Structure elucidation of glycosphingolipids and gangliosides using high-performance tandem mass spectrometry

Glycosphingolipids and gangliosides have been investigated by using fast atom bombardment high-performance tandem mass spectrometry (FABMS/MS). Homologous compounds have been investigated in order to ascertain the fragmentation. Collision-induced dissociation spectra of the molecular species in the positive ion mode mainly afford information on the ceramide constitution (aglycon as a whole, N-acyl residue, and long-chain base), whereas negative ion spectra show fragments informative of the sugar sequence and the degree of branching of the carbohydrate. In the case of gangliosides carrying a complex oligosaccharide moiety, collision spectra of fragment ions have been performed in order to gain additional structural data. The advantage of tandem mass spectrometry over conventional fast atom bombardment mass spectrometry (FABMS) consists in the fact that collision spectra of the individual components from mixtures, as usually encountered with these kinds of samples, can be recorded. Furthermore, the exclusion of most of the interfering signals from the matrix allows the identification of pertinent fragments at low mass.     

Modulation of DNA structure formation using small molecules

Genome integrity is essential for proper cell function such that genetic instability can result in cellular dysfunction and disease. Mutations in the human genome are not random, and occur more frequently at “hotspot” regions that often co-localize with sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures. Non-B DNA-forming sequences are mutagenic, can stimulate the formation of DNA double-strand breaks, and are highly enriched at mutation hotspots in human cancer genomes. Thus, small molecules that can modulate the conformations of these structure-forming sequences may prove beneficial in the prevention and/or treatment of genetic diseases. Further, the development of molecular probes to interrogate the roles of non-B DNA structures in modulating DNA function, such as genetic instability in cancer etiology are warranted. Here, we discuss reported non-B DNA stabilizers, destabilizers, and probes, recent assays to identify ligands, and the potential biological applications of these DNA structure-modulating molecules.     

Advances in mass spectrometry driven O-glycoproteomics

Background

Global analyses of proteins and their modifications by mass spectrometry are essential tools in cell biology and biomedical research. Analyses of glycoproteins represent particular challenges and we are only at the beginnings of the glycoproteomic era. Some of the challenges have been overcome with N-glycoproteins and proteome-wide analysis of N-glycosylation sites is accomplishable today but only by sacrificing information of structures at individual glycosites. More recently advances in analysis of O-glycoproteins have been made and proteome-wide analysis of O-glycosylation sites is becoming available as well.

Scope of review

Here we discuss the challenges of analysis of O-glycans and new O-glycoproteomics strategies focusing on O-GalNAc and O-Man glycoproteomes.

Major conclusions

A variety of strategies are now available for proteome-wide analysis of O-glycosylation sites enabling functional studies. However, further developments are still needed for complete analysis of glycan structures at individual sites for both N- and O-glycoproteomics strategies.

General significance

The advances in O-glycoproteomics have led to identification of new biological functions of O-glycosylation and a new understanding of the importance of where O-glycans are positioned on proteins.     

The application of mass spectrometry in the structural elucidation of glycosphingolipids

Mass spectrometry has been successfully applied to the analysis of permethylated glycosphingolipids, with and without reduction, as well as of permethylated gangliosides after reduction and silylation. The results obtained by several groups of workers are reviewed. From the data available it can be stated that, with the aid of mass spectrometry, conclusive evidence may be obtained concerning the carbohydrate sequence as far as the typo of sugar is concerned such as hexose, deoxyhexose, hexosamine, and neuraminic acid residues. Branching points can be recognized, and specific fragmentation products allow the differentiation between 1,3 and 1,4-substituted hexosamines of blood-group-active glycosphingolipids (type 1 and type 2 chains). The ceramide residue is documented by several characteristic ions that allow determination not only of sphingosine bases and fatty acid components but also of individual ceramide molecular species. Valuable structural information can thus be obtained to a certain extent also for mixtures of glycosphingolipids composed of different carbohydrate chains or different ceramide residues.     

Advances in membrane mimetics and mass spectrometry for understanding membrane structure and function

Membrane proteins and lipids play roles in regulating biological functions of cells. However, the analysis of interactions between membrane proteins and lipids in biological membranes remains challenging. Native membranes typically contain heterogenous lipid mixtures and low amounts of membrane proteins. This review presents recent developments in membrane mimetics and complementary mass spectrometry approaches for the investigation of membrane protein–lipid interactions after protein expression and purification. Furthermore, it is exemplified how delipidation knowledge on membrane mimetics can be used to gain insights into the role of lipids for protein structure and function. Because every technology has its strengths and weaknesses, it becomes apparent that integrated research approaches will facilitate the investigation of complex membrane environments in the future.     

Advances in proteome analysis by mass spectrometry

Proteome characterization using mass spectrometry is essential for the systematic investigation of biological systems and for the study of gene function. Recent advances in this multifaceted field have occurred in four general areas: protein and peptide separation methodologies; selective labeling chemistries for quantitative measurement of peptide and protein abundances; characterization of post-translational protein modifications; and instrumentation.     

Advances in mass spectrometry for proteome analysis

The most demanding problems in proteomics continue to challenge modern mass spectrometry. Recent developments in instrument design have led to lower limits of detection, while new ion activation techniques and improved understanding of gas-phase ion chemistry have enhanced the capabilities of tandem mass spectrometry for peptide and protein structure elucidation. Future developments must address the., understanding of protein-protein interactions and the characterisation of the dynamic proteome.     

Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation.

A new method based on protein fragmentation and directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC-FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2-3, 0 degrees C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC-FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h-1) to very slow (k < 0.002 h-1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC-FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC-FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.     

Advances in coupling droplet microfluidics to mass spectrometry

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Study of the inclusion complexes of aromatic molecules with cyclodextrins using ionspray mass spectrometry

The formation of inclusion complexes between cyclodextrins (cyclohexa-, cyclohepta-, and cyclooctamylose) and either 1-anilinonaphthalene-8-sulfonate or 2-p-toluidinylnaphthalene-6-sulfonate was investigated by ionspray mass spectrometry operated both in the positive and in the negative ion mode. This soft ionisation technique allowed the detection of the inclusion complexes; the presence of false positives was excluded by increasing the voltage at the orifice which caused breakage of the electrostatic adducts and some fragmentation of the free cyclodextrin molecules, but left the inclusion complexes intact. The spectra recorded in the negative mode showed the presence of complexes formed by two cyclodextrin molecules and one aromatic molecule; such stoichiometry was not detected in the positive mode.     

Glycomics using mass spectrometry

Mass spectrometry plays an increasingly important role in structural glycomics. This review provides an overview on currently used mass spectrometric approaches such as the characterization of glycans, the analysis of glycopeptides obtained by proteolytic cleavage of proteins and the analysis of glycosphingolipids. The given examples are demonstrating the application of mass spectrometry to study glycosylation changes associated with congenital disorders of glycosylation, lysosomal storage diseases, autoimmune diseases and cancer.     

Protein primary structure using orthogonal fragmentation techniques in Fourier transform mass spectrometry

Proteomics analysis using tandem mass spectrometry requires informative backbone fragmentation of peptide ions. Collision-activated dissociation (CAD) of cations alone is not sufficiently informative to satisfy all requirements. Thus, there is a need to supplement CAD with a complementary fragmentation technique. Electron capture dissociation (ECD) is complementary to collisional excitation in terms of the cleavage of a different bond (N-C_α versus C-N bond) and other properties. CAD-ECD combination improves protein identification and enables high-throughput de novo sequencing of peptides. ECD and its variants are also useful in mapping labile post-translational modifications in proteins and isomer differentiation; for example, distinguishing Ile from Leu, iso-Asp from Asp and even D- from L-amino acid residues.     

Protein primary structure using orthogonal fragmentation techniques in Fourier transform mass spectrometry

Proteomics analysis using tandem mass spectrometry requires informative backbone fragmentation of peptide ions. Collision-activated dissociation (CAD) of cations alone is not sufficiently informative to satisfy all requirements. Thus, there is a need to supplement CAD with a complementary fragmentation technique. Electron capture dissociation (ECD) is complementary to collisional excitation in terms of the cleavage of a different bond (N-Calpha versus C-N bond) and other properties. CAD-ECD combination improves protein identification and enables high-throughput de novo sequencing of peptides. ECD and its variants are also useful in mapping labile post-translational modifications in proteins and isomer differentiation; for example, distinguishing Ile from Leu, iso-Asp from Asp and even D- from L-amino acid residues.     

Quantitation of retinaldehyde in small biological samples using ultrahigh-performance liquid chromatography tandem mass spectrometry

We report an ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method to quantify all-trans-retinal in biological samples of limited size (15–35 mg), which is especially advantageous for use with adipose. To facilitate recovery, retinal and the internal standard 3,4-didehydroretinal were derivatized in situ into their O-ethyloximes. UHPLC resolution combined with high sensitivity and specificity of MS/MS allowed quantification of retinal-O-ethyloximes with a 5-fmol lower limit of detection and a linear range from 5 fmol to 1 pmol. This assay revealed that extraocular concentrations of retinal range from approximately 2 to 40 pmol/g in multiple tissues—the same range as all-trans-retinoic acid. All-trans-retinoic acid has high affinity (k_d ? 0.4 nM) for its nuclear receptors (RARα, -β, and -γ), whereas retinal has low (if any) affinity for these receptors, making it unlikely that these retinal concentrations would activate RAR. We also show that the copious amount of vitamin A used in chow diets increases retinal in adipose depots 2- to 5-fold relative to levels in adipose of mice fed a vitamin A-sufficient diet, as recommended for laboratory rodents. This assay also is proficient for quantifying conversion of retinol into retinal in vitro and, therefore, provides an efficient method to study metabolism of retinol in vivo and in vitro.     

Advances in quantitative hepcidin measurements by time-of-flight mass spectrometry

Assays for the detection of the iron regulatory hormone hepcidin in plasma or urine have not yet been widely available, whereas quantitative comparisons between hepcidin levels in these different matrices were thus far even impossible due to technical restrictions. To circumvent these limitations, we here describe several advances in time-of flight mass spectrometry (TOF MS), the most important of which concerned spiking of a synthetic hepcidin analogue as internal standard into serum and urine samples. This serves both as a control for experimental variation, such as recovery and matrix-dependent ionization and ion suppression, and at the same time allows value assignment to the measured hepcidin peak intensities. The assay improvements were clinically evaluated using samples from various patients groups and its relevance was further underscored by the significant correlation of serum hepcidin levels with serum iron indices in healthy individuals. Most importantly, this approach allowed kinetic studies as illustrated by the paired analyses of serum and urine samples, showing that more than 97% of the freely filtered serum hepcidin can be reabsorbed in the kidney. Thus, the here reported advances in TOF MS-based hepcidin measurements represent critical steps in the accurate quantification of hepcidin in various body fluids and pave the way for clinical studies on the kinetic behavior of hepcidin in both healthy and diseased states.     

Quantitative proteomics using mass spectrometry

The use of stable isotopes as internal standards in mass spectrometry has opened a new era for quantitative proteomics. Depending on the point at which the label is introduced, most procedures can be classified as in vivo labeling, in vitro pre-digestion labeling or in vitro post-digestion labeling. In vivo labeling has been used for cells that can be grown in culture and has the advantage of being more accurate. The pre-digestion and post-digestion labeling procedures are suitable for all types of sample including human body fluids and biopsies. Several new mass spectrometric strategies mark significant achievements in determining relative protein concentrations and in quantifying post-translational modifications. However, further technology developments are needed for understanding the complexity of a dynamic system like the proteome.