Secondary ion mass spectrometry for sulfoglycolipids: application of negative ion detection

A series of underivatized sulfoglycolipids (SM4g, lyso-SM4g, SM4s, SM3, SM2, SB2, and SB1a) from various tissues were analyzed by both positive (POS-SI-MS) and negative (NEG-SI-MS) secondary ion mass spectrometry. By POS-SI-MS were detected the molecular ions of sulfoglycolipids in the form with sodium or potassium together with some fragment ions useful for the carbohydrate sequence determination. The analysis of monosulfogangliotriaosyl- or monosulfogangliotetraosylceramide and bis-sulfoglycolipid was difficult due to noise in the high mass region. On the other hand, NEG-SI-MS of sulfoglycolipids gave more intense signals from molecular ion of (M-H)- for monosulfoglycolipids and [M-H+Na)-H)- for bis-sulfoglycolipid. Many fragment ions useful for the elucidation of the carbohydrate sequences were also obtained with significant intensities. The fragmentation was assessed to occur at the glycosidic linkages to form ions of the oligosaccharides with or without ceramide. These ions were useful for sugar sequencing and also for distinguishing the differences in the position of the sulfate group. The intensities of saccharide ions without sulfate were lower than those with sulfates. In the case of SB2 and SB1a, containing 2 mol of sulfate ester groups, the molecular ion was detected as [M-H+Na)-H)-. Also, fragment ions with 2 mol of sulfate were detected as the sodium-additive form. It was concluded that NEG-SI-MS is a very useful technique for the structural elucidation of higher sulfoglycolipids.     

Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates

Applicability of negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) was examined in trace mixture analyses and structural assignments of some isoprenoid diphosphates. Negative ion FAB-MS spectra using a glycerol matrix of these isoprenoid diphosphates showed predominantly molecular ions (M-H)- together with fragment ions at m/z 177 (H3P2O7)-, 176 (H2P2O7)-, 159 (HP2O6)-, and 79 (PO3)- which were characteristic of the diphosphate ester moiety. The molecular ions did not overlap with peaks arising from any impurities even when crude sample such as butanol extracts from enzymatic reaction mixtures were directly analyzed without any purification. Moreover, collisionally activated dissociation spectra of the molecular ion showed many structurally significant fragment ions which enabled us to elucidate the structures of such irregular alkyl chain moieties as those having a homoisoprenoid skeleton or substituted structures. These studies indicate that negative ion FAB-MS/MS is a simple and useful technique for trace mixture analysis and structure elucidation of isoprenoid diphosphates.     

Secondary ion mass spectrometry analysis of the copper distribution in Drosophila melanogaster chronically intoxicated with Bordeaux mixture

The fungicides used intensively in agriculture may affect non-target organisms. The concentrations of copper sulfate-based fungicide, Bordeaux mixture, normally used in agriculture, can significantly reduce both the life span and breeding rate of Drosophila melanogaster. The present study examines the distribution of copper in organ sections of fruit flies intoxicated with Bordeaux mixture, by secondary ion mass spectrometry. The organs of most control flies contained no copper. In contrast, copper accumulated in the cytoplasm of all the mesenteron and Malpighian tubule epithelial cells of the treated flies. There were also copper deposits in the fat body and the epithelia of the seminal receptacle and accessory glands of some flies, but there was little or no copper in the ovaries. The mesenteron and Malpighian tubules are generally responsible for detoxification by accumulation of ingested metal salts in insects. The high concentration of Bordeaux mixture used saturated these organs and resulted in excess copper being deposited in other sites, such as the fat body and the reproductive system.     

Secondary ion mass spectrometry and Raman spectroscopy for tissue engineering applications

1. Download : Download high-res image (250KB)
2. Download : Download full-size image

     

High performance liquid chromatography and time-of-flight secondary ion mass spectrometry: a new dimension in structural analysis of apolipoproteins

We report the isolation and characterization of an apolipoprotein A-I mutant using a new technique for structural analysis of apolipoproteins based upon the combined techniques of protein isolation by isoelectric focusing in immobilized pH-gradients, reversed-phase HPLC of tryptic peptides, and subsequent molecular weight analysis of isolated peptides by time-of-flight secondary ion mass spectrometry (TOF-SIMS). The particular advantages of the TOF-SIMS procedure in the characterization of proteolytic peptides are the detection limits in the picomole range, the accuracy of molecular weight determination (up to 3000 +/- 1 D), the speed of analysis, and the wide range of applications for involatile biomolecules. The described procedure for the analysis of apolipoproteins requires only 2 ml of serum as starting material. This method can be used to monitor for genetic polymorphisms and posttranslational modifications on a microscale basis. Applying these techniques, we characterized a new apolipoprotein A-I mutant with an amino acid exchange arginine177 by histidine.     

Secondary structural characterization of oligonucleotide strands using electrospray ionization mass spectrometry

Differences in charge state distributions of hairpin versus linear strands of oligonucleotides are analyzed using electrospray ionization mass spectrometry (ESI-MS) in the negative ion detection mode. It is observed that the linear structures show lower charge state distribution than the hairpin strands of the same composition. The concentration of ammonium acetate and the cone voltage are major factors that cause the shift of the negative ions in the charge states. The ESI data presented here are supported by UV spectra of strands acquired at 260 nm wavelength in aqueous ammonium acetate solution. We will show that the strands that demonstrate a higher charge state distribution in the gas phase also have a higher melting temperature in solution.     

Secondary ion mass spectrometry (SIMS) of standards for analysis of soft biological tissue.

Gelatin films containing water-soluble salts of lithium, rubidium, strontium, or copper were analyzed by secondary ion mass spectrometry. Calcium and vanadium organometallic compounds in an epoxy resin were similarly analyzed. A linear relationship between positive secondary ion intensity and ion concentration was observed over several decades of ion concentration and at absolute concentrations as low as 1 wt ppm. These standards can be used for quantitative analysis of tissue or other biological material in epoxy resins, providing a highly sensitive method for simultaneous quantitation and localization of elements.     

Introduction to ion trap mass spectrometry: Application to the structural characterization of plant oligosaccharides

This review will focus on ion trap mass spectrometry (ITMS) and the application of this technique to the structural analysis of carbohydrates. The basic principles of operation of the electrostatic ion traps are briefly described and the applicability of the technique to the structural characterization of carbohydrates is illustrated with the analysis of arabinoxylan oligosaccharides by ion trap mass spectrometry.     

The human erythrocyte proteome: analysis by ion trap mass spectrometry

This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins. Both fractions were divided into subfractions, and proteins were identified in each fraction separately through tryptic digestion. Membrane protein digests were collected from externally exposed proteins, internally exposed proteins, "spectrin extract" mainly consisting of membrane skeleton proteins, and membrane proteins minus spectrin extract. Cytoplasmic proteins were divided into 21 fractions based on molecular mass by size exclusion chromatography. The tryptic peptides were separated by reverse-phase high-performance liquid chromatography and identified by ion trap tandem mass spectrometry. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions. Glyceraldehyde-3-phosphate dehydrogenase was identified with high sequence coverage in both membrane and cytoplasmic fractions. Identified proteins include membrane skeletal proteins, metabolic enzymes, transporters and channel proteins, adhesion proteins, hemoglobins, cellular defense proteins, proteins of the ubiquitin-proteasome system, G-proteins of the Ras family, kinases, chaperone proteins, proteases, translation initiation factors, and others. In addition to the known proteins, there were 43 proteins whose identification was not determined.     

离子阱串联质谱仪质荷比测量误差统计分析

离子阱串联质谱仪是蛋白质组研究中一种高通量,高灵敏度的分析仪器。目前对影响离子阱质谱仪质荷比测量误差的因素和数据产出后系统误差的校正方法还没有系统的分析。利用两批蛋白质标准品的数据和统计方法分析了离子阱质谱仪质荷比测量误差的分布规律,对测量的质荷比和信号强度对测量误差的影响进行了分析。在此基础上,提出了一种对质谱数据进行系统误差再校正的方法和一种根据信号强度确定误差容限的模型。     

Molecular species analysis of lipids by metastable ion mass spectrometry

A convenient universal and fast mass spectrometrical method designed for the molecular species analysis of natural lipids is described. In contrast to the commonly employed procedures the method does not require chemical or enzymatic treatment and does not include chromatographic steps. The method relies on the recognition of ions characteristic of individual molecular species in the mass spectrum of a particular lipid fraction, that is accomplished on the basis of metastable ion spectra. The efficiency of this approach is demonstrated with a variety of natural lipids: triglycerides, glycerophospholipids, sphingomyelin and ornithinolipids. The advantages and limitations of the method as well as possible further developments are discussed.     

Protein surface mapping by chemical oxidation: structural analysis by mass spectrometry

The solvent-accessible surface area of proteins is important in biological function for many reasons, including protein-protein interactions, protein folding, and catalytic sites. Here we present a chemical technique to oxidize amino acid side chains in a model protein, apomyoglobin, and subsequent elucidation of the effect of solvent accessibility on the sites of oxidation. Under conditions of low protein oxidation (zero to three oxygen atoms added per apomyoglobin molecule), we have positively identified five oxidation sites by liquid chromatography-tandem mass spectrometry and high-resolution Fourier transform mass spectrometry. Our results indicate that all oxidized amino acids, with the exception of methionine, have highly solvent-accessible side chains, but the rate of oxidation may not be dictated solely by solvent accessibility and amino acid identity.     

Phosphoproteomics by mass spectrometry: insights,implications, applications and limitations

Phosphorylation of proteins is a predominant, reversible post-translational modification. It is central to a wide variety of physiological responses and signaling mechanisms. Recent advances have allowed the global scope of phosphorylation to be addressed by mass spectrometry using phosphoproteomic approaches. In this perspective, we discuss four aspects of phosphoproteomics: the insights and implications from recently published phosphoproteomic studies and the applications and limitations of current phosphoproteomic strategies. Since approximately 50,000 known phosphorylation sites do not yet have any ascribed function, we present our perspectives on a major function of protein phosphorylation that may be of predictive value in hypothesis-based investigations. Finally, we discuss strategies to measure the stoichiometry of phosphorylation in a proteome-wide manner that is not provided by current phosphoproteomic approaches.     

Proteomic analysis of glycosylation: structural determination of N- and O-linked glycans by mass spectrometry

This review summarizes the methods, mainly based on mass spectrometry, for the structural determination of N- and O-linked carbohydrates that are post-translationally attached to a large number of proteins and which play a key role in determining the function and biophysical properties of these compounds. Analysis of these carbohydrates has proved difficult in the past due to their structural complexity. However, modern analytical methods such as mass spectrometry have the ability to elucidate most structural details at the concentration levels required for proteomics. This review describes methods for direct examination of glycoproteins by mass spectrometry, the release of N- and O-linked glycans from glycoproteins separated in sodium dodecyl sulfate polyacrylamide electrophoresis gels, and the analysis of these compounds by techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry provides the most rapid method for comparing glycan profiles and is probably most appropriate for clinical studies. One of the most promising techniques for determining the structures of N-glycans in proteomic studies is negative ion fragmentation of electrosprayed ions. This technique combines high throughput with ease of structural interpretation and provides structural details that are difficult to obtain by classical methods.     

Nonredundant mass spectrometry: a strategy to integrate mass spectrometry acquisition and analysis

Protein identification using automated data-dependent tandem mass spectrometry (MS/MS) is now a standard procedure. However, in many cases data-dependent acquisition becomes redundant acquisition as many different peptides from the same protein are fragmented, whilst only a few are needed for unambiguous identification. To increase the quality of information but decrease the amount of information, a nonredundant MS (nrMS) strategy has been developed. With nrMS, data analysis is an integral part of the overall MS acquisition and analysis, and not an endpoint as typically performed. In this nrMS workflow a matrix assisted laser desorption/ionization-time of flight-time of flight (MALDI-TOF/TOF) instrument is used. MS and restricted MS/MS data are searched and identified proteins are used to generate an "exclusion list", after in silico digestion. Peptide fragmentation is then restricted to only the most intense ions not present in the exclusion list. This process is repeated until all peaks are accounted for or the sample is consumed. Compared to nanoLC-MS/MS, nrMS yielded similar results for the analysis of six pooled two-dimensional electrophoresis (2-DE) spots. In comparison to standard data-dependent MALDI-MS/MS for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel band analysis, nrMS dramatically increased the number of identified proteins. It was also found that this new workflow significantly increased sequence coverage by identifying unexpected peptides, which can result from post-translational modifications.     

Expanding the organismal scope of proteomics: cross-species protein identification by mass spectrometry and its implications

Due to the limited applicability of conventional protein identification methods to the proteomes of organisms with unsequenced genomes, researchers have developed approaches to identify proteins using mass spectrometry and sequence similarity database searches. Both the integration of mass spectrometry with bioinformatics and genomic sequencing drive the expanding organismal scope of proteomics.     

Negative ion electrospray mass spectrometry of neoflavonoids

The electrospray ionisation mass spectra of the neoflavanoids brazilin and hematoxylin are reported in both their reduced (1 and 2, respectively) and their oxidised forms (3 and 4, respectively). In the reduced forms, breakdown pathways under collision induced decomposition (CID) conditions produce fragments characteristic of rings A and C; in their oxidised forms, the fragments are characteristic of rings B and D. The structural assignments of the fragments are substantiated by recording the spectra after deuterium exchange at the hydroxyl groups.     

Fast-atom-bombardment mass spectrometry and electron-impact direct-probe mass spectrometry in the structural analysis of oligosaccharides with special reference to the poly(N-acetyl-lactosamine) series.

A comparison has been made of positive- and negative-ion fast-atom-bombardment (FAB) and electron-impact (EI) mass spectrometry for analysis of oligosaccharides and alditols containing alternating and consecutive sequences of neutral and acetamido sugars. Among these were novel chemically synthesized tetrasaccharides with Ii antigen activities. FAB ionization has the advantage that it is applicable to non-derivatized oligosaccharides and it can determine Mr. However, the abundance of fragment ions providing structural information and the amount of material required for analysis (1-50 nmol) varied from sample to sample. In contrast, EI mass spectrometry of 5 nmol of permethylated or peracetylated oligosaccharides reliably gives all the fragment ions formed by cleavage across the glycosidic bonds.     

C-5 modifications in N-acetyl-neuraminic acid: scope and limitations

Glycoconjugates containing sialic acid are involved in a large variety of biological phenomena, including cell-cell adhesion, recognition by viruses and bacteria, and oncogenesis. Therefore, they are important synthetic targets for the design of drugs and vaccines. In the last decades, different methodologies that improve yield and stereoselectivity in sialylation reactions have been investigated. This review summarizes the latest developments in the synthesis of C-5 modified sialic acid glycosyl donors and glycosyl acceptors and their application in the synthesis of alpha-sialosides.