Size exclusion chromatography/mass spectrometry applied to the analysis of polysaccharides.

A novel method for analysing polysaccharide materials is described which employs size-exclusion chromatography (SEC) followed by detection by on-line electrospray ionisation mass spectrometry (ESI-MS) and off-line matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). It is demonstrated through SEC/ESI ion trap mass spectrometry that the formation of multiply charged oligomer ions, which bind up to five sodium cations, allows the rapid analysis of polysaccharide ions with molecular weights in excess of 9 kDa. MALDI spectra generated from fractionation of the effluent collected from the same SEC separation are shown to be in good agreement with the ESI spectra with respect to molecular weight distributions and types of ions generated. ESI and MALDI mass spectra of samples obtained from sequential graded ethanol precipitation and SEC fractionation of acid and enzymatically digested arabinoxylan polysaccharides show important structural differences between polysaccharide fragments. In addition, a comparison is made between the mass spectra of native and permethylated SEC-separated fragments of acid and enzymatically treated arabinogalactan. Linkage information of the permethylated arabinogalactan oligomers can be rapidly established through the use of on-line SEC/ESI-MS( n) experiments.     

ESI-MS analyses of lake dissolved organic matter in light of supramolecular assembly

A high-performance size-exclusion chromatography (SEC) system was coupled on-line to an electrospray ionization (ESI) interface to detect gas-phase ions by an API 365 LC/MS/MS triple quadrupole analyzer. The SEC fractions of a strongly coloured freshwater solution containing dissolved organic matter-humic substances (DOM-HS) were screened both by UV₂₅₄ and by ESI mass spectrometry (ESI-MS) in the full-scan mode within the m/z range of 100–2,900 amu in negative and positive polarities. The ESI-MS spectra were also collected by direct infusion of the DOM-HS solution in both polarities. ESI-MS spectra did not primarily favour low mass compounds, and negative and positive total ion chromatograms were parallel to the SEC elution profile obtained by UV₂₅₄ detection from DOM-HS solution. The UV₂₅₄ detection overestimated the SEC portion of higher size/mass solutes and underestimated that of solutes of smaller sizes/masses as compared with the total ion chromatogram intensities in negative or positive polarities. The change of mass-weighted and number-weighted average sizes/masses (M _w and M _n) of different SEC fractions was fairly small, in contrast to UV₂₅₄ detection, with increasing elution volume. A reasonable explanation for the great differences between M _w and M _n values, obtained by UV₂₅₄ and ESI-MS detections for eight different SEC fractions, seems to be a supramolecular-type association of relatively small components through weak dispersive forces. M _n values obtained by vapour-pressure osmometry for different SEC fractions were to some extent analogous with those of negative and positive ESI-MS. The shapes obtained by either negative or positive polarities and calculated M _w and M _n values indicated a close structural similarity between each SEC fraction. Positive ion and negative ion spectra of different humic fractions represented quite similar components, and there was no evidence for noteworthy occurrence of multiply charged ions being able to lower mass distributions of negative ion spectra. The effect of nitrogen on the mass spectra seemed to be unimportant, and the weak ions observed at even m/z values correspond most likely to the ¹³C counterparts of the more abundant ¹²C odd ions. No uncontrolled ESI fragmentation was observable and humic solutes seemed to be quite heat-resistant. Direct infusion of the untreated DOM-HS solution and statistical calculation verified that the SEC-separated different fractions really represent distinct entities of the original DOM-HS mixture. ESI-MS results support the opinion that the structural composition of humic solutes in their original combined mixture resembles supramolecular-type associations of smaller molecular size entities possessing similar structural functionalities.     

Characterization of poly(butylene terephthalate) by size-exclusion chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Poly(butylene terephthalate) (PBT) samples have been analyzed with size-exclusion chromatography (SEC) using a mixed solvent of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and chloroform as the mobile phase. Several matrices and different sample deposition methods have been investigated to analyze PBT with matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Optimum results have been acquired by depositing PBT on top of a 2,4,6-trihydroxyacetophenone matrix. The found MALDI-TOF-MS method can be used to analyze the end group functionalities of PBT, as demonstrated with the samples at hand. By combining SEC (off-line) with MALDI-TOF-MS, absolute molecular masses of PBT can be measured, and these have been found to be considerably lower than those determined with SEC using polystyrene standards.     

Applications of mass spectrometry to food proteins and peptides

The application of mass spectrometry (MS) to large biomolecules has been revolutionized in the past decade with the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) techniques. ESI and MALDI permit solvent evaporation and sublimation of large biomolecules into the gaseous phase, respectively. The coupling of ESI or MALDI to an appropriate mass spectrometer has allowed the determination of accurate molecular mass and the detection of chemical modification at high sensitivity (picomole to femtomole). The interface of mass spectrometry hardware with computers and new extended mass spectrometric methods has resulted in the use of MS for protein sequencing, post-translational modifications, protein conformations (native, denatured, folding intermediates), protein folding/unfolding, and protein-protein or protein-ligand interactions. In this review, applications of MS, particularly ESI-MS and MALDI time-of-flight MS, to food proteins and peptides are described.     

Combined techniques for the characterization of linear–hyperbranched hybrid poly(butylene adipate) copolymers

Linear–hyperbranched hybrid poly(butylene adipate) (HPBA) copolymers were synthesized through a branching reaction between the linear tailored prepolymer terminated with methyl ester groups and different mol percents of the 1,1,1‐tris(hydroxymethyl) propane (TMP) as branching agent, using the titanium(IV) isopropoxide as catalyst, at 180 °C under vacuum for different times. All samples were characterized by NMR and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF MS). In particular, MALDI‐TOF mass spectra of the unfractionated and size exclusion chromatography (SEC)‐fractionated hyperbranched (HB) samples gave information on their composition, on the end groups as well as on the TMP units present in each family of HB macromolecules. HB chains containing cyclic branches and ether bonds formed by intermolecular transesterification and intramolecular and intermolecular transetherification side reactions, respectively, were also revealed by MALDI‐TOF MS analysis. All samples were also investigated by SEC. The average molar masses (MMs) evaluated by SEC calibrated with the polystyrene (PS) narrow standards were overestimated with respect to those calculated by the SEC/MALDI‐TOF MS self‐ calibration method, which gave reliable values. Moreover, it also showed that the hydrodynamic volume of the HPBA polymers was higher than that of the linear PSs with similar MMs. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Characterization of implant device materials using size-exclusion chromatography with mass spectrometry and with triple detection

A more complete understanding of the raw materials used for making implant device materials becomes increasingly important in the medical device industry. Often such detailed information requires utilization of a combination of analytical techniques. In this work, we characterize a poly(dimethyl siloxane) (PDMS) material using on-line size exclusion chromatography (SEC) with electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS) techniques. Here, we obtain detailed molecular compositional information such as repeat units, end group chemistry, and identification of impurities in both the high and low mass range. SEC with light scattering, viscosity, and refractive index detection (triple detection) is used to obtain information on a small quantity of high mass impurity that was undetected by both SEC-ESI and MALDI MS techniques. SEC with triple detection measures absolute molecular weights and molecular weight distributions. We compare average molecular weight values of the implantable device polymer obtained by SEC with triple detection, SEC-ESI, and SEC-MALDI MS techniques.     

有机硼化合物的质谱分析

对有机硼化合物进行质谱分析,考察不同种类仪器对样品分析的影响,确定使用电喷雾质谱仪效果最好。进一步考察了流动相、正负离子检测模式、离子化试剂及其浓度等因素的影响。实验发现:样品浓度为1×10-4mol/L,使用V(甲醇):V(H2O)=1:1的混合溶剂为流动相,正离子检测,0.1mol/LNa 为离子化试剂条件下可得到满意的谱图。     

大鼠肾脏汞结合金属硫蛋白的联用技术表征研究

通过凝胶色谱(SEC)及反相液相色谱(RPC)分别与电感耦合等离子体质谱(ICP-MS)和电喷雾电离质谱(ESI-MS)联用的技术，对灌喂HgCl₂大鼠的肾组织中诱导的多金属结合金属硫蛋白(MT)的结构进行表征分析。通过SEC-ICP-MS曲线上的金属信号及紫外检测的MT光谱吸收特征，可确认组织提取液中MT在色谱曲线上的位置。与对照组相比，灌喂HgCl₂大鼠肾中Hg-MT的诱导量显著增加，与此同时，Cu-MT在染毒组的诱导量也相应增加，说明MT在染汞大鼠肾脏中起着对汞的解毒和对铜的调节作用。MT样品提取物经葡聚糖凝胶(G-75)柱分离纯化并用透析法脱盐后，用细内径反相色谱柱分离，进行梯度洗脱，并与ESI-MS联用，可获得总离子流色谱图及与色谱峰对应的质谱图，通过对质谱信号的解析及参考脱金属MT构型的有关报道，对汞诱导大鼠肾组织MT的不同亚型和次亚型分子结构进行推断。结果证明，在灌喂HgCl₂大鼠的肾组织中存在着一系列汞结合的MT形态。     

Off-line size-exclusion chromatographic fractionation-matrix-assisted laser desorption ionization time-of-flight mass spectrometry for polymer characterization. Theoretical and experimental study

The parameters affecting the fractionation performance in size-exclusion chromatography (SEC) of broad polymer samples were investigated. Some equations were derived which enable the prediction of polydispersity (PD) in an SEC fraction. Good agreements were obtained between the calculated data and the experimental values. Based on these equations, SEC fractionation conditions were optimized. In the off-line SEC-matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), two different modes can be employed, i.e., using MALDI-MS to provide an absolute calibration curve for SEC, or using SEC as a sample preparation step for MALDI-MS measurements. It was demonstrated that it is more reliable to use the latter combination, because most problems inherent in SEC can be circumvented. Some guidelines for the optimization of off-line SEC fractionation-MALDI-TOF-MS were given. It was found that under optimized conditions normally only a few SEC fractions are already sufficient to separate a highly polydisperse sample into portions of low PD that can accurately be measured by MALDI-TOF-MS.     

Analysis of serum protein precipitated with antiserum by matrix-assisted laser desorption ionization/time-of-flight and electrospray ionization mass spectrometry as a clinical laboratory test

Serum transferrin precipitated with specific antisera was analyzed by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS) and electrospray ionization-mass spectrometry (ESI-MS). When analyzed by MALDI, transferrin showed signal peaks that clearly could be separated from ions of IgG present in the immunoprecipitate. By ESI-MS, when the immunoprecipitates were loaded through a microcapillary polymeric reversed-phase column connected to the electrospray ionization probe, the mass spectra of transferrin were observed with a high signal-to-noise ratio and good resolution. By MALDI/TOF-MS, the observed molecular weight of normal transferrin was ～ 1.2 ku smaller when analyzed in the reflectron mode than in the linear mode. The observed molecular weight of transferrin treated with sialidase was approximately the same in both modes. A comparison between the results obtained in both modes may help to estimate the number of sialic acids on the protein molecule. A transferrin isoform with a molecular weight of ～2.2 ku less than the normal species was identified in the serum of patients with a carbohydrate-deficient glycoprotein syndrome as well as in heavy alcohol consumers.     

Exploration of cardanol-based phenolated and epoxidized resins by size exclusion chromatography and MALDI mass spectrometry

Cardanol and cardanol derivatives are among the most important biobased materials currently investigated in green chemistry, as renewable and promising building blocks in lieu of traditional raw materials from non renewable resources, in particular owing to the olefinic linkages on the C15 alkyl side-chain. Despite the increasing interest they arouse, analytical chemistry dedicated to cardanol and associated resins has been rarely reported in the literature, found even poorer when dealing with chromatography and mass spectrometry. In this work, a thorough molecular characterization was conducted using matrix assisted laser desorption ionization (MALDI) mass spectrometry, size exclusion chromatography (SEC), and SEC–MALDI coupling to gain insights into the composition of phenolated, epoxidized, and epoxidized phenolated cardanol. A nomenclature was proposed to properly describe the numerous species found in these materials, while simulations of the unsaturation patterns and their comparison with the detected patterns in MALDI-MS gave useful details about the phenolation treatment expected to occur on the polyunsaturated C15 side chain. Finally, the SEC–MALDI off-line coupling allowed SEC peaks to be deconvoluted by mass spectrometry and MALDI artefacts related to matrix adduction to be pointed out.     

Direct sample fraction deposition using electrospray in narrow-bore size-exclusion chromatography/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for polymer characterization

A direct sample fraction deposition method was developed for off-line size-exclusion chromatography (SEC)/matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. By using electrospray, the SEC eluent, together with a suitable matrix solution added coaxially, was directly deposited on the MALDI plate. Owing to the formation of very small droplets in electrospray, solvent evaporation is much faster. The fractionation volume in narrow-bore SEC, which can directly be collected in one MALDI spot, can easily be optimized in the range of a few microlitres. In addition, fairly homogeneous sample spots were obtained. The possible influence of composition variation of the SEC effluent on the analytical results using direct fraction deposition was investigated; no substantial effects were observed. The applicability of the method was demonstrated by characterizing a broad poly(methyl methacrylate) sample. Copyright 2000 John Wiley & Sons, Ltd.     

Electrospray and matrix-assisted laser desorption/ionization mass spectral characterization of linear single nylon-6 oligomers

Synthetic nylon-6 single molecular mass oligomers were studied by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. These oligomers, considered as model compounds for the study of nylon-6 polymers, gave good mass spectrometric results using both MALDI and ESI. In spite of the gentle nature of both techniques, the MALDI and ESI spectra showed evidence of end-group cleavage from the oligomer chains. MALDI-MS was found to give similar fragmentation patterns for all of the oligomer samples. An increase in doubly charged ion signals with increasing oligomer mass was observed in the ESI mass spectra, as was end-group fragmentation. Signals from oligomer clusters were observed in ESI-MS for the dimer, tetramer and hexamer, most likely due to non-covalent bonding among the low-mass oligomer molecules.     

On particle ionization/enrichment of multifunctional nanoprobes: washing/separation-free, acceleration and enrichment of microwave-assisted tryptic digestion of proteins via bare TiO2 nanoparticles in ESI-MS and comparing to MALDI-MS

A simple, rapid, straightforward and washing/separation free of in-solution digestion method for microwave-assisted tryptic digestion of proteins (cytochrome c, lysozyme and myoglobin) using bare TiO(2) nanoparticles (NPs) prepared in aqueous solution to serve as multifunctional nanoprobes in electrospray ionization mass spectrometry (ESI-MS) was demonstrated. The current approach is termed as 'on particle ionization/enrichment (OPIE)' and it can be applied in ESI-MS, atmospheric pressure-matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The bare TiO(2) NPs can assist, accelerate and effectively enhance the digestion efficiency, sequence coverage and detection sensitivity of peptides for the microwave-assisted tryptic digestion of proteins in ESI-MS. The reason is attributed to the fact that proteins or partially digested proteins are easily attracted or concentrated onto the surface of TiO(2) NPs, resulting in higher efficiency of digestion reactions in the microwave experiments. Besides, the TiO(2) NPs could act as a microwave absorber to accelerate and enrich the protein fragments in a short period of time (40-60 s) from the microwave experiments in ESI-MS. Furthermore, the bare TiO(2) NPs prepared in aqueous solution exhibit high adsorption capability toward the protein fragments (peptides); thus, the OPIE approach for detecting the digested protein fragments via ESI and MALDI ionization could be achieved. The current technique is also a washing and separation-free technique for accelerating and enriching microwave-assisted tryptic digestion of proteins in the ESI-MS and MALDI-MS. It exhibits potential to be widely applied to biotechnology and proteome research in the near future.     

Structural studies of the allelic wheat glutenin subunits 1Bx7 and 1Bx20 by matrix-assisted laser desorption/ionization mass spectrometry and high-performance liquid chromatography/electrospray ionization mass spectrometry

Structural studies of the high molecular mass (HMM) glutenin subunits 1Bx7 (from cvs Hereward and Galatea) and 1Bx20 (from cv. Bidi17) of bread wheat were conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). For all three proteins, MALDI-TOFMS analysis showed that the isolated fractions contained a second component with a mass about 650 Da lower than the major component. The testing and correction of the gene-derived amino acid sequences of the three proteins were performed by direct MALDI-TOFMS analysis of their tryptic peptide mixture. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimizing the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP-HPLC/ESI-MS analysis of the tryptic digest. This resulted in coverage of about 98% of the sequences. In contrast to the gene-derived data, the results obtained demonstrate the insertion of the sequence QPGQGQ between Trp716 and Gln717 of subunit 1Bx7 (cv. Galatea) and a possible single amino acid substitution within the T20 peptide of subunit 1Bx20. Moreover, the mass spectrometric data demonstrated that the lower mass components present in all the fractions correspond to the major components but lack about six amino acid residues, which are probably lost from the protein C-terminus. Finally, the results obtained provide evidence for the lack of glycosylation or other post-translational modifications of these subunits.     

Theoretical investigation of low detection sensitivity for underivatized carbohydrates in ESI and MALDI

Electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) mainly generate protonated ions from peptides and proteins but sodiated (or potassiated) ions from carbohydrates. The ion intensities of sodiated (or potassiated) carbohydrates generated by ESI and MALDI are generally lower than those of protonated peptides and proteins. Ab initio calculations and transition state theory were used to investigate the reasons for the low detection sensitivity for underivatized carbohydrates. We used glucose and cellobiose as examples and showed that the low detection sensitivity is partly attributable to the following factors. First, glucose exhibits a low proton affinity. Most protons generated by ESI or MALDI attach to water clusters and matrix molecules. Second, protonated glucose and cellobiose can easily undergo dehydration reactions. Third, the sodiation affinities of glucose and cellobiose are small. Some sodiated glucose and cellobiose dissociate into the sodium cations and neutral carbohydrates during ESI or MALDI process. The increase of detection sensitivity of carbohydrates in mass spectrometry by various methods can be rationalized according to these factors. Copyright © 2016 John Wiley & Sons, Ltd.     

Imaging of Noncovalent Complexes by MALDI-MS

Noncovalent interactions govern how molecules communicate. Mass spectrometry is an important and versatile tool for the analysis of noncovalent complexes (NCX). Electrospray mass spectrometry (ESI-MS) is the most widely used MS technique for the study of NCXs because of its softer ionization and easy compatibility with the solution phase of NCX mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used to study NCXs. However, successful analysis depends upon several experimental factors, such as matrix selection, solution pH, and instrumental parameters. In this study, we employ MALDI imaging mass spectrometry to investigate the location and formation of NCXs, involving both peptides and proteins, in a MALDI sample spot.

Hydrocarbon polymer analysis by external MALDI fourier transform and reflectron time of flight mass spectrometry

The traditional solvent-based matrix-assisted laser desorption ionization (MALDI) preparation method has been used to analyze nonpolar polymers of various molecular weights. High resolution silver cationized oligomers with masses of up to 12 KDa were measured using 9.4 tesla Fourier transform mass spectrometry (FTMS) with an external ionization source. It was observed that when time-of-flight mass spectrometry was used, the spectra of polyethylene polymers showed abundant low mass fragment ions. However, these fragments were absent from the FTMS spectra.     

Methylating peptides to prevent adduct ion formation also directs cleavage in collision-induced dissociation mass spectrometry

We investigated the effect of N-terminal amino group and carboxyl group methylation on peptide analysis by electrospray mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Permethylation of the N-terminal amino group and the carboxyl groups can reduce metal ion adducts but does not enhance sensitivity in electrospray as previously observed for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. N-terminal trimethylated peptides exhibit collision-induced dissociation (CID) tandem mass spectra that differ from their unmodified analogs; the results support the mobile proton hypothesis of peptide fragmentation. A permanent positive charge at the N-terminus leads to competition between permanent-charge directed processes and loss of the N-terminal trimethyl amino group. Carboxyl methylation has no effect on fragmentation behavior other than to shift the mass of fragments containing methylated carboxyl groups. Comparison of regular and tandem mass spectra of different methylated peptides allowed probing the location of incomplete methylation, the proton displaced by alkali metal ions and the purity of a mass-selected methylated peptide ion.     

Control of polymer topology through transition-metal catalysis: synthesis of hyperbranched polymers by cobalt-mediated free radical polymerization

A novel approach was demonstrated for the synthesis of hyperbranched polymers by direct free radical polymerization of divinyl monomers controlled by a cobalt chain transfer catalyst (1). By controlling the competition between propagation and chain transfer with 1, the free radical polymerization of ethylene glycol dimethacrylate (3) afforded soluble hyperbranched polymers in one pot. The structure of the hyperbranched polymers was confirmed by (1)H and (13)C NMR. The molecular weight and intrinsic viscosity of the hyperbranched polymers were measured by matrix-assisted laser desorption ionization (MALDI) mass spectrometry and size exclusion chromatography (SEC) equipped with triple detectors. The intrinsic viscosities of the hyperbranched polymers are much lower than those of their linear analogues and do not show molecular weight dependence. The unique structure and properties of these hyperbranched polymers combined with the commercial availability of many divinyl monomers and the robustness of free radical polymerization make this new approach attractive for the preparation of new functional materials.