Simultaneous glycosylation analysis of human serum glycoproteins by high-performance liquid chromatography/tandem mass spectrometry

Changes in the glycosylation of some serum proteins are associated with certain diseases. In this study, we performed simultaneous site-specific glycosylation analysis of abundant serum glycoproteins by LC/Qq-TOF MS of human serum tryptic digest, the albumin of which was depleted. The glycopeptide peaks on the chromatogram were basically assigned by database searching with modified peak-list text files of MS/MS spectra and then based on mass differences of glycan units from characterized glycopeptides. Glycopeptide of IgG, haptoglobin and ceruloplasmin were confirmed by means of a comparison of their retention times and m/z values with those obtained by LC/MS of commercially available glycoproteins. Mass spectrometric carbohydrate heterogeneity in the assigned glycopeptides was analyzed by an additional LC/MS. We successfully demonstrated site-specific glycosylation of 23 sites in abundant serum glycoproteins.     

Separation of proteins by high-performance anion-exchange chromatography

The chromatographic separation of four proteins, cytochrome c, alpha 1-acid glycoprotein, ovalbumin, and beta-lactoglobulin, was achieved on a 4.6 X 250-mm wide-pore polyethyleneimine (PEI)-silica gel column (5-micron particles, 330-A pore size) with essentially baseline resolution using a 20-min linear gradient from 0.025 M potassium phosphate, pH 6.80, to 0.50 M potassium phosphate, pH 6.80. The back pressure of this anion-exchange column was 1000 psi at a flow rate of 1.0 ml/min. Protein recoveries averaged over 95% and protein capacity exceeded 33 mg for a single protein. Isocratic elution (0.040 M potassium phosphate, pH 6.8; flow rate, 0.50 ml/min) of ovalbumin gave a column efficiency of 15,700 plates/m with a peak asymmetry factor of 1.27. Resolution of these same four proteins on a 4.6 X 50-mm PEI-silica gel column occurred within 2 min. Nucleoside monophosphates were separated on the short PEI-silica column within 1 min with 0.01 M potassium phosphate, pH 2.58, at a flow rate of 6 ml/min which generated a column back pressure of 2000 psi.     

Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry

Carbohydrates display a large diversity of structures and their analysis presents many obstacles as the result of properties such as isomeric diversity, existence of branched structures and the lack of chromophores or fluorophores. Consequently, many analytical approaches depend on the application of chemical modifications such as hydrolysis or derivative formation. This review covers various aspects of derivatization that are used for such approaches as improving thermal stability and volatility for gas-phase analyses, introduction of fluorophores for optical detectors, introduction of charge for mass spectral analyses and attachment of bioaffinity tags for bioactivity studies. Reducing carbohydrates contain, in addition to multiple hydroxyl groups, several other sites for derivatization such as the single anomeric site that has been used in numerous methods for attaching various property-enhancing tags. Other sites are restricted to specific carbohydrates but include carboxy groups in sialic acids and amino groups in glycosylamines. All of these groups have been the targets of derivatization and this review attempts to summarise the main methods used for these various functional groups.     

Structural fingerprinting of Asn-linked carbohydrates from specific attachment sites in glycoproteins by mass spectrometry: application to tissue plasminogen activator

A sensitive and specific strategy has been developed for determining the sites of attachment of Asn-linked carbohydrates in glycoproteins, and defining the compositions and molecular heterogeneity of carbohydrates at each specific attachment site. In this carbohydrate 'fingerprinting' strategy, potential glycopeptides are identified by comparing the high pressure liquid chromatography (HPLC) chromatograms of proteolytic digests of a glycoprotein obtained before and after digestion with a glycosidase, usually peptide:N-glycosidase F (PNGase F). The glycopeptide-containing HPLC fractions are analyzed by fast atom bombardment mass spectrometry (FAB MS) prior to and after digestion with PNGase F to identify the former glycosylation site peptide and its sequence location (Carr and Roberts, (1986) Anal. Biochem. 157, 396-406). Carbohydrates are extracted from these fractions as the peracetates which are then permethylated and analyzed by FAB MS. The spectra exhibit molecular weight-related ions for each of the parent oligosaccharides present in the fraction which provide composition in terms of hexose, deoxyhexose, N-acetylhexosamine and sialic acid. The relative ratios of these peaks reflect the relative abundances of the various carbohydrate homologs present in the mixture. The derivatives formed are directly amenable to methylation analysis for determination of linkage. This strategy enables the structural classes of carbohydrates at specific attachment sites to be determined using only a few nmol of glycoprotein. The carbohydrate fingerprinting strategy has been applied to a number of glycoproteins including tissue plasminogen activator, the results for which are described herein.     

Oligosaccharide analysis by capillary-scale high-pH anion-exchange chromatography with on-line ion-trap mass spectrometry

A capillary-scale high-pH anion-exchange chromatography (HPAEC) system for the analysis of carbohydrates was developed, in combination with two parallel on-line detection methods of sub-picomolar sensitivity: (1) pulsed amperometric detection (PAD); (2) capillary-scale desalting followed by electrospray ion-trap (IT) mass spectrometry (MS). The capillary chromatographic system combined the superb selectivity of HPAEC that allows routine separation of isomeric oligosaccharides with the information on monosaccharide sequence and linkage positions obtained by MS/MS fragmentation using the IT-MS. The applicability of the system in biomedical research was demonstrated by its use for the analysis of a urine sample of a GM1-gangliosidosis patient. Isomeric glycans in the sample could be resolved by HPAEC and assigned on the basis of the monosaccharide linkage information revealed by on-line IT-MS/MS.     

Gas chromatography and mass spectrometry of disaccharides from glycoproteins.

Partial acid hydrolysis and methanolysis released disaccharides and disaccharide methylglycosides from the glycoproteins, ovomucoid and porcine gastric mucin in amounts of 0.5--7 microgram disaccharide per mg of glycoprotein. These disaccharides were fractionated by gas chromatography as the trimethylsilyl (Me3Si) derivatives. The composition of recovered disaccharides has been determined by hydrolysis and rechromatography of the Me3Si monosaccharides. The intersaccharide linkages of the disaccharides have been determined by electron impact mass spectrometry. This simple and rapid method can give structural information on small glycoprotein samples.     

Analysis of nonstructural carbohydrates in storage organs of 30 ornamental geophytes by high-performance anion-exchange chromatography with pulsed amperometric detection

A comprehensive analysis of nonstructural carbohydrates in storage organs (bulbs and corms) of 30 ornamental geophytes was conducted by employing a variety of extraction techniques followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Among species, starch, fructan, glucomannan and soluble sugars accounted for 50-80% of storage organ dry weight (DW). Starch ranged from 24 to 760 mg g(-1) DW, fructan (commonly occurring with starch) from 25 to 500 mg g(-1) DW, and glucomannan from 15 to 145 mg g(-1) DW. An acid hydrolysis protocol for concurrent determination of fructan and glucomannan was developed. The average degree of polymerization (DP) of ethanol and water-soluble fructan and the man : glu ratio of glucomannan also varied between species. The 80% ethanol fraction contained soluble sugars and short-chain fructans (< 25 DP), whereas water extracts contained soluble sugars, fructans (both short- and long-chain, 相似文献   

Screening of eltanolone metabolites in dog urine by anion-exchange/reversed-phase liquid chromatography and mass spectrometry

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated ¹⁴C-labelled eltanolone (5β-Pregnan-3α-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of ¹⁴C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC–ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography–mass spectrometry with an electron impact ion source (GC–MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).     

Structural characterization of fucose-containing oligosaccharides by high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Eight pyridylamino (PA) derivatives of fucose-containing oligosaccharides, which occur as free oligosaccharides in human milk and also are derived from glycosphingolipids, have been analyzed by high-performance liquid chromatography (HPLC) on normal-phase and reversed-phase columns, and by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Six out of eight PA-oligosaccharides were clearly separated by both normal- and reversed-phase HPLC at a column temperature of 40 degrees C, but two PA-oligosaccharides, lacto-N-fucopentaose II [Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-3Gal beta1-4GIcPA] and lacto-N-fucopentaose III [Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-3Gal beta1-4GIcPA], were not separated. The two unresolved PA-oligosaccharides were finally separated by reversed-phase HPLC at a column temperature of 11 degrees C. MALDI-TOF mass spectra of PA-oligosaccharides demonstrated pseudo-molecular ions as the predominant signals, therefore information about the molecular mass of each PA-oligosaccharide was easily obtained. Post-source decay (PSD) MALDI-TOF mass spectra of PA-oligosaccharides gave information about the carbohydrate sequences and carbohydrate species of each PA-oligosaccharide by detecting the ions responsible for the cleavage of the glycosidic bonds. The detection limits of the PA-oligosaccharides by HPLC, MALDI-TOF mass spectrometry, and PSD MALDI-TOF mass spectrometry were 20 fmol, 20 fmol, and 2 pmol, respectively. These results suggest that a system including HPLC and MALDI-TOF mass spectrometry or HPLC and PSD MALDI-TOF mass spectrometry is quite useful for the structural characterization of sub-pmol or pmol levels of fucose-containing oligosaccharides, and that these methods could be used for the analysis of various types of oligosaccharides derived from glycoproteins and glycosphingolipids.     

Variables in the high-performance anion-exchange chromatography of proteins

The effect of mobile phase velocity, separation time, support pore diameter, column length, and temperature on resolution and loading capacity of a new commercially available high-performance anion-exchange support, SynChropak AX-300, has been examined. This material is a macroporous spherical silica of 10 μm particle size with a bonded polymeric amine layer. It was found that the heterogeneity of ovalbumin samples, combined with bovine serum albumin, make them useful probes in evaluation of anion-exchange supports. In the columns of 4.1 mm i.d., the highest resolutions of proteins were achieved at a flow rate of 0.25 ml/min. Up to 10 mg of protein per injection could be applied on a 4.1 × 250 mm AX-300 column with good resolution. Columns of 50 mm length had one-tenth the protein load capacity of a 250-mm column, retaining approximately 75% of the resolution.     

Identification of N-linked glycoproteins in human milk by hydrophilic interaction liquid chromatography and mass spectrometry

Breastfeeding is now generally recognized as a critical factor in protecting newborns against infections. An important mechanism responsible for the antibacterial and antiviral effects of breast milk is the prevention of pathogen adhesion to host cell membranes mediated by a number of glycoconjugates, also including glycoproteins. A number of approaches to describe the complexity of human milk proteome have provided only a partial characterization of restricted classes of N-linked glycoproteins. To achieve this objective, profiling N-linked glycoproteins of human milk was performed by Hydrophilic Interaction LC (HILIC) and MS analysis. Glycopeptides were selectively enriched from the protein tryptic digest of human milk samples. Oligosaccharide-free peptides obtained by peptide N-glycosidase F (PNGase F) treatment were characterized by a shotgun MS-based approach, allowing the identification of N-glycosylated sites localized on proteins. Using this strategy, 32 different glycoproteins were identified and 63 N-glycosylated sites encrypted in them were located. The glycoproteins include immunocompetent factors, membrane fat globule-associated proteins, enzymes involved in lipid degradation and cell differentiation, specific receptors, and other gene products with still unknown functions.     

Structural analysis of hydroperoxy- and epoxy-triacylglycerols by liquid chromatography mass spectrometry

Oxidation of triacylglycerols (TAGs) containing oleic acid leads to the formation of several products. This study characterizes hydroperoxy- and epoxy-TAGs including their regio-isomers. For this purpose, epoxy- and hydroperoxy-TAGs, formed by oxidation of 1,2-dipalmitoyl-3-oleoyl-glycerol (PPO) and 1,3-dipalmitoyl-2-oleoyl-glycerol (POP) under air and , were analysed by reverse phase liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) using a triple quadrupole mass analyser, in positive ion mode. Post-column infusion of ammonium formiate was used to obtain intense molecular ion adducts. Pure 1,2-dipalmitoyl-3-epoxystearoyl-glycerol (PPEs) and 1,3-dipalmitoyl-2-epoxystearoyl-glycerol (PEsP), synthesized by epoxidation of the corresponding monounsaturated TAGs, were used to confirm MS/MS identification. The use of oxidation experiments permitted unambiguous identification of MS/MS fragmentation pathways of both hydroperoxide and epoxy-TAGs. Fragmentation of hydroperoxy-TAGs are very distinct from their epoxy-TAGs homologues and consist of simultaneous losses of hydrogen peroxide (34 a.m.u.) and water (18 a.m.u.).     

The identification of serum tuftsin by reverse-phase high-performance liquid chromatography and mass spectrometry

We describe for the first time a method for unambiguously identifying the phagocytic stimulating tetrapeptide tuftsin from trypsinized human serum. The method consists of separating serum tuftsin by reverse-phase (RP)-HPLC, collecting HPLC fractions corresponding to the synthetic tuftsin retention time, and then subjecting those fractions to mass spectrometry/mass spectrometry (MS/MS) analysis, which provides optimal molecular specificity to the measurement. Although quantification was not the goal, it was estimated that the amount of tuftsin found by RP-HPLC and MS/MS was in the hundreds of nanograms per milliliter.     

Analysis of oligosaccharides by on-line high-performance liquid chromatography and ion-spray mass spectrometry.

Oligosaccharides were analyzed by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). First, oligosaccharides labeled with 2-aminopyridine were studied to see if they could be analyzed by MS under the conditions used for separation by HPLC. Pyridylamino (PA)-oligosaccharides could be analyzed under these conditions, although the mass spectra were affected. Then, liquid chromatography-mass spectrometry was used to analyze a PA-oligosaccharide mixture derived from human immunoglobulin G. The PA-oligosaccharides were separated on a reversed-phase column and mass-analyzed directly. The observed molecular weights were close to or identical to those expected from the structures, which were estimated from the elution position on HPLC. This method is rapid and simple, as the mass spectrometer can give the accurate molecular weight of each PA-oligosaccharide in one chromatography run, even if the HPLC separation is incomplete. This method can be used to extend the so-called two-dimensional mapping of PA-oligosaccharides. The structure can be studied in greater detail by tandem MS.     

Determination of andrographolide in human plasma by high-performance liquid chromatography/mass spectrometry

In this paper, a rapid method based on high-performance liquid chromatography/electrospray-mass spectrometry (HPLC/ESI-MS) method for the quantitative determination of andrographolide (AND) in human plasma has been developed and validated. A liquid-liquid extraction (LLE) procedure was selected to isolate AND from biological matrixes. Isosorbide-5-mononitrate (IS-5-MN) was selected as the internal standard (IS). The correlation coefficient of the calibration curve was 0.998, in the range of 9.9-320.0 ng/mL. The validated method may be used to assess the bioavailability and pharmacokinetics of the drug.     

Determination of carbohydrates in fermentation processes by high-performance liquid chromatography

Summary HPLC is a universal, fast, accurate and selective method for the quantification of carbohydrates during fermentation processes. HPLC is not affected by complex constituents of fermentation media, such as meat extract, soybean meal or distillers solubles. The detection limit of the different investigated carbohydrates by refractive index monitoring ranges between 20 and 40 mg/l using a cation-exchange resin and between 50 and 100 mg/l using amino- or diol-bonded phases.Offprint requests to: H.-P. Fiedler     

Analysis of recombinat glycoproteins by mass spectrometry

The advent of new technologies for analysis of biopolymers by mass spectrometry has revolutionised strategies for recombinant protein characterization. The principal recent developments have been matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Using these tools, accurate molecular mass determinations can now be obtained routinely-often using minute (picomole-femtomole) quantities of protein or protein fragments. These techniques have proved indispensible for detailed characterization of the post-translational modifications of recombinant proteins produced by eukaryotic systems. Glycosylation is arguably the most important and complex of these modifications and has prompted widespread use of these new techniques. In this mini-review article I describe recent advances in the use of mass spectrometry for analysis of recombinant glycoproteins.     

Analysis of tetracyclines in raw urine by column-switching high-performance liquid chromatography and tandem mass spectrometry

The aim of the project was to develop a fast and reliable method for the quantification of the three tetracyclines: tetracycline, oxytetracycline and chlortetracycline in urine. The method is based on column-switching high-performance liquid chromatography with detection by MS–MS. Buffer is added to the sample before it is injected into the chromatographic system, and the first column which is an internal surface reversed-phase column separates the tetracyclines from the bulk of other compounds in urine. The tetracyclines are collected and concentrated on the analytical column before they are separated and eluted into the mass spectrometer in which the tetracycline are detected. The mass spectrometer is a triple quadrupole instrument and is equipped with an electrospray ion source. The MH⁺ ions are selected in the first quadrupole and collisionally activated in the collision cell. Upon collision, activation all three tetracyclines form fragment ions which could be assigned as: [M+H–H₂O–NH₃]⁺ which are selected in the sond mass filter. The detection limits for all three tetracyclines are about 10 ppb, and the calibration curves are linear from 10 to 1000 ppb.     

Large-scale purification of plasmid DNA by anion-exchange high-performance liquid chromatography.

Numerous methods have previously been reported for the final steps in the large-scale purification of plasmid DNA. Although gel permeation and reverse-phase high-performance liquid chromatography have been utilized for this procedure in the past, the limited capacity of these systems often necessitated multiple rounds of chromatography, especially with the high copy number plasmids commonly in use today. In this paper, the use of the high-capacity, high-resolution Protein-Pak DEAE 8HR column is presented for the large-scale isolation of highly purified plasmid DNA from crude E. coli cell lysates. Up to 5 mg of plasmid DNA have been purified in a single 50-minute chromatography run. The purified DNA demonstrated excellent biological activity as demonstrated by restriction endonuclease digestion, E. coli transformation and DNA-mediated gene transfection of eukaryotic cells.