Imaging lipids with secondary ion mass spectrometry

This review discusses the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and magnetic sector SIMS with high lateral resolution performed on a Cameca NanoSIMS 50(L) to imaging lipids. The similarities between the two SIMS approaches and the differences that impart them with complementary strengths are described, and various strategies for sample preparation and to optimize the quality of the SIMS data are presented. Recent reports that demonstrate the new insight into lipid biochemistry that can be acquired with SIMS are also highlighted. This article is part of a Special Issue entitled Tools to study lipid functions.     

Imaging of metabolites using secondary ion mass spectrometry

This article provides an overview of the technique of secondary ion mass spectrometry imaging and highlights some current and future areas of application relevant to the field of metabolomics. The approach benefits from label-free analysis of molecular species up to ~1500 Da with minimal sample preparation. Offering the highest spatial resolution of current mass spectrometry imaging methodologies, the technique is well-suited to metabolite imaging in both biological tissue and cells, in both 2D and 3D.     

Imaging of metabolites using secondary ion mass spectrometry

This article provides an overview of the technique of secondary ion mass spectrometry imaging and highlights some current and future areas of application relevant to the field of metabolomics. The approach benefits from label-free analysis of molecular species up to ~1500 Da with minimal sample preparation. Offering the highest spatial resolution of current mass spectrometry imaging methodologies, the technique is well-suited to metabolite imaging in both biological tissue and cells, in both 2D and 3D.

     

Derivatization of hydrophilic peptides for liquid secondary ion mass spectrometry at the picomole level

A simple procedure for preparing alkyl and benzyl esters of peptides is described. The procedure can provide an increase in the secondary ion yield of a factor of 25 or more in the liquid secondary ion mass spectra of hydrophilic peptides. The procedure allows rapid in situ derivatization of, e.g., collected, lyophilized HPLC fractions. No sample transfers are required and excess reagents are easily removed. Mass spectrometry of such fractions is typically required to prepare a mass map of the peptides produced by proteolytic digestion of a protein. However, small hydrophilic peptides are often not detected because their low secondary ion yield. Relative yields of MH+ ions from peptides esterified with various alcohols are compared: methanol, 2-propanol, 1-butanol, 1-hexanol, 1-octanol, and benzyl alcohol. The best combination of ion yield and ease of reagent removal is obtained with 1-hexanol. The degree of improvement depends on the specific peptide; the greatest improvement is generally observed with the most hydrophilic peptides. The procedure does not affect side-chain amides. Partial derivatization is sometimes observed with peptides containing more than one carboxyl group. Hexylation is shown to have a leveling effect on the mass spectra of peptide mixtures, allowing detection of surface-inactive peptides in the presence of surface-active ones. Benzyl alcohol is useful for derivatizing peptides that are not retained or that elute very early from reverse-phase HPLC columns. The derivatives have longer retention times and greater uv molar absorptivity and are more easily detected by subsequent mass spectrometry than the underivatized peptides.     

Structural characterization of intact, branched oligosaccharides by high performance liquid chromatography and liquid secondary ion mass spectrometry

We report results of a mass-spectrometric-based strategy for determining the detailed structural features of N-linked oligosaccharides from glycoproteins. The method was used to characterize a series of intact, high mannose oligosaccharides isolated from human immunoglobulin M (IgM). The IgM was purified from a patient with Waldenstrom's macroglobulinemia. The strategy included releasing the oligosaccharides by digestion of the purified glycoprotein with endoglycosidase H, separating the released oligosaccharides by high resolution gel filtration, and derivatizing the resulting reducing termini with the uv-absorbing moiety, ethyl p-aminobenzoate. This particular derivative facilitates HPLC detection and provides centers for protonation and deprotonation enhancing liquid secondary ion mass spectra. Positive and negative ion spectra contained molecular species of similar abundance. However, fragment ion peaks yielding sequence information were significantly more prominent in the negative ion mass spectra. Furthermore, it was obvious that the fragmentation patterns differed substantially for linear and branched oligomers. For linear oligosaccharides, a smooth envelope of fragment ions was observed; from low to high mass there was an ordered decrease in ion abundance from both the reducing and nonreducing termini. This pattern of fragment ions was not observed for branched oligosaccharides since in these cases fragments at certain masses could not arise by single bond cleavages. Therefore, these fragments were either significantly reduced in abundance or absent as compared with identical fragments formed from linear molecules. Importantly, 200 pmol of an oligosaccharide could be derivatized, separated, and detected by mass spectrometry, allowing identification of previously unreported minor components of the IgM oligosaccharides. Therefore, this experimental strategy is particularly useful for the purification and detailed structural characterization of low abundance oligosaccharides isolated from heterogeneous biological samples.     

Quantification of cytokinin o-glucosides by negative ion mass spectrometry

Pulsed positive ion-negative ion chemical ionization mass spectra of O-glucosyl-zeatin and -dihydrozeatin, their ribosides, and their N-9 2-cyanoethyl and 2-chloro-2-cyanoethyl derivatives are reported. By methods based on these spectra, the levels of the glucosides were determined in soybean (Glycine max) leaves.     

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.     

High resolution imaging by organic secondary ion mass spectrometry

Secondary-ion mass spectrometry (SIMS) is based on the acceleration of high-energy primary ions onto a target. Secondary electrons, neutrals and ions are emitted from the target, reflecting its chemical composition. This enables simultaneous analysis and localization of target molecules, giving valuable information that is difficult or impossible to obtain with other analytical methods. The secondary ions can be extracted and detected by any type of mass analyzer. SIMS is unique in its ability to detect several target molecules simultaneously in small samples and to image their localization at subcellular resolution. The recent development of bioimaging SIMS opens up new possibilities in biotechnology and biological research with applications in biomedicine and pathology. The current development of this technique has the potential to become as important for biotechnology as the advent of the electron microscope, confocal microscope or in situ hybridization.     

High-performance liquid chromatography coupled with negative ion tandem mass spectrometry for determination of pravastatin in human plasma

A new method, using high-performance liquid chromatography/ion electrospray (negative ion) mass spectrometry, has been developed for the determination of a hydrophilic liver-specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase, pravastatin in human plasma. In this method, plasma samples were prepared by a solid-phase extraction on C(18) Bond Elut cartridge. Chromatography was carried out with a Zorbax C(8) column. Simple isocratic chromatography conditions were used. The method has been validated in a linear range of 0.25-300 ng/ml with a coefficient of variation of 0.6-3.4%. The overall recovery was 90.5% for pravastatin and 90.8% for the internal standard beta-hydroxy-lovastatin. The method is simple and reliable with a total run time of less than 2 min.     

Positive ion fast atom bombardment mass spectrometry of some small oligosaccharides

Positive ion fast atom bombardment mass spectrometry appears to be a very useful method for the determination of molecular weight and composition of underivatized di- and trisaccharides. Information on the identity of the monosaccharide units can be obtained from the metastable ion and collisional activation spectra of selected ions. The type of linkage between the monosaccharides is reflected in some spectral characteristics, but the differences are relatively small and do not always allow an unambiguous identification. The position of a fructose unit in a trisaccharide molecule is shown by the collisional activation spectra of the [M + H]+ ion, as an anhydrofructose molecule is easily eliminated from the ions in which fructose is in a terminal position.     

Isolation and mass spectrometry characterization of molecular species of lactosylceramides using liquid chromatography-electrospray ion trap mass spectrometry

Reverse-phase liquid chromatography/electrospray ion trap mass spectrometry (LC-ESI-MSn) was established for identification of the molecular species of lactosylceramides. Lactosylceramides derived from porcine blood cells were separated on a CapcellPak C8 column using a mixture of methanol and 1 mM ammonium formate from the C16 to C26 fatty acyl chains based on the length of total carbon chains and the nature of sphingoid bases (w') and fatty acyl chains (Y0'-w') was identified by MS3 as their [M+H]+ ions. The same number of fatty acyl moieties appeared in the order of unsaturated, (2-)hydroxylated, and saturated components. The molecular species of lactosylceramides derived from porcine blood cells totaled more than 33 and included mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1 in addition to 28 minor species from C16:0 to C26:0 fatty acyl moieties. The molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells (70% were differentiated into macrophage-lineage cells) were identified as C24:0-d18:1, C24:1-d18:1, C22:0-d18:1, C16:0-d18:1, and more than 21 other minor species. Our results suggest that reverse-phase LC-ESI-MSn is a useful and simple method for identification of lactosylceramide molecular species.     

Application of nanoscale secondary ion mass spectrometry to plant cell research

Imaging resource flow in soil-plant systems remains central to understanding plant development and interactions with the environment. Typically, subcellular resolution is required to fully elucidate the compartmentation, behavior, and mode of action of organic compounds and mineral elements within plants. For many situations this has been limited by the poor spatial resolution of imaging techniques and the inability to undertake studies in situ. Here we demonstrate the potential of Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS), which is capable of the quantitative high-resolution spatial imaging of stable isotopes (e.g., ¹²C, ¹³C, ¹⁴N, ¹⁵N, ¹⁶O, ¹⁸O, ³¹P, ³⁴S) within intact plant-microbial-soil systems. We present examples showing how the approach can be used to investigate competition for ¹⁵N-labelled nitrogen compounds between plant roots and soil microorganisms living in the rhizosphere and the spatial imaging of ³¹P in roots. We conclude that NanoSIMS has great potential to elucidate the flow of isotopically-labelled compounds in complex media (e.g., soil) and opens up countless new opportunities for studying plant responses to abiotic stress (e.g., ¹⁸O₃, elevated ¹³CO₂), signal exchange, nutrient flow and plant-microbial interactions.Key words: mass spectrometry, NanoSIMS, rhizosphere, isotope labelling, soil, nitrogen, carbon, phosphorus, ¹⁵N, ¹³C, ³¹PWe have used the NanoSIMS technique to investigate the flow of nutrients between microbial and plant cells within the rhizosphere. Secondary Ion Mass Spectrometry (SIMS) involves bombarding a sample with a high-energy ion beam, which sputters atoms, molecules and electrons from the sample surface. Ionized species (secondary ions) are extracted to a mass spectrometer, sorted according to their energy and their mass-to-charge ratio, and counted. NanoSIMS, a recent development in SIMS, combines high sensitivity with high spatial resolution (typically 100 nm) to allow elemental and isotopic imaging of secondary ions, such as ¹²C-, ¹⁶O- and ¹²C¹⁴N-, on a range of biological materials at the sub-cellular scale (Fig. 1A and B). An element map is obtained by scanning the primary ion beam over the sample surface and measuring the secondary ion intensities of any given ion species, at each pixel in the image. The intrinsically high mass resolution allows the separation of different ion species at the same nominal atomic mass (e.g., ¹²C¹⁵N- from ¹³C¹⁴N- at mass 27), while the multi-collection capability allows the simultaneous measurement of up to five ion species. This makes it possible to obtain images of different isotopes from the same area simultaneously, from which quantitative isotope ratios from individual components can then be extracted. As such, NanoSIMS offers a means of elucidating processes involved in the transport of ions and molecules into cells and their distribution within cells, at scales and sensitivities not attainable by other methods.^1–5Open in a separate windowFigure 1(A) ¹²C¹⁴N^- and (B) ³¹P^- images of a wheat root cell nucleus from NanoSIMS illustrating the potential to map different elements at the sub-cellular scale; (C) TEM image of two bacteria attached to a cortical cell wall; (D) corresponding ¹⁵N/¹⁴N ratio image from NanoSIMS of the same bacteria. The differential uptake of ¹⁵N is illustrated by the color scale; ranging from natural abundance (blue) to a ¹⁵N/¹⁴N ratio = 1.0 (i.e., 50 at% ¹⁵N) (pink) for the plant cell and bacteria, respectively; (E) Linescan (3.5 µm) illustrating the variation in ¹⁵N/¹⁴N across an enriched bacterium and an un-enriched plant cell wall (line in D). Error bars are based on the Poisson counting statistics for each pixel.We previously demonstrated the use of NanoSIMS to image and map the location of ¹⁵N-labelled bacterial communities artificially introduced into soil microhabitats.^6,7 We extended this approach to a natural ecosystem, by examining the differential partitioning of ¹⁵N-labelled ammonium (¹⁵NH₄⁺) between plant roots and soil microbial communities at the nanometer scale (Fig. 1C and D).⁸ It was shown that introduced ¹⁵N could be detected, and more importantly, mapped, in individual bacterial cells found in the soil matrix, within the rhizosphere, within root hairs, and intra-cellular within the root. The ¹⁵N/¹⁴N ratio data (determined as the ratio between the ¹²C¹⁵N- and the ¹²C¹⁴N- signals) could then be extracted from specific regions of interest—groups of pixels bounding a particular feature, such as a bacterium or a root cell wall, or linescans (Fig. 1E). This unique approach allows the visualization of nutrient flows and metabolic pathways through complex, multi-component ecosystems. Here we consider further the application of the technique to study nutrient availability in plant cell research.     

Visualization of acetaminophen-induced liver injury by time-of-flight secondary ion mass spectrometry

Time-of-flight secondary ion mass spectrometry (MS) provides secondary ion images that reflect distributions of substances with sub-micrometer spatial resolution. To evaluate the use of time-of-flight secondary ion MS to capture subcellular chemical changes in a tissue specimen, we visualized cellular damage showing a three-zone distribution in mouse liver tissue injured by acetaminophen overdose. First, we selected two types of ion peaks related to the hepatocyte nucleus and cytoplasm using control mouse liver. Acetaminophen-overdosed mouse liver was then classified into three areas using the time-of-flight secondary ion MS image of the two types of peaks, which roughly corresponded to established histopathological features. The ion peaks related to the cytoplasm decreased as the injury became more severe, and their origin was assumed to be mostly glycogen based on comparison with periodic acid–Schiff staining images and reference compound spectra. This indicated that the time-of-flight secondary ion MS image of the acetaminophen-overdosed mouse liver represented the chemical changes mainly corresponding to glycogen depletion on a subcellular scale. In addition, this technique also provided information on lipid species related to the injury. These results suggest that time-of-flight secondary ion MS has potential utility in histopathological applications.     

纳米二次离子质谱技术(NanoSIMS)在微生物生态学研究中的应用

超高分辨率显微镜成像技术与同位素示踪技术相结合的纳米二次离子质谱技术(NanoSIMS)具有较高的灵敏度和离子传输效率、极高的质量分辨率和空间分辨率(＜ 50 nm),代表着当今离子探针成像技术的最高水平.利用稳定性或者放射性同位素在原位或者微宇宙条件下示踪目标微生物,然后将样品进行固定、脱水、树脂包埋或者导电镀膜处理,制备成可供二次离子质谱分析的薄片,进一步通过NanoSIMS成像分析,不仅能够在单细胞水平上提供微生物的生理生态特征信息,而且能够准确识别复杂环境样品中的代谢活跃的微生物细胞及其系统分类信息,对于认识微生物介导的元素生物地球化学循环机制具有重要意义.介绍了纳米二次离子质谱技术的工作原理和技术路线,及其与同位素示踪技术、透射电子显微镜(TEM)、扫描电子显微镜(SEM)、荧光原位杂交技术(FISH)、催化报告沉积荧光原位杂交技术(CARD-FISH)、卤素原位杂交技术(Halogen In Situ Hybridization,HISH)等联合使用在微生物生态学研究方面的应用.     

Orientation of membrane-bound melittin studied by a combination of HPLC and liquid secondary ion mass spectrometry (LSIMS)

Combining two analytical techniques, HPLC and liquid secondary ion mass spectrometry, the orientation of liposomal membrane-bound melittin was analyzed through its trypsin-digested products. We found that trypsin can access all proteolytic sites of the membrane-bound melittin when the liposomes have no transmembrane potential, whereas the proteolytic site near the N terminus of melittin is blocked when the liposomes have a negative transmembrane potential. The results suggest that the negative transmembrane potential may induce the melittin molecules to insert into the membrane perpendicularly, whereas melittin lies flat on the membrane surface in the absence of a negative potential.     

Revision of the blocked N terminus of rat heart fatty acid-binding protein by liquid secondary ion mass spectrometry

The primary structure of rat heart muscle fatty acid-binding protein was investigated by liquid secondary ion mass spectrometry. The protein was digested with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease and the resulting peptides were separated by reverse phase high performance liquid chromatography. The masses of the protonated molecular ions (MH+) of the tryptic, chymotryptic, and S. aureus protease peptides were determined by liquid secondary ion mass spectrometry analysis using 20-500 pmol of material. From the tryptic digest, two peptides with MH+ 1036 and 861 were initially found that did not match the published primary sequence (Sacchettini, J. C., Meininger, T. A., Lowe, J. B., Gordon, J. I., and Banaszak, L. J. (1987) J. Biol. Chem. 262, 5428-5430). The amino acid sequences of these two peptides were determined by a combination of mass spectrometry, B/E-linked scanning, and high performance tandem mass spectrometric techniques to be: (Formula: see text). These new data require that corrections be made to the previously published sequence, involving residues 1-4 and 51-52. The corrected amino sequence for rat m-FABP reveals greater homology with myelin P2, mouse adipocyte p422 protein, and intestinal fatty acid-binding protein than was previously demonstrated.     

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.     

Lipid imaging with time-of-flight secondary ion mass spectrometry (ToF-SIMS)

Fundamental advances in secondary ion mass spectrometry (SIMS) now allow for the examination and characterization of lipids directly from biological materials. The successful application of SIMS-based imaging in the investigation of lipids directly from tissue and cells are demonstrated. Common complications and technical pitfalls are discussed. In this review, we examine the use of cluster ion sources and cryogenically compatible sample handling for improved ion yields and to expand the application potential of SIMS. Methodological improvements, including pre-treating the sample to improve ion yields and protocol development for 3-dimensional analyses (i.e. molecular depth profiling), are also included in this discussion. New high performance SIMS instruments showcasing the most advanced instrumental developments, including tandem MS capabilities and continuous ion beam compatibility, are described and the future direction for SIMS in lipid imaging is evaluated.     

Comparative study of acidic glycosphingolipids by field desorption and secondary ion mass spectrometry

Acidic glycosphingolipids were analyzed by field desorption (FD-MS) and secondary ion mass spectrometry (SI-MS) using the primary ion Xe+ with a glycerol matrix. In the analysis of underivatized gangliosides by FD-MS, the fragment corresponding to the asialo residue resulting from the cationized cluster ion (M + Na)+ was the base peak, and ions due to cleavage at the glycosidic linkages were detected, as in the neutral glycosphingolipids. In the case of sulfatide, the ceramide fragment showed the highest intensity in the spectrum. In SI-MS spectra of acidic glycosphingolipids, (M + Na)+, (M + 2Na-H)+, and (M + K)+ were continuously detected as relatively high intensity ions during analysis of gangliosides and sulfatide. Other ions were mostly similar to those obtained by FD-MS. In FD-MS spectra of permethylated gangliosides, the cationized molecular ion (M + Na)+ was the base peak, and fragment ions due to asialo gangliosides were prominent. Other peaks were hard to detect. In SI-MS, molecular ions (M + H)+ and (M + H-32)+ and other ions due to cleavage of the glycosidic linkages were clearly detected. In this case, the sensitivity was greatly improved. Ions due to the non reducing end sugars were clearly detected, because of the relatively low intensity of ion peaks due to the glycerol matrix. It is concluded that the combination with FD-MS and SI-MS is particularly useful for the determination of molecular weight, sugar sequence and ceramide structure with sample amounting to only a few micrograms order.