds to GCSF conjugated to only one chain of PEG (Table 1). On the other hand, the RPHPLC analysis of the TGase-mediated PEGylation of hGH showed four chromatographic peaks (Figure 3B). When analyzed by ESI-TOF mass spectrometry, these products gave masses corresponding to hGH conjugated to one or two chains of PEG (peaks 1 and 2, respectively), while the protein material of peak 3 corresponded to a mixture of hGH dimer and of hGH covalently bound to mono-PEGylated hGH; the protein material of peak 4 was a dimer of mono-PEGylated hGH (see Table 1 and Figure 3B). PEGylated apoMb gave a mass of 17491.28 Da corresponding to a mono-PEGylated apoMb (Figure 3C). Of interest, we have observed that, by conducting the TGase-mediated reaction under identical experimental conditions and using a high molecular weight PEG (20 kDa), the same degree of protein modification was achieved with GCSF, hGH, and apoMb (data not shown). Identification of the Sites of PEGylation by ESI-MS. In order to determine the sites of conjugation by TGase, the cysteine residues of PEG-GCSF and of native GCSF were
Technical Notes
Bioconjugate Chem., Vol. 20, No. 2, 2009 387 Table 2. Molecular Masses of the Fragments Obtained upon Digestion with V8-Protease of GCSF and PEG-GCSF and with Trypsin of hGH and PEG-hGH and of apoMb and PEG-apoMba molecular mass (Da) fragmentb
calculatedc
foundd
124-162 (GCSF) 124-162 (PEG-GCSF) 39-41 (hGH) 39-41 (PEG-hGH) 141-145 (hGH) 141-145 (PEG-hGH) 80-96 (apoMb) 80-96 (PEG-apoMb) 79-96 (apoMb) 79-96 (PEG-apoMb)
4025.08 4564.41 403.21 942.54 625.31 1164.64 1852.95 2392.28 1981.05 2520.38
4024.69 4563.96 e
942.67 e
1164.80 1853.01 2392.39 1981.10 2520.50
a
Only the molecular masses of the fragments which contain the Gln residues derivatized with PEG are reported. b Peptides obtained by proteolysis of GCSF and PEG-GCSF with V8-protease and of hGH and PEG-hGH and apoMb and PEG-apoMb with trypsin. c Monoisotopic molecular masses calculated from the amino acid sequence of GCSF, hGH, and apoMb. d Experimental molecular masses determined by ESI-MS. e These very short and rather hydrophilic peptides were not detected, since likely they were lost in the desalting step of the sample digest (see Experimental Procedures).
Figure 3. RP-HPLC analyses of GCSF (A), hGH (B), apoMb (C), and of the reaction mixtures of the PEGylated derivatives. A dashed line and a straight line indicate the chromatograms of native and conjugated proteins, respectively. Table 1. Molecular Masses of Native Proteins, PEG-NH2, and PEGylated Proteins molecular mass (Da) Boc-PEG-NH2 GCSF PEG-GCSF hGH PEG-hGH (PEG)2-hGH hGH dimer PEG-(hGH)2 (PEG)2-(hGH)2 apoMb PEG-apoMb
calculateda
foundb
556.36 18667.68 19207.34 22125.07 22664.73 23204.39 44216.08 44755.74 45295.40 16951.50 17491.16
556.4 18666.3 19207.2 22124.7 22665.9 23203.5 44216.0 44756.0 45294.0 16951.51 17491.28
a Calculated molecular masses. determined by ESI-MS.
b
Experimental molecular masses
reduced and S-carboxamidomethylated, and the two proteins were then digested with V8-protease. This enzyme was used instead of trypsin, since it allowed us to obtain peptide fragments with a lower molecular mass. Indeed, proteolysis with trypsin of GCSF leads to a fragment of 11.3 kDa (residues 41-146), which is not suitable for MS/MS analysis for the identification of the site of modification. Analysis by ESI-MS of the digests of PEG-GCSF and of native GCSF obtained with V8-protease led to a sequence coverage for the protein of 95%, with 10 peptide fragments identified over the 12 expected. The only fragment that was not detected by MS and that contains one Gln residue corresponds to residues 28-33 (sequence GAALQE) (Figure 1). Interestingly, all identified fragments had the same molecular mass in the digests of both PEG-GCSF and native GCSF, except peptide 124-162. Indeed, this peptide fragment deriving from the digest of conjugated GCSF showed an increase in mass corresponding to the addition of one molecule of PEG, thus indicating that PEGylation occurred at the level of this
region of the protein chain (Table 2). The derivatization of the peptide 124-162 appears to be quantitative, since in the MS spectrum of the digest of PEG-GCSF, the m/z signals corresponding to a nonconjugated fragment were not observed. This is evident from an analysis of the region of the spectrum between 1000 and 1200 m/z, which contains signals of the ion [M + 4H]4+ of this peptide (Figure 4A). In the MS spectrum of GCSF, fragment 124-162 shows a signal at 1007.18 m/z (data not shown). This signal is absent in the spectrum of the digest of PEG-GCSF, where only the signal at 1142.00 m/z is observed, which corresponds to the ion with four charges of the modified fragment. Since the GCSF fragment 124-162 contains four Gln residues (see Figure 1), the ion [M + 4H]4+ of the derivatized peptide was subjected to MS/MS analysis in order to determine which Gln residue was modified (Figure 4B). For comparison, the nonmodified peptide was also analyzed by MS/MS (data not shown). In the case of both derivatized and non-derivatized peptides, the analysis of the MS/MS spectra indicates that the most intense product ions of the series b and y are generated from the fourteen N-terminal residues of the fragment, a region that contains two of the four Gln residues. Nevertheless, it was possible to determine the site of specific modification. In the MS/MS spectrum of the PEGylated peptide, fragment ions that contain Gln145 and Gln158 (y25-28) and Gln131 (b8) have m/z values expected for the nonmodified peptide, indicating that these Gln residues are not conjugated. On the other hand, all product ions containing Gln134 (y35-29) show an increase in mass of 439.28 Da, corresponding to the addition of a BocPEG-NH2 with the N-protecting group (Boc) removed. Indeed, this group is conjugated to PEG via an urethane moiety, which is easily cleaved off under the conditions of the MS/MS analysis. Overall, sequence analysis by MS/MS of the modified GCSF peptide 124-162 clearly demonstrated that the TGase-mediated PEGylation occurs at Gln134. In the case of hGH, the reduced and S-carboxamidomethylated hGH and mono- and bis-PEGylated hGH conjugates were digested with trypsin. The digested samples were analyzed by ESI-MS, and most of the tryptic fragments were identified, resulting in a sequence coverage of 88%. The only peptide fragments that were not detected in the digests were fragments 17-19, 128-134, and 179-191, with the last fragment having one Gln residue. By comparing the mass spectra of the digests of hGH and of conjugated hGH, it was observed that only the
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Figure 4. Electrospray mass spectrum of the digest of PEG-GCSF with V8-protease and MS/MS analysis of peptide 124-162. (A) The region of the mass spectrum between 1000 and 1200 m/z is reported in order to show the derivatization with PEG of peptide 124-162 of GCSF. The signals at 1142.00 m/z and at 1147.72 m/z of ions with four charges are indicated. (B) Electrospray MS/MS mass spectrum of the ion at 1142.00 m/z of peptide 124-162 of GCSF-PEG. (Top) Fragments of the series b and y that were identified in the MS/MS spectrum are indicated on the sequence of the peptide. Gln residues are shown in bold, and Gln134 is indicated in bold as QPEG. (Bottom) In the MS/MS spectrum, ions assigned to fragments of the series y and b are indicated. Some internal fragments and immonium ions are also labeled with their sequence in one-letter amino acid code. Fragments containing Gln134 show a mass increment corresponding to the conjugation with PEG-NH2 without the Boc moiety, since the N-protecting group dissociates in the MS/MS analysis (-Boc).
spectrum of PEG-hGH displayed a signal at 472.34 m/z, corresponding to the triply charged ion of fragment 39-41 linked to one chain of PEG, and a signal at 583.41 m/z, corresponding to the ion with three charges of the monoPEGylated fragment 141-145 (Figure 5A,B). Of interest, signals of the conjugated peptides without Boc were also detected in the mass spectrum, since the urethane bond, as reported above, is easily fragmented under the conditions of the ESI ionization. The detection by ESI-MS of Boc removal can be used as a further probe of the PEGylation of the peptide. Since fragments 39-41 and 141-145 contain only one Gln residue, it can be concluded that the TGase-mediated PEGylation of hGH took place at Gln40 and Gln141 in the bis-PEGylated form of the hormone, while the mono-PEGylated species was a mixture of the conjugates at Gln40 and Gln141. In the case of apoMb, samples of native and PEGylated protein were subjected to trypsin digestion. All tryptic fragments were identified for both native apoMb and PEG-apoMb species with a sequence coverage of 98%. However, in the digest of PEG-apoMb, the tryptic fragments 80-96 and 79-96 gave only MS signals corresponding to their PEGylated species. Indeed, in the spectral region between 760-810 m/z, a signal can be
observed at 794.48 m/z, which corresponds to the ion with three charges of fragment 80-96 modified with a single chain of PEG (Figure 5C). Since fragments 80-96 and 79-96 contain the single Gln91 residue, these data indicate that TGase catalyzes the conjugation of PEG at the level of Gln91 of apoMb.
CONCLUSION The results here reported demonstrate that the use of a low molecular weight monodisperse PEG (Boc-PEG-NH2) allows for an accurate identification of the sites of TGase-mediated PEGylation by ESI-MS and MS/MS analyses. The technique requires a small amount of protein and a limited number of analytical steps, at variance from the more laborious analyses so far employed in the characterization of proteins conjugated to high molecular weight polydisperse PEGs. A striking observation of this study is that the use of TGase in the enzymatic PEGylation yields to the specific modification of only few Gln residues, giving rise to mono- or bis-PEG derivatives. Even if GCSF, hGH, and apoMb contain 17, 11, and 6 Gln residues, respectively, just one Gln in GCSF and apoMb and two in hGH were selectively PEGylated. The reason for this selectivity resides in the fact that a flexible polypeptide substrate
Technical Notes
Bioconjugate Chem., Vol. 20, No. 2, 2009 389
ACKNOWLEDGMENT This research was supported in part by the Italian Ministry of University and Research (PRIN-2006 No. 03035 and FIRB2003 No. RBNEOPX83). The authors are grateful to BioKer Srl (Pula, Italy) for a generous supply of protein samples of GCSF and hGH. The excellent technical assistance of Marcello Zambonin is also gratefully acknowledged.
LITERATURE CITED
Figure 5. Electrospray mass spectra of the digests of PEG-hGH and PEG-apoMb with trypsin. (A,B) The regions between 420-500 m/z and 520-600 m/z, respectively, of the mass spectrum of the digest of PEG-hGH are shown. The signals of the modified peptides 39-41 and 141-145 are indicated. (C) The signals of PEGylated peptide 80-96 and of peptide 17-31 are indicated in the region 760-810 m/z of the mass spectrum of the digest of PEG-apoMb.
is required for a productive binding and adaptation at the active site of TGase. Indeed, the modified Gln residues of the three proteins herewith investigated are all located in disordered regions of the proteins (16). The method described here considers that the modification of proteins mediated by TGase with the low molecular weight PEG occurs at the same Gln residues as those derivatized with the high molecular weight polydisperse polymer. This assumption appears to be valid, since the TGase-modified Gln residues are dictated by the structure and dynamics of the protein substrate and not by features of the primary amines used as acyl acceptors (16). Indeed, the results of this study, based on monodisperse PEG, are in agreement with those of recent reports showing that GCSF is selectively PEGylated at the level of Gln134 (17) and hGH at Gln40 and Gln141 (18) by TGase and high molecular weight PEGs (20 kDa). Therefore, overall our results indicate that BocPEG-NH2 can find a general application for the identification by ESI/MS and MS/MS of the site(s) of protein PEGylation mediated by TGase. Finally, the selectivity of the TGasemediated reactions suggests a wider application of this enzyme for protein modification, besides PEGylation. While the TGasemediated products can be homogeneous, a complex mixture of isomers is instead usually obtained by other chemical processes.
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