Ultraperformance ion-pair liquid chromatography coupled to electrospray time-of-flight mass spectrometry for compositional profiling and quantification of heparin and heparan sulfate

Heparin and heparan sulfate (HS) are important pharmaceutical targets because they bind a large number of proteins, including growth factors and cytokines, mediating many biological processes. Because of their biological significance and complexity, there is a need for development of rapid and sensitive analytical techniques for the characterization and compositional analysis of heparin and HS at the disaccharide level, as well as for the structure elucidation of larger glycosaminoglycan (GAG) sequences important for protein binding. In this work, we present a rapid method for analysis of disaccharide composition using reversed-phase ion-pairing ultraperformance liquid chromatography coupled with electrospray time-of-flight mass spectrometry ((RPIP)-UPLC-MS). Heparin disaccharide standards were eluted in less than 5 min. The method was used to determine the constituents of GAGs from unfractionated heparin/HS from various bovine and porcine tissues, and the results were compared with literature values.     

Heparin sequencing using enzymatic digestion and ESI-MSn with HOST: a heparin/HS oligosaccharide sequencing tool

Mass spectrometry, and specifically sequential stages of mass spectrometry (MSn), is an established tool for the analysis of carbohydrates, proteins, and more recently glycosaminoglycans. As this trend continues, the development of algorithms for the rapid and automatic interpretation of mass spectra to identify glycan structure is also expected to grow as an active field of research. The methodology described herein utilizes a combination of enzymatic digestion, ESI-MS, and MSn for the sequencing of small heparin oligosaccharides. The heparin oligosaccharide sequencing tool (HOST) is a basic software application that was developed to aid in the integration and analysis of the data generated from these experiments, facilitating the process involved in arriving at sequence information. The sequences of several heparin oligosaccharides were determined using this method to illustrate proof of principle. Tandem MS is a very rapid and efficient tool for oligosaccharide analysis when limited amounts of material are available. Having a means, such as HOST, for automating the interpretation of the MSn data generated from glycosaminoglycans, provides a practical methodology for the future analysis of heparin/HS oligosaccharides of unknown structure.     

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS(1) spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS(2) and MS(3) spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods.     

Online reverse phase-high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry separation and characterization of heparan sulfate, heparin, and low-molecular weight-heparin disaccharides derivatized with 2-aminoacridone

A high-resolution online reverse-phase-high-performance liquid chromatography (RP-HPLC)-fluorescence detector (Fd)-electrospray ionization-mass spectrometry (ESI-MS) separation and structural characterization of disaccharides prepared from heparin (Hep), heparan sulfate (HS), and various low-molecular-weight (LMW)-Hep using heparin lyases and derivatization with 2-aminoacridone (AMAC) are described. A total of 12 commercially available Hep/HS-derived unsaturated disaccharides were separated and unambiguously identified on the basis of their retention times and mass spectra. The constituent disaccharides of various samples, including unfractionated Hep/HS, fast-moving and slow-moving Hep components, and several marketed products, were characterized. Furthermore, for the first time, the saturated trisulfated disaccharide belonging to the nonreducing end of Heps was detected as being approximately 2% in unfractionated samples and ~15-21% in LMW-Heps prepared by nitrous acid depolymerization. No desalting of the commercial products prior to enzymatic digestion or prepurification steps to eliminate any excess of AMAC reagent or interference from proteins, peptides, and other sample impurities before RP-HPLC-Fd-ESI-MS injection were necessary. This method has applicability for the rapid differentiation of pharmaceutical Heps and LMW-Heps prepared by means of different depolymerization processes and for compositional analysis of small amounts of samples derived from biological sources by using the highly sensitive fluorescence detector.     

Hadamard transform measurement of tandem Fourier-transform mass spectra

The simultaneous collection of multiple spectra using tandem (MS/MS) and multidimensional (MS/MS/MS) mass spectrometry from multiple precursors is demonstrated to yield correspondingly enhanced sensitivity. This approach utilizes Hadamard transform deconvolution and takes advantage of the multichannel dissociation capability of Fourier-transform mass spectrometry. By application of this to an 11-component mixture, the 11 spectra of the products of dissociating 11 different combinations of six of the component molecular ions are measured; Hadamard transformation yields individual spectra of the precursor ions exhibiting a signal-to-noise improvement of 1.8x over spectra measured separately, as predicted by theory. Precursor ion selection with high specificity and product formation with high abundance reproducibility are critical; spurious peaks resulting from imperfect reproducibility can be minimized by using simultaneous equation coefficients reflecting the degree of precursor dissociation. Extension of this technique to MSn spectra is demonstrated with simultaneous MS/MS/MS monitoring of three precursors and three daughters yielding nine spectra representing the nine possible dissociation pathways. For MSn spectra, coding the product relationships for each additional step (e.g., precursor----daughter, daughter----granddaughter) requires elimination of half of the remaining ions. No ions are lost for coding in an improved Hadamard approach in which the combined daughter spectrum of the selected half of the precursors is subtracted from that of the other half.     

Improved liquid chromatography-MS/MS of heparan sulfate oligosaccharides via chip-based pulsed makeup flow

Microfluidic chip-based hydrophilic interaction chromatography (HILIC) is a useful separation system for liquid chromatography-mass spectrometry (LC-MS) in compositional profiling of heparan sulfate (HS) oligosaccharides; however, ions observed using HILIC LC-MS are low in charge. Tandem MS of HS oligosaccharide ions with low charge results in undesirable losses of SO(3) from precursor ions during collision induced dissociation. One solution is to add metal cations to stabilize sulfate groups. Another is to add a nonvolatile, polar compound such as sulfolane, a molecule known to supercharge proteins, to produce a similar effect for oligosaccharides. We demonstrate use of a novel pulsed makeup flow (MUF) HPLC-chip. The chip enables controlled application of additives during specified chromatographic time windows and thus minimizes the extent to which nonvolatile additives build up in the ion source. The pulsed MUF system was applied to LC-MS/MS of HS oligosaccharides. Metal cations and sulfolane were tested as additives. The most promising results were obtained for sulfolane, for which supercharging of the oligosaccharide ions increased their signal strengths relative to controls. Tandem MS of these supercharged precursor ions showed decreased abundances of product ions from sulfate losses yet more abundant product ions from backbone cleavages.     

Reduction in matrix-related signal suppression effects in electrospray ionization mass spectrometry using on-line two-dimensional liquid chromatography.

The effect of liquid chromatographic separation on matrix-related signal suppression in electrospray ionization mass spectrometry (LC-ESI-MS) was investigated. A method incorporating on-line two-dimensional liquid chromatography mass spectrometry (LC/LC-MS) was developed to compensate for matrix effects and signal suppression in qualitative and quantitative analysis. The LC/LC-MS(MS) approach was successfully applied for single-component and multicomponent analysis in a variety of complex matrixes. It was demonstrated that matrix-related signal suppression could be induced solely by (i) column overload, (ii) matrix component-analyte coelution, or a combination of each. Application of on-line orthogonal LC/LC separations can be effective in reducing both causes of matrix-related signal suppression effects i.e., column overload and matrix-analyte coelution for a variety of LCn/MSn applications.     

Characterization of sulfated oligosaccharides in mucopolysaccharidosis type IIIA by electrospray ionization mass spectrometry

Heparan sulfate is a linear glycosaminoglycan with considerable structural diversity that binds a myriad of growth factors and proteins that play pivotal roles in a variety of biological processes. We have investigated the structural complexity of partially degraded fragments of heparan sulfate in mucopolysaccharidosis type IIIA in which there is a defect in heparan sulfate catabolism. Mono- to hexadecasaccharides were isolated from the urine of a mucopolysaccharidosis IIIA patient and shown to have non-reducing end glucosamine N-sulfate residues, reflecting the catabolic deficiency in heparan N-sulfatase (sulfamidase) activity. The use of nitrous acid digestion (pH 1.5) combined with separation by reverse-phase high-performance liquid chromatography and analysis by electrospray ionization-mass spectrometry identified multiple forms of these oligosaccharides with some N-acetylated glucosamine residues and one to three sulfates per disaccharide. Furthermore, we demonstrated that each oligosaccharide existed in multiple sulfated forms. Many structural isomers were present, suggesting a complex mixture of oligosaccharides present in the urine as a consequence of a defect in heparan sulfate degradation.     

Colloidal graphite-assisted laser desorption/ionization mass spectrometry and MSn of small molecules. 1. Imaging of cerebrosides directly from rat brain tissue

Graphite-assisted laser desorption/ionization (GALDI) mass spectrometry (MS) was investigated for analysis of cerebrosides in a complex total brain lipid extract. Conventional MALDI MS and GALDI MS were compared regarding lipid analysis by using high-vacuum (HV, <10-6 Torr) LDI time-of-flight mass spectrometry and intermediate-pressure (IP, 0.17 Torr) linear ion trap mass spectrometry. Cerebrosides were not detected or detected with low sensitivity in MALDI MS because of other dominant phospholipids. By using GALDI, cerebrosides were detected as intense mass peaks without prior separation from other lipid species while mass peaks corresponding to phosphatidylcholines (PCs) were weak. The signal increase for cerebrosides and the signal decrease for PCs in GALDI MS were more significant in HV than in IP. MSn experiments of precursor ions corresponding to cerebrosides and PCs in brain lipid extract were performed to identify the detected species and distinguish isobaric ions. Twenty-two cerebroside species were detected by GALDI whereas eight cerebroside species were detected by MALDI. Sulfatides in brain lipid extract were also easily detected by GALDI MS in the negative ion mode. By forming a colloidal graphite thin film on rat brain tissue, direct lipid profiling by imaging mass spectrometry (IMS) was performed. Chemically selective images for cerebrosides and sulfatides were successfully obtained. Imaging tandem mass spectrometry (IMS/MS) was performed to generate images of specific product ions from isobaric species.     

Desorption electrospray ionization mass spectrometry of glycosaminoglycans and their protein noncovalent complex

Glycosaminoglycans heparin and heparan sulfate are biologically active polysulfated carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. Fragmentation of oligosaccharides and sulfate loss are important hindrance to their analysis by mass spectrometry (MS), requiring thus soft ionization methods. The recently introduced soft ionization method desorption electrospray ionization (DESI) has been applied here to heparin and heparan sulfate oligosaccharides, showing that DESI-MS is well suited for the detection of such fragile biomolecules in their intact form. Characterization of complicated oligosaccharides such as synthetic heparin octadecasulfated dodecasaccharide was successfully achieved. The use of water for a spray solvent instead of denaturing organic solvents allowed the first DESI-MS detection of noncovalent biomolecular complexes between heparin oligosaccharides and the chemokine Stromal Cell-derived Factor-1. The hyphenation of the DESI ion source with the high-resolution LTQ-Orbitrap MS analyzer led to high accuracy of mass measurement and enabled unambiguous determination of the protein-bound sulfated oligosaccharide.     

Distinguishing glucuronic from iduronic acid in glycosaminoglycan tetrasaccharides by using electron detachment dissociation

Distinguishing the epimers iduronic acid (IdoA) and glucuronic acid (GlcA) has been a long-standing challenge for the mass spectrometry analysis of glycosaminoglycan (GAG) oligosaccharides. In this work, electron detachment dissociation (EDD) and Fourier transform ion cyclotron resonance mass spectrometry is shown to provide mass spectral features that can distinguish GlcA from IdoA in heparan sulfate (HS) tetrasaccharides. EDD of HS tetrasaccharide dianions produces a radical species that fragments to produce information-rich glycosidic and cross-ring product ions which can be used to determine the sites of acetylation/sulfation. More significantly, EDD of HS tetrasaccharide epimers produces diagnostic product ions that can be used to distinguish IdoA from GlcA. These diagnostic product ions are not observed in the tandem mass spectra obtained by collisionally activated dissociation or infrared multiphoton dissociation of the tetrasaccharides, suggesting a radical-initiated mechanism for their formation. Differences in the observed product ions obtained by EDD of the tetrasaccharide epimers can be rationalized by simple alpha-cleavage of an oxy radical located at C2 or C3 or a radical at C3 or C4. These radicals are proposed to arise from a hydrogen rearrangement in which a hydrogen atom is transferred from the C2 or C3 hydroxyl group or C3 or C4 to a carboxy radical at C5. This hydrogen transfer depends on the proximity of the carboxy radical to the hydroxyl group on C2 or C3 or the hydrogen on C3 or C4 and is thus influenced by C5 stereochemistry. These epimer-sensitive fragmentations should allow this approach to be applied to the structural analysis of a wide variety of GAG oligosaccharides.     

A strategy for identification of oligosaccharide structures using observational multistage mass spectral library

Glycosylation is the most widespread posttranslational modification in eukaryotes; however, the role of oligosaccharides attached to proteins has been little studied because of the lack of a sensitive and easy analytical method for oligosaccharide structures. Recently, tandem mass spectrometric techniques have been revealing that oligosaccharides might have characteristic signal intensity profiles. We describe here a strategy for the rapid and accurate identification of the oligosaccharide structures on glycoproteins using only mass spectrometry. It is based on a comparison of the signal intensity profiles of multistage tandem mass (MSn) spectra between the analyte and a library of observational mass spectra acquired from structurally defined oligosaccharides prepared using glycosyltransferases. To smartly identify the oligosaccharides released from biological materials, a computer suggests which ion among the fragment ions in the MS/MS spectrum should yield the most informative MS3 spectrum to distinguish similar oligosaccharides. Using this strategy, we were able to identify the structure of N-linked oligosaccharides in immunoglobulin G as an example.     

Comparative study of photodissociation and surface-induced dissociation by laser desorption Fourier transform mass spectrometry.

Photodissociation (PD) and surface-induced dissociation (SID) are compared for structural analysis of several nonvolatile compounds analyzed by laser desorption Fourier transform mass spectrometry (LD/FTMS). SID and PD of a porphyrin and two metalloporphyrins were investigated using a variety of experimental conditions. Optimum structural information is obtained from PD when parent ions are irradiated for relatively long times (10-30 s) using 575-nm radiation and short times (0.5-1 s) using 308- or 388-nm radiation. Shorter irradiation times in the visible region resulted in less efficient production of structurally significant product ions, while longer times in the ultraviolet region produced more nonspecific fragment ions, apparently at the expense of more structurally significant fragment ions. SID conversion efficiencies for the porphyrins are estimated for collision energies from 25 to 360 eV, with maximum conversion efficiency found using 62- and 115-eV collision energies for the two porphyrins studied. Results from a concurrent study on the combined use of PD and SID for MS/MS/MS are discussed in the context of these results. The MS3 ion spectra generated by the two dissociation techniques differ more significantly than MS2 product ion spectra. These data suggest some general guidelines for MSn studies of nonvolatile compounds analyzed by LD/FTMS, employing PD and SID for ion activation.     

Mass imaging and identification of biomolecules with MALDI-QIT-TOF-based system

Imaging mass spectrometry is becoming a popular visualization technique in the medical and biological sciences. For its continued development, the ability to both visualize and identify molecules directly on the tissue surface using tandem mass spectrometry (MSn) is essential. We established an imaging system based on a matrix-assisted laser/desorption ionization quadrupole ion trap time-of-flight type instrument (AXIMA-QIT, Shimadzu, Kyoto, Japan), which was compatible with both imaging and highly sensitive MSn. In this paper, we present the operating conditions of the AXIMA-QIT as an imaging instrument and introduce the data converter we developed that is available free of charge. The converted data can be applied to Biomap, the commonly used visualization software. For the feasibility experiments, we demonstrated the visualization of phospholipids, glycolipid, and tryptic-digested proteins in the mouse cerebellum. The visualized lipids were successfully identified by MSn directly on the tissue surface, with a strong ability to isolate precursor ions. In the analysis of tryptic-digested proteins, we compared the product ion spectra between AXIMA-QIT and a tandem TOF-type instrument. The results confirmed that AXIMA-QIT can provide a high quality of product ion spectra even on the tissue surface.     

A comprehensive compositional analysis of heparin/heparan sulfate-derived disaccharides from human serum

The analysis of heparan sulfate glycosaminoglycans (HSGAGs) variations in human serum at the disaccharide level has a great potential for disease diagnosis and prognosis. However, the lack of available analytical methodology for the compositional analysis of HSGAGs in human serum remains to be addressed to delineate the possible role of HSGAGs on the onset and/or progression of a disease. In this study, we have developed a method for the in-depth compositional analysis of the 12 heparin/HS-derived disaccharides from human serum using a combination of technologies--fractionation, exhaustive digestion, solid phase extraction, and LC-MS/MS. The method exhibits high recovery (72-110%) and good reproducibility (standard deviation of less than 5%) with a low limit of detection and quantification. Errors from the method validation were within 1.1%. Nondetectable non- or low-sulfated disaccharides in human serum were also detected using the optimized protocol. Further applying this method, the comprehensive analysis of HSGAGs compositions in human sera from female donors showed considerable variations in disaccharide patterns and compositions.     

Multistage mass spectrometric sequencing of keratan sulfate-related oligosaccharides

To establish a universal protocol for sequencing keratan sulfate (KS) using mass spectrometry (MS), systematic electrospray ionization-MSn fragmentation experiments were carried out for 10 KS-related oligosaccharides of defined structure. Under the experimental conditions employed, fully charged molecular-related ions were observed as dominant peaks in all MS(1) spectra, which clearly reflected the number of sulfates and sialic acids in the oligosaccharide structures. In the subsequent MS2, almost all of the oligosaccharides gave fragment ions corresponding to their dehydrated molecular-related ions as well as (0,2)A(r) scission ions (according to the nomenclature developed by Domon and Costello, where "r" represents the reducing end in this study). Further fragmentation of the (0,2)A(r) ions in MS3 predominantly yielded the corresponding (2,4)A(r) ions. Finally, in MS(4), these (2,4)A(r) ions were subjected to extensive glycosidic cleavage. Hence, the MS4 data of KS oligosaccharides provided sufficient information for their sequence determination. In addition, some important features of MSn fragmentation became evident. These findings should lead to the establishment of consensus rules applied for KS oligosaccharides, including those previously unidentified, and also accelerate functional studies on KS, i.e., KS-related glycosaminoglycomics.     

Inlet ionization: a new highly sensitive approach for liquid chromatography/mass spectrometry of small and large molecules

Inlet ionization is a new approach for ionizing both small and large molecules in solids or liquid solvents with high sensitivity. The utility of solvent based inlet ionization mass spectrometry (MS) as a method for analysis of volatile and nonvolatile compounds eluting from a liquid chromatography (LC) column is demonstrated. This new LC/MS approach uses reverse phase solvent systems common to electrospray ionization MS. The first LC/MS analyses using this novel approach produced sharp chromatographic peaks and good quality full mass range mass spectra for over 25 peptides from injection of only 1 pmol of a tryptic digest of bovine serum albumin using an eluent flow rate of 55 μL min(-1). Similarly, full acquisition LC/MS/MS of the MH(+) ion of the drug clozapine, using the same solvent flow rate, produced a signal-to-noise ratio of 54 for the major fragment ion with injection of only 1 μL of a 2 ppb solution. LC/MS results were acquired on two different manufacturer's mass spectrometers using a Waters Corporation NanoAcquity liquid chromatograph.     

Carbohydrate structural isomers analyzed by sequential mass spectrometry

Consistent with the goals of a comprehensive carbohydrate sequencing strategy, we extend earlier reports to include the characterization of structural (constitutional) isomers. Protocols were developed around ion trap instrumentation providing sequential mass spectrometry (MSn) and supported with automation and related computational tools. These strategies have been built on the principle that for a single structure all product spectra upon sequential fragmentation are reproducible and each stage represents a rational spectrum of its precursor; i.e., all major fragments should be accounted for. Anomalous ions at any stage are clues indicating the presence of structural isomers. Gas-phase isolation and subsequent fragmentation of such ions provide an opportunity to specifically resolve selected structures for their detailed characterization. Importantly, some isomers were not detected following MS2 and required multiple (MSn>2) stages for their characterization. Derivatization remains critical to position substructures in a glycan array since product ions carry fragmentation "scars" throughout the MSn tree. Equally as important are the pathway relationships between each stage and the greater yield of fragments with the smaller number of oscillators. Applications were directed to the structural isomers in ovalbumin and IgG, where, in the latter case, several previously unreported glycans were detected. Procedures were supported with bioinformatics tools for assimilating structure from the MSn data sets.     

STAT: a saccharide topology analysis tool used in combination with tandem mass spectrometry

Sequential stages of mass spectrometry (MSn) have the potential to provide a great deal of structural information in glycan analysis. The saccharide topology analysis tool (STAT) presented here is a Web-based computational program that can quickly extract sequence information from a set of MSn spectra for an oligosaccharide of up to 10 residues. After information such as precursor ion mass, possible monosaccharide moieties, charge carrier, and product ion mass has been input, all possible connectivities are generated and evaluated against the MSn data. The list of possible structures is given a rating based on the likelihood that it is the correct sequence. Examples are given to demonstrate the feasibility of applying STAT to MSn data generated from bacterial lipooligosaccharides and an N-linked glycan. The major advantage of STAT is that the list of possible structures is generated quickly and the rating system pushes the more likely structures to the top of the list. Combining the data generated by STAT with data on the branching patterns of the glycan serves to eliminate all but a handful of structures. These remaining structures could then be used to guide further structural analysis.