Elucidation of PEGylation site with a combined approach of in-source fragmentation and CID MS/MS

Poly(ethylene glycol) (PEG)ylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG heterogeneity, site of addition and number of attached PEGylated moieties. Recently, we published a novel LC/MS methodology with a post-column addition of amines to obtain accurate masses of PEGylated peptides and proteins. The accurate masses can be used to assign the structures and number of attached PEGs [15], but the PEGylation site remains unclear in situations where multiple potential attachments are involved. Here, we present a methodology combining in-source fragmentation (ISF) with CID-MS/MS to elucidate the PEGylated sites in PEGylated products. All PEGylated samples, either prepared in acidic solution, or collected from a RP-HPLC stream, were first ionized via ISF to produce products containing small PEG fragment attachment, and then those fragment ions obtained were sequenced via CID MS/MS to deduce the PEGylation site. The methodology was successfully applied to PEGylated glucagon and IgG4 antibody light chain, which demonstrated that the small PEG fragments attached were stable during the CID activation.     

Direct Quantitative Analysis of a 20 kDa PEGylated Human Calcitonin Gene Peptide Antagonist in Cynomolgus Monkey Serum Using In-Source CID and UPLC-MS/MS

PEGylation is a successful strategy to improve the pharmacokinetic and pharmaceutical properties of therapeutic peptides. However, quantitative analysis of PEGylated peptides in biomatrix by LC-MS/MS poses significant analytical challenge due to the polydispersity of the polyethylene glycol (PEG), and the multiple charge states observed for both the peptide and PEG moieties. In this report, a novel LC-MS/MS method for direct quantitative analysis of 20 kDa PEGylated CGRP[Cit, Cit] in cynomolgus monkey serum is presented. CGRP[Cit, Cit] is an investigational human calcitonin gene peptide receptor antagonist with amino acid sequence Ac -WVTH[Cit]LAGLLS[Cit]SGGVVRKNFVPT DVGPFAF-NH ₂ . In-source collision-induced dissociation (in-source CID) of 20 kDa PEGylated peptide was used to generate CGRP[Cit, Cit] fragment ions, among which the most abundant b ₈⁺ ion was selected and measured as a surrogate for the 20 kDa PEGylated peptide. A solid phase extraction (SPE) method was used to extract the PEGylated peptides from the biomatrix prior to the UPLC-MS/MS analysis. This method achieved a lower limit of quantitation (LLOQ) of 5.00 ng/mL with a serum sample volume of 100 μL, and was linear over the calibration range of 5.00 to 500 ng/mL in cynomolgus monkey serum. Intraday and interday accuracy and precision from QC samples were within ±15%. This method was successfully applied to a pharmacokinetic study of the 20 kDa PEGylated CGRP[Cit, Cit] in cynomolgus monkeys.     

胸腺素α1类似物的定点PEG修饰及其免疫活性评价

PEG修饰是改善蛋白质及肽类药物药代动力学特性的有效途径。然而与蛋白质相比，肽类化合物的分子较小，PEG的分子体积较大，其长链很可能会遮蔽肽的活性位点。因此，肽类化合物PEG修饰的位置和数量对于保持肽的生物活性至关重要。为阐明PEG修饰的位置与肽生物活性之间的关系，对肽类药物日达仙（胸腺素α1，Tα1）进行了定点修饰。Tα1具有α-螺旋、β-转角和无规卷曲的结构区域。分别在这些区域选择不同的位点进行PEG修饰。PEG的定点修饰是通过引入Cys，利用其-SH与mPEG-MAL的特异性反应而实现的。Con A刺激下的脾细胞产生IFN-γ试验的初步结果表明，PEG修饰对活性的影响与修饰的位置有一定的关系，大多数情况下，PEG修饰能保持Tα1的免疫活性。PEG修饰的位点对于保持肽的生物活性是很重要的。     

N-terminal H3/D3-acetylation for improved high-throughput peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer

A novel method for peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) is presented. A stable isotope label introduced in the peptide N-terminus by derivatization, using a 1:1 mixture of acetic anhydride and deuterated acetic anhydride, allows for easy and unambiguous identification of ions belonging either to the N- or the C-terminal ion series in the product ion spectrum, making sequence assignment significantly simplified. The good performance of this technique was shown by successful sequencing of the contents of several peptide maps. A similar approach was recently applied to nanoelectrospray ionization (nanoESI) and nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS). The MALDI-TOF/TOF technique allows for fast, direct sequencing of modified peptides in proteomics samples, and is complementary to the nanoESI and nanoLC/MS/MS approaches.     

反相高效液相色谱/基质辅助激光解吸电离飞行时间质谱 分离鉴定螺蛳血管紧张素转换酶抑制肽

运用反相高效液相色谱(RP-HPLC)对酶解螺蛳腹足肌得到的血管紧张素转换酶(ACE)抑制肽进行两步分离提纯,第一步主要得到8个组分;选取其中活性最高的组分进一步分离,得到2个组分,其中活性较高组分的ACE半抑制浓度为43.5 μmol/L,基本为单一肽组分。对提纯的组分分别使用高效液相色谱/电喷雾离子质谱法(HPLC/ESI-MS)和基质辅助激光解吸电离飞行时间质谱法(MALDI-TOF MS)进行分析,同时结合氨基酸组成分析结果,最终得到的肽链一级结构为Lys-Glu-Ile-Trp(KEIW),符合已知的高活性ACE抑制肽的结构规律。经过对两种方法分析过程的比较,认为ESI-MS可以得到多方面的信息,但无法确定肽的序列;MALDI-TOF MS可以得到精确的二级质谱图(m/z精确至0.0001),从而可以得到确定的肽的序列。     

Synthesis,Photophysics and PDT Evaluation of Mono‐, Di‐, Tri‐ and Hexa‐PEG Chlorins for Pointsource Photodynamic Therapy

Pointsource photodynamic therapy (PSPDT) is a newly developed fiber optic method aimed at the delivery of photosensitizer, light and oxygen to a diseased site. Because of a need for developing photosensitizers with desirable properties for PSPDT, we have carried out a synthetic, photophysical and phototoxicity study on a series of PEGylated sensitizers. Chlorin and pheophorbide sensitizers were readily amenable to our synthetic PEGylation strategy to reach triPEG and hexaPEG galloyl pheophorbides and mono‐, di‐, triPEG chlorins. On screening these PEG sensitizers, we found that increasing the number of PEG groups, except for hexaPEGylation, increases phototoxicity. We found that three PEG groups but not less or more were optimal. Of the series tested, a triPEG gallyol pheophorbide and a triPEG chlorin were the most efficient at generating singlet oxygen, and produced the highest phototoxicity and lowest dark toxicity to Jurkat cells. A detailed kinetic analysis of the PEGylated sensitizers in solution and cell culture and media is also presented. The data provide us with steps in the development of PSPDT to add to the PDT tools we have in general.     

Protein PEGylation attenuates adsorption and aggregation on a negatively charged and moderately hydrophobic polymer surface

Covalent grafting of poly(ethylene glycol) chains to proteins ("PEGylation") is emerging as an effective technique to increase the in vivo circulation time and efficacy of protein drugs. PEGylated protein adsorption at a variety of solid/aqueous interfaces is a critical aspect of their manufacture, storage, and delivery. A special category of block copolymer, PEGylated proteins have one or more water-soluble linear polymer (PEG) blocks and a single globular protein block that each exert distinct intermolecular and surface interaction forces. We report the impact of PEGylation on protein adsorption at the interface between aqueous solutions and solid films of poly(lactide-co-glycolide) (PLG), a moderately hydrophobic and negatively charged polymer. Using the model protein lysozyme with controlled degrees of PEGylation, we employ total internal reflection fluorescence techniques to measure adsorption isotherms, adsorption reversibility, and the extent of surface-induced aggregation. Lysozyme PEGylation reduces the extent of protein adsorption and surface-induced aggregation and increases the reversibility of adsorption compared to the unconjugated protein. Results are interpreted in terms of steric forces among grafted PEG chains and their effects on protein-protein interactions and protein orientation on the surface.     

Automated protein identification using atmospheric-pressure matrix-assisted laser desorption/ionization

Atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) ion trap mass spectrometry (ITMS) has been evaluated for automated protein identification. By using signal averaging and long ion-injection times, protein identification limits in the 50-fmol range are achieved for standard protein digests. Data acquisition requires 7.5 min or less per sample and the MS/MS spectra files are automatically processed using the SEQUEST database searching algorithm. AP-MALDI-ITMS was compared with the widely used methods of microLC/MS/MS (ion trap) and automated MALDI-TOF peptide mass mapping. Sample throughput is 10-fold greater using AP-MALDI compared with microcapillary liquid chromatography/tandem mass spectrometry (microLC/MS/MS). The protein sequence coverage obtained from AP-MALDI-MS/MS spectra matched by SEQUEST is lower compared with microLC/MS/MS and MALDI-TOF mass mapping. However, by using the AP-MALDI full-scan peptide mass fingerprint spectrum, sequence coverage is increased. AP-MALDI-ITMS was applied for the analysis of Coomassie blue stained gels and was found to be a useful platform for rapid protein identification.     

Site-specific PEGylation of Human Growth Hormone by Mutated Sortase A

Human growth hormone(hGH), a classic therapeutic protein, which promotes growth and wound healing, is released from the pituitary gland. As a protein drug, its short half-life is its main barrier to therapeutic efficacy. Various strategies have been designed to prolong its serum half-life, the most common of which is the conjugation with polyethylene glycol(PEG), as this has been shown to significantly extend protein's serum half-life. However, PEGylation often results in random conjugation, which can lead to impaired protein function and hinder purification, characterization and evaluation of the PEGylated protein. Therefore, site specific PEGylation is a promising direction for PEG-protein conjugation. Here we took advantages of the mutated sortase A(7M) enzyme, which can enzymatically ligate the universal α-amino acids to a C-terminal tagged protein. This then allows specific modification of the C-terminal of hGH with PEG. This site-specific bound PEG-hGH has similar efficacy, receptor binding and cell proliferation as wild-type hGH; however, pharmacokinetic analysis demonstrates that its serum half-life is almost 24 times that of wild-type hGH. Herein, we provided a promising advancement in the development of site specific PEGylated therapeutic proteins.     

Design of poly(ethylene glycol)/streptavidin coimmobilized upconversion nanophosphors and their application to fluorescence biolabeling

Infrared-to-visible upconversion phosphors (i.e., rare earth ion-doped Y2O3 nanoparticles (UNPs)) were synthesized by the homogeneous precipitation method. Because the charge on the erbium (Er) ion-doped Y2O3 (Y2O3:Er) NP (UNP1) surface is positive under neutral conditions, the UNP1 surface was electrostatically PEGylated using negatively charged poly(ethylene glycol)- b-poly(acrylic acid) (PEG- b-PAAc). The adsorption of PEG- b-PAAc was confirmed by Fourier transform infrared (FT-IR) measurements and thermal gravimetric analysis (TGA). The surface charge of the PEGylated UNP1s (PEG-UNP1s) was effectively shielded by the PEGylation. The dispersion stability of the UNP1s was also significantly improved by the PEGylation. The PEG-UNP1s were dispersed over 1 week under physiological conditions as a result of the steric repulsion between the PEG chains on the UNP1 surface. The upconversion emission spectrum of PEG-UNP1s was observed under physiological conditions and was confirmed by near-infrared excited fluorescence microscope observation. Streptavidin (SA)-installed ytterbium (Yb) and Er ion-codoped Y2O3 (Y2O3:Yb,Er) NPs (UNP2s) were prepared by the coimmobilization of PEG- b-PAAc and streptavidin. The PEG/SA coimmobilized UNP2s (PEG/SA-UNP2s) specifically recognized biotinylated antibodies and emitted strong upconversion luminescence upon near-infrared excitation. The obtained PEG/streptavidin coimmobilized UNPs are promising as high-performance near-infrared biolabeling materials.     

A novel approach enables imaged capillary isoelectric focusing analysis of PEGylated proteins

PEGylation has been used as a strategy to enhance pharmacokinetic properties of therapeutic proteins by pharmaceutical industry. Imaged CIEF (iCIEF) is the current industry standard technology for pI determination and charge variant quantification of proteins and antibodies. However, the charge variants of PEGylated proteins merge into one broad peak during iCIEF, most likely due to masking of proteins by the surrounding PEG chain as well as the increased hydrodynamic volume due to PEGylation. Here, we report our novel matrix formula with a combination of glycine and taurine that significantly improved the separation of charge variants in PEGylated proteins. As a result, it is no longer necessary to conduct IEF of proteins prior to PEGylation, which does not reflect the changes caused by PEGylation and purification processes. The novel matrix (glycine and taurine) enables iCIEF analysis of PEGylated proteins in their real conjugated states.     

De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature

De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.     

Native PAGE eliminates the problem of PEG-SDS interaction in SDS-PAGE and provides an alternative to HPLC in characterization of protein PEGylation

PEGylation of proteins has become an increasingly important technology in recent years. However, determination and characterization of the PEGylation products are problematic especially for the reaction mixture containing various modified proteins, unreacted PEG, and unmodified protein. A comparative study was carried out with two HPLC methods and two electrophoresis methods for characterization of the reaction mixture in PEGylation of HSA with PEG 5000, 10000, and 20000. RP-HPLC fails to give the correct information about the reaction of PEG 20000. Size-exclusion HPLC (SE-HPLC) produced very poor resolution on the PEG 5000 reaction. SDS-PAGE can run multiple samples of all PEGylation but the bands were smeared or broadened probably due to the interaction between PEG and SDS. On the other hand, native PAGE eliminates the problem of PEG-SDS interaction and provides better resolutions for all samples. Various PEGylated products and unmodified protein migrate differentially in native PAGE under nondenatured conditions. The results demonstrated that native PAGE could be a good alternative to HPLC and SDS-PAGE for the analysis of PEG-protein conjugates especially for characterization of the PEGylation mixture.     

NMR structure of two novel polyethylene glycol conjugates of the human growth hormone-releasing factor,hGRF(1-29)-NH2

Two novel mono-PEGylated derivatives of hGRF(1-29)-NH(2) [human growth hormone-releasing factor, fragment 1-29] have been synthesized by regio-specific conjugation of Lys(12) or Lys(21) to a monomethoxy-PEG(5000) chain (compounds Lys(12)PEG-GRF and Lys(21)PEG-GRF). The PEG moiety has been covalently linked at the amino group of a norleucine residue via a carbamate bond. The Lys(12)PEG-GRF regioisomer was found to be slightly less active in vitro than both the unmodified peptide and Lys(21)PEG-GRF. To assess whether the differences in the biological activity of the PEGylated analogues could be related to conformational rearrangements induced by the PEG moiety, the structure of these PEGylated derivatives has been worked out (TFE solution) by means of NMR spectroscopy and molecular dynamics. Secondary structure shifts, hydrogen/deuterium exchange kinetics, temperature coefficients of amide protons, and NOE-based molecular models point out that hGRF(1-29)-NH(2), Lys(21)PEG-GRF and Lys(12)PEG-GRF share a remarkably similar pattern of secondary structure. All three compounds adopt an alpha-helix conformation which spans the whole length of the molecule, and which becomes increasingly rigid on going from the N-terminus to the C-terminus. Residues Lys(12) and Lys(21) are enclosed in all the compounds considered into well-defined alpha-helical domains, indicating that PEGylation either at Lys(12) or Lys(21) does not alter the tendency of the peptide to adopt a stable alpha-helix conformation, nor does it induce appreciable conformational mobility in the proximity of the PEGylation sites. No significant variation of the amphiphilic organization of the alpha-helix is observed among the three peptides. Therefore, the different biological activities observed for the PEGylated analogues are not due to conformational effects, but are rather due to sterical hindrance effects. The relationship between the biological activitiy of the mono-PEGylated derivatives and sterical hindrance is discussed in terms of the topology of interaction between hGRF(1-29)-NH(2) and its receptor.     

Optimization of matrix-assisted laser desorption/ionization time-of-flight collision-induced dissociation using poly(ethylene glycol)

Details of the optimization of the collision-induced dissociation (CID) process, using a collision cell on a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer, are described using poly(ethylene glycol) 1000 (PEG 1000) as a model analyte. The effects of collision gas identity (helium, air, and argon), as well as collision gas pressure, on the resulting MS/MS data were investigated. With PEG 1000, helium was found to give the best results with respect to signal-to-noise (S/N) ratio. The optimum pressure for each gas was found to be in the range where the precursor ion signal was attenuated to approximately 30-50% for helium and 40-60% for argon. The effect of cation choice (Li, Na, and K) on the CID of PEG was also studied. CID spectra were produced for each, but PEG cationized with lithium was found to produce the spectra with the highest S/N ratio. The MALDI-TOF CID spectra that were generated for PEG were compared with the high-energy and low-energy MS/MS spectra obtained from a sector mass spectrometer and from a triple quadrupole mass spectrometer, respectively. The results observed for PEG confirm that CID on a MALDI-TOF mass spectrometer is a high-energy MS/MS technique.     

Separation of positional isomers of mono-poly(ethylene glycol)-modified octreotides by reversed-phase high-performance liquid chromatography

The purpose of this study was to develop a reversed-phase high-performance liquid chromatographic method (RP-HPLC) for separating each positional isomer from low- to high-molecular-weight mono-PEGylated octreotides prepared by polyethylene glycol (PEG) derivatives with various molecular weights (2, 5, or 20 kDa). In the gradient elution using acetonitrile and 10 mM phosphate buffer at pH 7.0 on a Phenomenex Gemini C-18 column (250 mm × 4.6 mm id, 5 μm), each positional isomer of the mono-PEGylated octreotides was completely resolved with good resolution (PEG-2K: 7.6, PEG-5K: 6.6, and PEG-20K: 3.1). The optimal RP-HPLC condition also resolved the degradation products of mono-PEG-octreotide isomers in thermal stability studies at 55 °C and enzymatic stability studies with trypsin. In conclusion, the developed RP-HPLC method will be valuable for studying the effect of PEGylation site and the attached PEG size on the physicochemical and pharmacological properties of PEGylated octreotides.     

Interactions of ADP-stimulated human platelets with PEGylated polystyrene substrates prepared by surface amidation

A study primarily focused on the interactions between ADP-stimulated human platelets and PEGylated polystyrene substrates is described in this paper. The platelet–surface interactions were investigated using colorimetric acid phosphatase assay. Two types of amine-containing polymeric hydrogel materials based on poly(ethylene glycol) (PEG), H₂N–PEG–OCH₃ and H₂N–PEG–NH₂, were used to PEGylate polystyrene surfaces derivatized with maleic anhydride by amidation at alkaline pH. In addition, comparative studies using surfaces non-covalently adsorbed by bovine serum albumin (BSA) or fibrinogen (Fg) were also conducted. The assay results showed that no significant platelet adhesion was observed when PEGylated surfaces or BSA-coated surfaces were exposed to unstimulated gel-filtered platelets (GFP). However, upon ADP-stimulation, platelet adhesion to the surfaces under investigation in this study all increased to varying degrees. Most importantly, the results showed that polystyrene surfaces PEGylated using H₂N–PEG–NH₂ were most effective in resisting platelet adhesion when assays were performed using ADP-stimulated GFP. By PEGylating the surfaces of polystyrene microtiter wells via the amidation reaction described in this paper, it is demonstrated that (i) higher degree of surface PEGylation is favored at more alkaline pH and (ii) polystyrene substrates capable of more effectively resisting the adhesion of ADP-stimulated GFP can be obtained by the PEGylation reaction carried out at pH 9.1 using H₂N–PEG–NH₂.     

Mass calibration of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer including the rise time of the delayed extraction pulse

To utilize fully modern MALDI-TOF and TOF/TOF mass spectrometers with mass resolution exceeding 10,000 and 2 ppm precision of flight time measurements for high mass accuracy, the model of ion motion used in the mass calibration equation must be expanded. The standard three-term equation providing up to 5-10 ppm (rms) mass accuracy with internal standards was modified with an additional term accounting for the finite rise time of the high-voltage extraction pulse. This new four-term calibration equation minimizes the effect of systematic error resulting from the fact that ion velocities are mass dependent due to the rise time of the extraction pulse. Applying this new calibration equation to a mass spectrum obtained in an axial MALDI-TOF MS containing 70 peaks (sodiated PEG), each with a signal-to-noise ratio greater than 100, a mass accuracy of 1.6 ppm (rms) was obtained over the mass range 1.0-4.0 kDa compared with 3.6 ppm (rms) with the standard three-term equation. The physical basis of the effects of the finite extraction pulse rise time on mass calibration is examined for axial MALDI-TOF mass spectrometers, as well as for orthogonal acceleration TOF mass spectrometers.     

Using Hydrogen/Deuterium Exchange Mass Spectrometry to Study Conformational Changes in Granulocyte Colony Stimulating Factor upon PEGylation

PEGylation is the covalent attachment of polyethylene glycol to proteins, and it can be used to alter immunogenicity, circulating half life and other properties of therapeutic proteins. To determine the impact of PEGylation on protein conformation, we applied hydrogen/deuterium exchange mass spectrometry (HDX MS) to analyze granulocyte colony stimulating factor (G-CSF) upon PEGylation as a model system. The combined use of HDX automation technology and data analysis software allowed reproducible and robust measurements of the deuterium incorporation levels for peptic peptides of both PEGylated and non-PEGylated G-CSF. The results indicated that significant differences in deuterium incorporation were induced by PEGylation of G-CSF, although the overall changes observed were quite small. PEGylation did not result in gross conformational rearrangement of G-CSF. The data complexity often encountered in HDX MS measurements was greatly reduced through a data processing and presentation format designed to facilitate the comparison process. This study demonstrates the practical utility of HDX MS for comparability studies, process monitoring, and protein therapeutic characterization in the biopharmaceutical industry.     

Microcalorimetric study of the adsorption of PEGylated lysozyme on a strong cation exchange resin

Adsorption of native as well as mono-, di-, and tri-PEGylated lysozyme on Toyopearl Gigacap S-650M in sodium phosphate buffer is studied by isothermal titration calorimetry and by independent adsorption equilibrium measurements at pH 6 and 25°C. The production and separation of PEGylated lysozyme is discussed. Two different PEG sizes are used (5 kDa and 10 kDa) which leads to six different forms of PEGylated lysozyme which were systematically studied. The sodium chloride concentration is varied according to the elution conditions in the production process. The specific enthalpy of adsorption Δh(p)(ads) is determined from the calorimetric and the adsorption equilibrium data. It was found to be exothermal and constant with increasing adsorber loading for native lysozyme. For all PEGylated forms there is an influence of the adsorber loading on Δh(p)(ads) which is found to become more important with increasing PEGylation degree (total molecular weight of conjugated PEG). At low loadings the adsorption of all PEGylated lysozyme forms is exothermal. With increasing loading the adsorption becomes less exothermal and for the species with higher PEGylation degree also endothermal effects are observed at higher loadings. A thermodynamic analysis is carried out by which the enthalpic and entropic contributions to the binding constants are quantified. The findings are discussed on a molecular level. The results provide insight into the adsorption mechanisms of polymer-modified proteins on chromatographic resins.