Capillary electrophoresis off-line matrix-assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic-coated capillaries

Capillary electrophoresis (CE) was coupled off-line with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analysis of proteins and peptides. CE fractions were collected directly on a matrix-coated MALDI target, using a sheath-flow interface. Protein adsorption during CE separations was prevented by coating the capillaries with the physically adsorbed, cationic polymer PolyE-323. The CE/MALDI-MS system was used for the analysis of model proteins and peptides at physiological pH as well as analysis of proteins in tear fluid. Moreover, tryptic on-target digestion of the collected protein fractions, with subsequent MALDI-MS and MS/MS peptide analysis, was demonstrated.     

Capillary electrophoresis using a surfactant-treated capillary coupled with offline matrix-assisted laser desorption ionization mass spectrometry for high efficiency and sensitivity detection of proteins.

A method of combining capillary electrophoresis (CE) using a surfactant-modified capillary with matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is described for protein analysis. The CE-MALDI-MS coupling is based on CE fraction collection of nanoliter volume samples in less than 5 microl of dilute acid. This offline coupling does not require any special instrumentation and can be readily performed with commercial instruments. Protein adsorption during CE separation is prevented by coating the capillary with the surfactant didodecyldimethylammonium bromide. This surfactant binds strongly with the capillary wall, hence it does not desorb significantly to interfere with subsequent MALDI-MS analysis. It is shown that the use of a dilute acid for CE fraction collection is advantageous in lowering the detection limit of MALDI-MS compared to using an electrophoretic buffer. The detection limit for proteins such as cytochrome c is 23 fmol injected for CE, or 1.2 fmol spotted for MALDI-MS. This sensitivity is comparable to alternative CE-MALDI-MS coupling techniques using direct CE sample deposition on the MALDI target. In addition, the fraction collection approach has the advantage of allowing multiple reactions to be carried out on the fractioned sample. These reactions are very important in protein identification and structure analysis.     

Capillary electrophoresis—matrix-assisted laser-desorption ionization mass spectrometry of proteins

An “off-line” combination of capillary electrophoresis (CE) with matrix-assisted laser-desorption mass spectrometry (MALDI-MS) has been developed for the structural characterization of CE-separated peptides and proteins. Using a sheath flow interface, similar to that developed for “on-line” CE—fast atom bombardment MS and CE—electrospray MS, an efficient sample isolation procedure has been developed which is applicable to bioorganic compounds in aqueous buffer solutions. This isolation procedure, with subsequent transfer to the MALDI-MS sample target, has been successfully used for the direct analysis of CE-separated proteins of M _r up to 67 000, and a mixture of apolipoprotein AII monomer and homodimer, using sample amounts of less than 1 pmol.     

Rapid identification of differentiation markers from whole epithelial cells by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and statistical analysis

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) was applied to identify markers for cellular differentiation. The differentiation of a human colon epithelial carcinoma T84 cell line was monitored over a period of 28 days by transepithelial electrical resistance (TER) measurements, alkaline phosphatase (AP) assay, and MALDI-TOF mass spectral fingerprints combined with statistical analysis. MALDI-MS generated specific mass spectral fingerprints characteristic of cell differentiation. Twenty-two ions were selected as diagnostic signals of fully differentiated T84 cells. Ten protein ion signals, detected by MALDI-MS and validated by statistical analysis, were proposed as T84 cell differentiation markers. Among these signals, ubiquitin was identified as a T84 cell differentiation marker by nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS). Moreover, depending on the concentration of the cells seeded on the growth support, it was possible to predict the timing of the exponential phase and of cellular differentiation by MALDI-MS-derived marker ions. MALDI-TOFMS was compared to other methods for the determination of cellular differentiation: TER measurements are rapid but yield limited information as to the cellular differentiation state. AP assays are more specific for the differentiation state but take more time. By contrast, MALDI-MS has been found to be a fast, sensitive and precise method for cell differentiation assessment and provides the opportunity for multiplexing and high throughput. Moreover, the consumable costs per assay are very low.     

Characterization of polyether mixtures using thin-layer chromatography and matrix-assisted laser desorption/ionization mass spectrometry

Thin-layer chromatography (TLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were combined to achieve characterization of polyether mixtures. Three polyethers, polyethylene glycol (PEG), polypropylene glycol (PPG) and polytetramethylene glycol (PTMG), or mixtures of these compounds, were studied. One shortcoming of mixture analysis of synthetic polymers using MALDI-MS is that individual polymers in the mixture may display different detection sensitivities. For example, the MALDI mass spectrum of an equimolar mixture of PEG, PPG and PTMG displayed a high intensity of PPG ions, while no PTMG ions were detectable; however, PTMG ions were detected after the mixture had been separated by TLC. This combined TLC and MALDI-MS analysis of a PPG polymer bearing reactive epoxy groups showed that the polymer contained byproducts with different end-groups. These byproducts were identified as chloro-substituted polymers formed during polymer synthesis. Our study shows TLC to be a rapid and low-cost separation technique, and that it can be combined with MALDI-MS to achieve effective analysis of synthetic polymers.     

Characterization of cross-linking structures in UV-cured acrylic ester resin by MALDI-MS combined with supercritical methanolysis.

The cross-linking structure of the ultra violet (UV)-cured resin prepared from dipentaerithritol hexacrylate (DPHA) was characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with supercritical methanolysis. The MALDI-mass spectrum of the decomposition products obtained by supercritical methanolysis contained a series of peaks of sodium-cationized methyl acrylate (MA) oligomers up to around m/z = 4000 formed through selective cleavage and methylation occurred at ester linkages in UV-cured DPHA. Furthermore, in order to observe widely distributed sequence lengths in the cross-linking junctions, the decomposed products of the cured resin were then fractionated using size exclusion chromatography followed by the MALDI-MS measurements of the individual fractions. The MALDI-mass spectra of the lower molar mass fractions mainly consisted of a series of peaks of MA oligomers around m/z values of several thousands, whereas those of higher molecular weight showed a broad peak up to m/z ca. 180000. The observed distributions of the supercritical methanolysis products suggested that the network junctions in the given UV-cured resin were composed of up to around 2000 acrylate units.     

Generation of average mass values and end group information of polymers by means of a combination of matrix-assisted laser desorption/ionization-mass spectrometry and liquid secondary ion-tandem mass spectrometry

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and liquid secondary ion-tandem mass spectrometry (LSI-MS/MS) have been applied to the analysis of synthetic polymers to generate values for the average mass and the mass of the end groups. The average mass values were calculated for polymethylmethacrylate and polystyrene standards from the MALDI-MS data. Abundant fragment ions of the polymers, generated by means of LSI-MS/MS, were consistent with the known structures of the end groups of the polymers. Furthermore, losses from the side chains of the polymers were also observed in the LSI-MS/MS spectra.     

Complementary analysis of lipids in whole bacteria cells by thermally assisted hydrolysis and methylation-GC and MALDI-MS combined with on-probe sample pretreatment

The molecular structure of lipids in whole bacteria cells was characterized in detail by using two different and complementarily direct analyses; thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with on-probe sample pretreatment. First, THM-GC in the presence of tetramethylammonium hydroxide (TMAH) was applied to compositional analysis of the fatty acid components of lipids in whole bacterial cells. On the resulting chromatograms, a series of fatty acid components in bacterial lipids were clearly observed as resolved peaks of their methyl esters. The fatty acid compositions determined on the basis of the peak intensities were in good agreement with those obtained by the conventional technique involving solvent extraction of the lipids in the samples. Furthermore, MALDI-MS combined with the on-probe sample treatment, using 2,5-dihydroxybenzoic acid (DHB) and sodium iodide (NaI) as matrix and cationization reagents, respectively, was used to detect intact phospholipids directly from whole bacterial cells. The MALDI spectra thus obtained showed an array of ions generated from bacterial phospholipids, such as phosphatidylethanolamines (PEs) and phosphatidylglycerols (PGs). Finally, the bacterial lipids were comprehensively characterized in terms of the acyl groups and the molecular structures by taking both of the results obtained by THM-GC and MALDI-MS into consideration.     

Analysis of glycans derived from glycoconjugates by capillary electrophoresis-mass spectrometry

The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful MS and MS/MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high-order MS/MS (MS(n) ). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion-exchange chromatography, hydrophilic interaction chromatography and GC. However, CE and microfluidics CE (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to MS.     

Analysis of small molecules by ultra thin-layer chromatography-atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

The feasibility of ultra thin-layer chromatography atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (UTLC-AP-MALDI-MS) has been studied in the analysis of small molecules. Because of a thinner adsorbent layer, the monolithic UTLC plates provide 10-100 times better sensitivity in MALDI analysis than conventional high performance thin-layer chromatography (HPTLC) plates. The limits of detection down to a low picomole range are demonstrated by UTLC-AP-MALDI-MS. Other advantages of UTLC over HPTLC include faster separations and lower solvent consumption. The performances of AP-MALDI-MS and vacuum MALDI-MS have been compared in the analysis of small drug molecules directly from the UTLC plates. The desorption from the irregular surface of UTLC plates with an external AP-MALDI ion source combined with an ion trap instrument provides clearly less variation in measurements of m/z values when compared with a vacuum MALDI-time-of-flight (TOF) instrument. The performance of the UTLC-AP-MALDI-MS method has been applied successfully to the purity analysis of synthesis products produced by solid-phase parallel synthesis method.     

Comparing MALDI-MS, RP-LC-MALDI-MS and RP-LC-ESI-MS glycomic profiles of permethylated N-glycans derived from model glycoproteins and human blood serum

The glycomic profiling of purified glycoproteins and biological specimen is routinely achieved through different analytical methods, but mainly through MS and LC-MS. The enhanced ionization efficiency and improved tandem MS interpretation of permethylated glycans have prompted the popularity of this approach. This study focuses on comparing the glycomic profiling of permethylated N-glycans derived from model glycoproteins and human blood serum using MALDI-MS as well as RP-LC-MALDI-MS and RP-LC-ESI-MS. In the case of model glycoproteins, the glycomic profiles acquired using the three methods were very comparable. However, this was not completely true in the case of glycans derived from blood serum. RP-LC-ESI-MS analysis of reduced and permethylated N-glycans derived from 250 nl of blood serum allowed the confident detection of 73 glycans (the structures of which were confirmed by mass accuracy and tandem MS), while 53 and 43 structures were identified in the case of RP-LC-MALDI-MS and MALDI-MS analyses of the same sample, respectively. RP-LC-ESI-MS analysis facilitates automated and sensitive tandem MS acquisitions. The glycan structures that were detected only in the RP-LC-ESI-MS analysis were glycans existing at low abundances. This is suggesting the higher detection sensitivity of RP-LC-ESI-MS analysis, originating from both reduced competitive ionization and saturation of detectors, facilitated by the chromatographic separation. The latter also permitted the separation of several structural isomers; however, isomeric separations pertaining to linkages were not detected.     

In situ structural characterization of phosphatidylcholines in brain tissue using MALDI-MS/MS

Phosphatidylcholine (PC) is one of the most abundant classes of phospholipids and is a major component of membranes in biological systems. Recently, PCs have been detected by direct tissue analysis using MALDI-TOFMS. However, these studies did not allow for the structural characterization of PCs in tissue. In the current study, an in situ method for detection and structural analysis of PC species in brain tissue was developed using a MALDI-TOF/TOF mass spectrometer. Initial profiling of lipids in tissue is performed by MALDI-TOFMS, which allows for the assignment of PC species. However, to confirm the structure of the PC species detected in tissue, MALDI-MS/MS analysis was employed. In this work, protonated, sodiated, and potassiated PC species were detected in brain tissue using DHA matrix. MALDI-MS/MS analysis of these species yielded fragments that verified a phosphocholine head group, but did not supply any fragments that would permit the identification of acyl substituents. To obtain more structural information, lithium adducts of PC species were produced using DHA matrix dissolved in 100 mM lithium chloride. MALDI-MS/MS analysis of lithiated PC species produced fragments that allowed for the identification and positional assignment of acyl groups in PC species.     

Derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate for enhancing the ionization yield of small peptides and glycopeptides in matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The characterization of glycosylation in proteins by mass spectrometry (MS) is often impeded by strong suppression of ionization of glycopeptides in the presence of non-glycosylated peptides. Glycopeptides with a large carbohydrate part and a short peptide backbone are particularly affected by this problem. To meet the goal of generating mass spectra exhibiting glycopeptide coverages as complete as possible, derivatization of glycopeptides offers a practical way to increase their ionization yield. This paper investigated derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) which is a rapid labeling technique commonly used for fluorescence detection in high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). As test samples we used peptides and glycopeptides obtained by enzymatic digestion of three different glycoproteins, i.e., human antithrombin, chicken ovalbumin, and bovine alpha1-acid-glycoprotein. It was found that AQC derivatization resulted in strongly increased signal intensities when analyzing small peptides and glycopeptides by matrix-assisted laser desorption/ionization (MALDI)-MS. For these compounds the limit of detection could be reduced to low fmol amounts. Without derivatization only glycopeptides containing large peptide backbones were detected by MALDI-MS. This effect was even significant when glycopeptides were pre-separated and enriched by means of lectin affinity chromatography before MALDI-MS analysis and when using electrospray ionization (ESI). This labeling method, applied in combination with MS detection for the first time, was found to be well suited for the enhancement of detection sensitivity for small glycopeptides in MALDI-MS analysis and thus for reducing the need for pre-separation steps.     

Tiopronin gold nanoparticle precursor forms aurophilic ring tetramer

In the two step synthesis of thiolate-monolayer protected clusters (MPCs), the first step of the reaction is a mild reduction of gold(III) by thiols that generates gold(I) thiolate complexes as intermediates. Using tiopronin (Tio) as the thiol reductant, the characterization of the intermediate Au(4)Tio(4) complex was accomplished with various analytical and structural techniques. Nuclear magnetic resonance (NMR), elemental analysis, thermogravimetric analysis (TGA), and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) were all consistent with a cyclic gold(I)-thiol tetramer structure, and final structural analysis was gathered through the use of powder diffraction and pair distribution functions (PDF). Crystallographic data has proved challenging for almost all previous gold(I)-thiolate complexes. Herein, a novel characterization technique when combined with standard analytical assessment to elucidate structure without crystallographic data proved invaluable to the study of these complexes. This in conjunction with other analytical techniques, in particular mass spectrometry, can elucidate a structure when crystallographic data is unavailable. In addition, luminescent properties provided evidence of aurophilicity within the molecule. The concept of aurophilicity has been introduced to describe a select group of gold-thiolate structures, which possess unique characteristics, mainly red photoluminescence and a distinct Au-Au intramolecular distance indicating a weak metal-metal bond as also evidenced by the structural model of the tetramer. Significant features of both the tetrameric and the aurophilic properties of the intermediate gold(I) tiopronin complex are retained after borohydride reduction to form the MPC, including gold(I) tiopronin partial rings as capping motifs, or "staples", and weak red photoluminescence that extends into the Near Infrared region.     

Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry

The structural characterization of Glycosyl-Inositol-Phospho-Ceramides (GIPCs), which are the main sphingolipids of plant tissues, is a critical step towards the understanding of their physiological function. After optimization of their extraction, numerous plant GIPCs have been characterized by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) full scan analysis of negative ions provides a quick overview of GIPC distribution. Clear differences were observed for the two plant models studied: six GIPC series bearing from two to seven saccharide units were detected in tobacco BY-2 cell extracts, whereas GIPCs extracted from A. thaliana cell cultures and leaves were less diverse, with a dominance of species containing only two saccharide units. The number of GIPC species was around 50 in A. thaliana and 120 in tobacco BY-2 cells. MALDI-MS/MS spectra gave access to detailed structural information relative to the ceramide moiety, the polar head, as well as the number and types of saccharide units. Once released from GIPCs, fatty acid chains and long-chain bases were analyzed by GC/MS to verify that all GIPC series were taken into account by the MALDI-MS/MS approach. ESI-MS/MS provided complementary information for the identification of isobaric species and fatty acid chains. Such a methodology, mostly relying on MALDI-MS/MS, should open new avenues to determine structure-function relationships between glycosphingolipids and membrane organization.     

Capillary electrophoretic and mass spectrometric analysis of a polydisperse fluorosurfactant

A fluorosurfactant has been studied using capillary electrophoresis and mass spectrometry. The fluorosurfactant, FC134, can be used as a buffer additive in capillary electrophoresis in order to decrease wall adsorption of proteins and in micellar electrokinetic chromatography. However, it has been discovered that this fluorosurfactant is polydisperse, thus containing substances with different lengths and structures. In this work, the fluorosurfactant sample components were separated by capillary electrophoresis. An uncoated as well as a poly(vinyl alcohol)-coated capillary were used with running electrolytes containing methanol and acetic acid. Following the capillary electrophoretic separation, fractions were collected for further analysis by MALDI-MS. Non-fractionated samples were also analyzed both by MALDI-MS and by ESI-MS.     

Combining capillary electrophoresis matrix-assisted laser desorption/ionization mass spectrometry and stable isotopic labeling techniques for comparative crustacean peptidomics

Herein we describe a sensitive and straightforward off-line capillary electrophoresis (CE) matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) interface in conjunction with stable isotopic labeling (SIL) technique for comparative neuropeptidomic analysis in crustacean model organisms. Two SIL schemes, including a binary H/D formaldehyde labeling technique and novel, laboratory-developed multiplexed dimethylated leucine-based isobaric tagging reagents, have been evaluated in these proof-of-concept experiments. We employ these isotopic labeling techniques in conjunction with CE-MALDI-MS for quantitative peptidomic analyses of the pericardial organs isolated from two crustacean species, the European green crab Carcinus maenas and the blue crab Callinectes sapidus. Isotopically labeled peptide pairs are found to co-migrate in CE fractions and quantitative changes in relative abundances of peptide pairs are obtained by comparing peak intensities of respective peptide pairs. Several neuropeptide families exhibit changes in response to salinity stress, suggesting potential physiological functions of these signaling peptides.     

Influence of a crown ether comonomer on the temperature-induced phase transition of poly(N-isopropylacrylamide) hydrogels

Copolymerization of thermosensitive hydrogels based on poly(N-isopropylacrylamide) (PNIPA) is a possible route to enhanced storage capacity of guest molecules. This article describes the synthesis of the amphiphilic crown ether N9-propenoyl-3,6,12,15-tetraoxa-9,21-diazabicyclo[15.3.1]heneicosa-1(21),17,19-triene (CE) and its incorporation into a PNIPA hydrogel (PNIPA/CE). Mechanical measurements on the gel show that the CE units contribute to the elasticity of the network, but the swelling ratio in water is reduced compared to the unmodified system. The comonomer reduces the temperature of the volume phase transition (VPT), TVPT, and broadens the transition. Both the enthalpy and the entropy associated with the VPT decrease. Scattering measurements indicate that the local structural features on the scale of 10 A are unchanged, but the CE units form large clusters, the size of which increases with rising temperature. In the phase-separated state above TVPT these clusters are distributed on the polymer-water interface.     

A novel approach for identification and measurement of hemoglobin adducts with 1,2,3,4-diepoxybutane by liquid chromatography/electrospray ionisation mass spectrometry and matrix-assisted laser desorption/ionisation tandem mass spectrometry

The structural characterisation of the adducts formed by in vitro interaction of hemoglobin (Hb) with 1,2,3,4-diepoxybutane (DEB), the most reactive 1,3-butadiene (BD) metabolite, was obtained by liquid chromatography/electrospray ionisation mass spectrometry (LC/ES-MS) analysis of modified tryptic peptides of human hemoglobin chains. The reactive sites of human hemoglobin towards DEB and its hydroxylated derivatives (trihydroxybutyl (THB)-derivatives) were identified through the characterisation of alkylated tryptic peptides by matrix-assisted laser desorption/ionisation tandem mass spectrometry (MALDI-MS/MS). Based on this characterisation, a procedure was set up to measure the Hb-adducts of THB-derivatives by isotope dilution mass spectrometry with the use of a deuterated peptide standard. The results obtained here could permit optimisation of molecular dosimetry of BD-adducts, and extension of the analysis to the biological monitoring of occupational exposure to butadiene.     

基质辅助激光解吸电离质谱分析糖类物质

基质辅助激光解吸电离质谱（MALDI-MS）是一种样品无需衍生、图谱解析简单、灵敏度高、快速便捷的分析生物样品结构的方法,已被广泛用于糖类物质的结构分析。此技术与HPLC、糖苷酶外切技术以及各种串联质谱等技术结合使用,可给出糖类物质详细的结构信息。本文介绍了基质辅助激光解吸（MALDI）离子化技术的原理、特点、与飞行时间质量分析器（TOF）联用时的相关技术和裂解方式,以及MALDI-MS在分析糖类物质时选用的基质、样品的制备、糖链碎片分析的方法和在不同糖型分析中的应用,展示了它的发展前景。随着MALDI对糖类物质分析时基质的改进、质谱分辨率的提高、质量检测范围的扩大,MALDI-MS技术必将成为糖类物质分析中强有力的工具。