Fingerprinting proteins coupled with polymers by mass spectrometry: Investigation of polyethylene glycol-conjugated superoxide dismutase

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry was investigated as a method for the rapid determination of the extent of polymer coupling in polyethylene glycol- (PEG) conjugated superoxide dismutase (SOD). PEG-conjugated SOD, an antioxidant with an extended in vivo circulation lifetime compared to that of superoxide dismutase, is being evaluated as an effective therapeutic agent for the treatment of injuries and arthritis. The mass spectra of a standard batch of PEG-conjugated bovine SOD showed the presence of identifiable and well resolved peaks that correspond to 0–7 PEG molecules attached to bovine SOD. The area of each of the peaks provides a determination of the amount of PEG-conjugated SOD with a given number of bound PEG groups. SOD is a noncovalent dimer of two identical subunits that dissociates in MALDI. The information obtained in the mass spectra thus corresponds to a monomer of SOD. Each SOD monomer contains 10 lysines, which are the sites of PEG-conjugation. Multiple MALDI determinations of two batches of samples indicated good reproducibility for routine determination of the extent of polymer content. The amount of PEG-conjugated SOD that contained a given number of PEG molecules, determined by MALDI, was compared with the value deduced from the amount of PEG-conjugation at each attachment site measured by a peptide mapping method. Agreement between the data obtained in the two techniques (MALDI and peptide mapping) indicates that MALDI may be used to obtain quantitative information on PEG-conjugated SOD to determine the amounts of PEG-conjugated protein each with a different number of PEG groups attached. Measurement of several batches of samples stored at a higher temperature showed a lower extent of PEG-conjugation in PEG-conjugated SOD. This reduction in the PEG content resulted from the PEG-deconjugation of PEG-conjugated SOD at a higher temperature. Thus, MALDI can be used to examine the stability of PEG-conjugated SOD. The high sensitivity, relatively straightforward data interpretation, speed of analyses, and good reproducibility in measurements make this technique a useful analytical tool for fingerprinting PEG-conjugated SOD as well as potentially other polymer-conjugated proteins.     

Separation and determination of polyethylene glycol fatty acid esters in cosmetics by a reversed-phase HPLC/ELSD

A comprehensive analytical method was established for the separation of polyethylene glycol (PEG) stearates according to the distribution of ethylene oxide (EO) and subsequent determination of the surfactants in cosmetic samples by using a high-performance liquid chromatography–evaporative light scattering detection. Separation of the PEG stearates comprising approximately up to 82 EO adducts was performed on a reversed-phase YMC-Pack C₈ column using water–acetonitrile gradient elution. The PEG oligomers were separated in order of the increasing number of EO adducts. Quantitation of the PEG fatty acid esters, which was separated as single peak per each component, was performed by chromatography on a reversed-phase Wakosil 10 C₁₈ column using water–methanol gradient elution. The standard curve to quantify the PEG stearates was constructed by the log–log plot, which showed good linearity with the correlation coefficients (R²) 0.998 and more. Working range, repeatability, limit of detection and recovery were acceptable for analysis of the surfactants in cosmetic products. The analytical methods were applied to characterize the PEG stearates according to the EO distributions, then to quantify the surfactants in cosmetic products.     

The influence of electrospray deposition in matrix-assisted laser desorption/ionization mass spectrometry sample preparation for synthetic polymers

Although electrospray sample deposition in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) sample preparation increases the repeatability of both the MALDI signal intensity and the measured molecular mass distribution (MMD), the electrospray sample deposition method may influence the apparent MMD of a synthetic polymer. The MMDs of three polymers of differing thermal stability, polystyrene (PS), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG), were studied by MALDI time-of-flight (TOF) MS as the electrospray deposition voltage was varied. The MMDs obtained using the electrospray deposition method were compared with those obtained for hand-spotted samples. No change was observed in the measured polymer MMD when the electrospray deposition voltage was varied in the analysis of PS, but those of PEG and PPG changed at higher electrospray voltages due to increased ion fragmentation. It was also shown that the fragmentation in the hand-spotted samples is dependent on the matrix used in sample preparation.     

The fragmentation of ethoxylated surfactants by AP-MALDI-QIT

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important technique to characterize the chemical structure of industrial polymer materials. MALDI methods have been developed to address a broad variety of different polymer materials containing different chemistries. One of the key aspects of the typical MALDI experiment is the generation of intact ions. The development of Atmospheric Pressure (AP) MALDI quadrupole ion trap (QIT) instruments has opened another channel to obtaining MS/MS experiments for polymer samples. These experiments provide a new method to obtain chemical structure information from MALDI experiments. Collision-Induced Dissociation (CID) provides an improved MALDI MS/MS experiment that can be done on readily available mass spectrometers. AP MALDI QIT techniques have been successfully applied to a variety of synthetic polymers. This work explores the applicability of AP MALDI QIT methods to relatively low molecular weight ethoxylated surfactants. In these experiments we show the CID fragmentation mass spectra on some ethoxylated surfactants, and demonstrate the existence of analyte matrix clusters.     

染料敏化太阳能电池用琼脂糖基磁性聚合物电解质的电化学性能

以琼脂糖为聚合物基质,N-甲基吡咯烷酮为溶剂,磁性纳米粒子四氧化三铁为无机纳米颗粒添加剂制备了用于染料敏化太阳能电池(DSSC)的磁性聚合物电解质.通过研究不同小分子表面活性剂,聚乙二醇(PEG200)、曲拉通(TritonX-100)、乙酰丙酮和三者混合的表面活性剂对掺杂有1%(w)Fe3O4的磁性聚合物电解质离子电导率的影响,发现PEG200的加入可有效提高琼脂糖基磁性聚合物电解质的离子电导率.同时,对不同PEG200浓度添加下的电解质进行离子电导率测试研究发现:当PEG200加入量为61.8%(w)时,电解质具有最佳的离子电导率(2.88×10-3S·cm-1);对染料敏化太阳能电池进行电化学交流阻抗谱(EIS)测试的结果表明:染料敏化太阳能电池的电子寿命和复合电阻随着PEG200浓度的增加是先增大后减小,最大的电子寿命和复合电阻出现在PEG200浓度为68.3%(w)处.     

Exact mass measurements using a 7 tesla fourier transform ion cyclotron resonance mass spectrometer in a good laboratory practices-regulated environment

Fourier transform ion cyclotron resonance mass spectrometry has been found to produce reliable exact mass measurements using two different internal calibration methods. For these measurements, electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) were utilized both individually and in tandem. For internal calibration with a co-dissolved polyethylene glycol standard, measurements of 41 compounds resulted in an average absolute mass determination error of 0.7 ppm, with a standard deviation of 0.9 ppm. For comparison, internal calibration was effected through the simultaneous use of ESI and MALDI, with the former being used for the introduction of analyte ions and the latter for formation of polymethylmethacrylate calibrant ions. This technique led to mass measurements with an average absolute error of 0.8 ppm and a standard deviation of 1.0 ppm. In addition, exact mass measurements of tandem mass spectrometry fragment ions were made for 35 compounds using external calibration with a single internal mass standard. The observed average absolute error was 0.7 ppm with a standard deviation of 1.0 ppm.     

The analysis of industrial synthetic polymers by electrospray droplet impact/secondary ion mass spectrometry

Electrospray droplet impact (EDI)/secondary ion mass spectrometry (SIMS) is a new desorption/ionization technique for mass spectrometry in which highly charged water clusters produced from the atmospheric‐pressure electrospray are accelerated in vacuum by several kV and impact the sample deposited on the metal substrate. In this study, several industrial synthetic polymers, e.g. polystyrene (PS) and polyethylene glycol (PEG) were analyzed by EDI/SIMS mass spectrometry. For higher molecular weight analytes, e.g. PS4000 and PEG4600, EDI/SIMS mass spectra could be obtained when cationization salts are added. For the polymers of lower molecular weights, e.g. PEG300 and PEG600, they could be readily detected as protonated ions without the addition of cationization agents. Anionized PS was also observed in the negative ion mode of operation when acetic acid was added to the charged droplet. Compared to matrix‐assisted laser desorption/ionization (MALDI), ion signal distribution with lower background signals could be obtained particularly for the low‐molecular weight polymers. Copyright © 2009 John Wiley & Sons, Ltd.     

Atmospheric pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry of synthetic polymers: a comparison with vacuum matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI/QIT) mass spectrometry has been investigated for the analysis of polyethylene glycol (PEG 1500) and a hyperbranched polymer (polyglycidol) in the presence of alkali-metal salts. Mass spectra of PEG 1500 obtained at atmospheric pressure showed dimetallated matrix/analyte adducts, in addition to the expected alkali-metal/PEG ions, for all matrix/alkali-metal salt combinations. The relative intensities of the desorbed ions were dependent on the matrix, the alkali-metal salt added to aid cationisation and the ion trap interface conditions [capillary temperature, in-source collisionally-induced dissociation (CID)]. These data indicate that the adducts are rapidly stabilised by collisional cooling enabling them to be transferred into the ion trap. Experiments using identical sample preparation conditions were carried out on a vacuum MALDI time-of-flight (ToF) mass spectrometer. In all cases, vacuum MALDI-ToF spectra showed only alkali-metal/PEG ions and no matrix/analyte adducts. The tandem mass spectrometry (MS/MS) capability of the ion trap has been demonstrated for a lithiated polyglycol yielding a rich fragment-ion spectrum. Analysis of the hyperbranched polymer polyglycidol by AP-MALDI/QIT reveals the characteristic ion series for these polymers as also observed under vacuum MALDI-ToF conditions.     

拥挤环境中表面活性剂胶束化过程的低场核磁共振研究

利用长弛豫低场核磁共振(LF-NMR)方法研究了由聚乙二醇(PEG)构建的拥挤溶液环境中表面活性剂的聚集行为。通过Carr-Purcell-Meiboom-Gill(CPMG)脉冲序列所得的回波数据及反演拟合数据对不同的表面活性剂-PEG体系进行分析,结果发现:不同分子量和不同浓度的PEG溶液可用低场核磁共振方法进行区分;高浓度的PEG溶液中均能监测到胶束形成过程,而较低浓度的PEG溶液不利于其胶束化过程监测;NaCl对含有离子型表面活性剂体系的影响较为明显,而对含有非离子型表面活性剂的体系几乎无影响。该研究丰富了低场核磁共振弛豫技术在表面活性剂胶束表征过程中的应用,同时对理解生物体系中分子的自组装及功能也具有重要意义。     

Melanogenesis by tyrosinase action on 3,4-dihydroxyphenylalanine (DOPA) in the presence of polyethylene glycol: a matrix-assisted laser desorption/ionization mass spectrometric investigation

The enzymatic reaction between DOPA and tyrosinase, the enzyme considered to be responsible for melanogenesis, was carried out in the presence of polyethylene glycol (PEG). This choice was made in order to increase the solubility of melanins, since these polymers are highly insoluble. The reaction mixtures were sampled at different times, immediately ultrafiltered to remove the enzyme, lyophilized, and analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The results were very different from those obtained in the absence of PEG. Only a few oligomers of dihydroxyindole (DHI) and dihydroxyindole-2-carboxylic acid (DHICA) were detected in low abundances, whereas new species originating from reaction of PEG with species belonging to the Raper-Mason pattern appeared. The results show that, in the presence of PEG, tyrosinase-catalyzed oligomerization of DOPA exhibits kinetics slower than those observed in the absence of the polymer. However, melanogenesis still takes place in the presence of PEG, as indicated by the formation of black pigments and by the detection of DHI and DHICA oligomers, considered to be the first intermediates in melanin formation.     

Separation and first structure elucidation of Cremophor EL-components by hyphenated capillary electrophoresis and delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry

The polyethoxylated heterogeneous components of the so far poorly characterized nonionic emulsifier Cremophor EL (polyoxyl 35 castor oil) (CrEL) were fractionated by cyclodextrin-modified micellar electrokinetic capillary chromatography (CD-MEKC). Due to the low UV absorbance of most of the CrEL-components an indirect UV detection was used with phenobarbital-sodium as background absorber. For a precise assignment of the resulting peaks to the corresponding components capillary electrophoresis (CE) had to be combined with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS) as detection system. For this purpose, the fractionating robot Probot was employed which enables both the on-line fractionation of the CE eluate on a MALDI target during the electrophoretic separation and the simultaneous dosage of the MALDI matrix solution. The applied CrEL amount was optimized by varying the CE injection parameters time, pressure and concentration of the sample in order to obtain homologue peak series of sufficient intensity without decreasing the separation efficiency. Evaluation of the mass spectra was performed by comparing the residue masses of the homologue peak series with the calculated residue masses of potential CrEL-components. However, the high number of polyethoxylated components leads to overlapping of homologue peak series with isobaric residue masses. These isobaric interferences were detected by a high mass accuracy of the measurements (obtained by internal calibration with polyethylene glycol (PEG) 1000 and by means of the residue mass plot, the newly developed evaluation method. The combination of these techniques allowed the first detailed structure analysis of the CrEL-components showing glycerol polyoxyethylene (POE) monoricinoleate and POE monoricinoleate to be the two main components of the emulsifier. Furthermore, the coupling of CE with DE-MALDI-TOF-MS is generally applicable to the fractionation and identification of polymers.     

Quantitative analysis of polydisperse systems via solvent-free matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Quantitative analysis of partially soluble and insoluble polydisperse materials is challenging due to the lack of both appropriate standards and reliable analytical techniques. To this end, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) incorporating a solvent-free sample preparation technique was investigated for the quantitative analysis of partially soluble, polydisperse, polycyclic aromatic hydrocarbon (PAH) oligomers. Molecular weight standards consisting of narrow molecular weight dimer and trimer oligomers of the starting M-50 petroleum pitch were produced using both dense-gas/supercritical extraction (DGE/SCE) and preparative-scale, gel permeation chromatography (GPC). The validity of a MALDI-based, quantitative analysis technique using solvent-free sample preparation was first demonstrated by applying the method of standard addition to a pitch of known composition. The standard addition method was then applied to the quantitative analysis of two insoluble petroleum pitch fractions of unknown oligomeric compositions, with both the dimer and trimer compositions of these fractions being accurately determined. To our knowledge, this study represents the first successful MALDI application of solvent-free quantitative analysis to insoluble, polydisperse materials.     

Solvent selection for matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analysis of synthetic polymers employing solubility parameters

The principle relating to the selection of a proper matrix, cationization reagent, and solvent for matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of synthetic polymers is still a topic of research. In this work we focused on the selection of a suitable MALDI solvent. Polystyrene PS7600 and poly(ethylene glycol) PEG4820 were analyzed by MALDI‐TOF MS using various solvents which were selected based on the Hansen solubility parameter system. For polystyrene (PS), dithranol was used as the matrix and silver trifluoroacetate as the cationization reagent whereas, for poly(ethylene glycol) (PEG), the combination of 2,5‐dihydroxybenzoic acid and sodium trifluoroacetate was used for all experiments. When employing solvents which dissolve PS and PEG, reliable MALDI mass spectra were obtained while samples in non‐solvents (solvents which are not able to dissolve the polymer) failed to provide spectra. It seems that the solubility of the matrix and the cationization reagent are less important than the polymer solubility. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification and quantification of polyethylene glycol types in polyethylene glycol methyl ether and polyethylene glycol vinyl ether

Quantitative determination of polyethylene glycol (PEG) impurities in two monofunctional polyglycol types, PEG methyl ether (M-PEG) and PEG vinyl ether (V-PEG), has been carried out by reversed-phase liquid chromatography with evaporative light scattering detection (ELSD). In addition to optimizing the resolution between PEG and monofunctional PEG peaks, the major focus has been to determine the molecular weights of PEG impurities in M-PEG and V-PEG of diverse molecular weights. The latter is achieved by examining liquid chromatography–mass spectrometry (LC–MS) mass spectra of both monofunctional PEG and PEG in several cases, and matching peak retention times with those of available PEG standards for all M-PEG and V-PEG sample types. This information is helpful in selecting the appropriate PEG standard to determine PEG content in each sample type. ELSD response factors for various PEG standards have also been compared. It has been found that PEG standards with molecular weights from 1000 Da to 8000 Da show responses that are within 10% of each other. However, a low molecular weight PEG such as PEG 400, provides approximately 30% less response compared to its higher molecular weight counterparts.     

Block length determination of the block copolymer mPEG‐b‐PS using MALDI‐TOF MS/MS

The molar mass determination of block copolymers, in particular amphiphilic block copolymers, has been challenging with chromatographic techniques. Therefore, methoxy poly(ethylene glycol)‐b‐poly(styrene) (mPEG‐b‐PS) was synthesized by atom transfer radical polymerization (ATRP) and characterized in detail not only by conventional chromatographic techniques, such as size exclusion chromatography (SEC), but also by matrix‐assisted laser/desorption ionization tandem mass spectrometry (MALDI‐TOF MS/MS). As expected, different molar mass values were obtained in the SEC measurements depending on the calibration standards (either PEG or PS). In contrast, MALDI‐TOF MS/MS analysis allowed the molar mass determination of each block, by the scission of the weakest point between the PEG and PS block. Thus, fragments of the individual blocks could be obtained. The PEG block showed a depolymerization reaction, while for the PS block fragments were obtained in the monomeric, dimeric, and trimeric regions as a result of multiple chain scissions. The block length of PEG and PS could be calculated from the fragments recorded in the MALDI‐TOF MS/MS spectrum. Furthermore, the assignment of the substructures of the individual blocks acquired by MALDI‐TOF MS/MS was accomplished with the help of the fragments that were obtained from the corresponding homopolymers. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Determination of polyethylene glycol in low-density polyethylene by large volume injection temperature gradient packed capillary liquid chromatography

Polyethylene glycol (PEG) 20000 in low-density polyethylene has been determined using column switching and inverse temperature programming in reversed-phase packed capillary liquid chromatography with evaporative light scattering detection. PEG 20000 was extracted into water from the polyethylene dissolved in toluene and PEG 35000 was added as an internal standard (I.S.). The samples in aliquots of 100 microl were reconcentrated on the enrichment column using a loading mobile phase of acetonitrile-water (3:97, v/v) at a flow-rate of 75 microl/min for 3 min, then back-flushed and separated on the analytical column with acetonitrile-THF-water (40:5:55, v/v) as mobile phase. The column temperature was reduced from 68 to 55 degrees C with a ramp of -1.5 degrees C/min, held constant for 3 min and then reduced further to 45 degrees C with a -1.5 degrees C/min ramp and kept constant for 1 min. The analysis runtime was 20 min. The recovery of PEG 20 000 was determined to 65.1% with 2.8% RSD and the mass limit of detection of PEG 20 000 was 1.25 microg. The within-assay and between day precision of the retention times of both PEG 20000 and PEG 35000 displayed RSD of less than 1.1% (n = 9), while the overall area ratio RSD of 100 microg/ml PEG 20000 over PEG 35000 was 1.3% (n = 9). The method was linear within the investigated concentration range 25-125 microg/ml (R2 = 0.9983). In addition, a mixture of PEG 4000, 8000, 10000, 20000 and 35000 was analysed on the system to demonstrate the possibility of analysing several PEGs in a sample with the use of temperature gradient elution.     

凝胶渗透色谱用聚丙烯酸标样的制备

以二氧六环为良溶剂,石油醚为沉淀剂,通过等温沉淀分级,制得8种不同分子量的聚丙烯酸标样。 采用渐近校正法测定了它们的峰值分子量和分子量分布,建立了GPC校正曲线,并将其用于聚丙烯酸试样的测定。 结果表明,由渐进校正法测得的粘均分子量与粘度法测得的分子量之间的平均相对误差为7%,由渐进校正法测得的数均分子量与端基滴定法测得分子量之间的平均相对误差为9%。 将上述标样用于实际聚丙烯酸试样的测定,测得试样的粘均分子量与粘度法测定结果之间平均相对误差小于10%,优于以聚乙二醇为标样测定的平均相对误差（30%左右）。 本标样可用于具有与聚丙烯酸相似结构的阴离子聚电解质分子量分布的测定。     

Selection of internal standards for quantitative analysis by matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry

The selection of an appropriate internal standard (IS) for quantification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is critical for the successful application of quantitative MALDI. Selection of the IS depends on the chemical similarity of the analyte and IS and the mass separation of the analyte and IS as a function of instrumental peak resolution. For the quantification of bovine insulin, a series of internal standards including horse heart cytochrome C, bovine insulin chain B, des-pentapeptide human insulin, and des-octapeptide porcine insulin was investigated. Des-pentapeptide human insulin was found to be the most appropriate internal standard (relative standard deviation of the standard curve slope = 2.99%, correlation coefficient = 0.988 in the range of 0.5-0.4 μmol/L). Two methods for measuring of the MALDI signal intensity were evaluated, direct peak integration following subtraction of a linear background and non-linear least squares curve fitting. The results obtained with these methods were equivalent.     

Improved matrix-assisted laser desorption/ionisation sample preparation of a partially depolymerised cellulose derivative by continuous spray deposition and interfacing with size-exclusion chromatography

Continuous spray deposition (CSD) of aqueous solutions of partially depolymerised methyl cellulose was found to improve matrix-assisted laser desorption/ionisation (MALDI) sample preparation. One feature was that the sensitivity in MALDI time-of-flight mass spectrometry increased up to an order of magnitude compared with the standard sample preparation method. Another feature was that CSD provided targets for MALDI with homogeneously distributed analyte. This resulted in a more even signal intensity and a higher reproducibility than in the standard method. High-mass discrimination was more pronounced in CSD than in the standard method. Size-exclusion chromatography with aqueous eluent was coupled online to CSD onto matrix-precoated foils. The suitability for determination of the molar mass distribution of methyl cellulose was investigated.     

电喷雾/飞行时间质谱法测定红霉素类抗生素的准 确质量

研究了聚乙二醇(PEG)为内标物的电喷雾(ESI)/飞行时间质谱(TOF-MS)准确质量测定方法,并应用于5个红霉类类抗生素质子化分子离子(MH^+)的质量测定。与理论值相比,相对误差均在5×10^-^6以内。PEG可与K^+,Na^+,H^+等形成三种加合离子形式,通过选择适当的实验条件,控制PEG仅以其中的一种加合离子形式出现,这一特点扩大了其应用范围,使之可作为一种普适性的内标物。另外讨论了扫描质量范围、扫描速度等因素对测定结果的影响,并且比较了采用多峰校正法和两峰校正法的结果。结果表明,以PEG为内标的ESI/TOF质谱法可对不稳定碱性化合物的化分子离子(MH^+)进行准确质量测定,而且简便、快速。