Liquid ionization mass spectrometry of phospholipids

Several phosphatidylcholines (PC) and a phosphatidylethanolamine (PE) were subjected to liquid ionization (LI) mass spectrometry, in which a sample is ionized through energy transfer from metastable argon atoms under atmospheric pressure. Commercially available and synthesized, saturated or unsaturated fatty acid containing phospholipids and their mixtures were studied. A sample either as a concentrated chloroform-methanol solution or with glycerol (matrix) gave characteristic peaks such as MH+ and four fragment ions. One of the fragment ions (e.g., m/z 551 of PC 16:0, 16:0) containing both fatty acid residues has been commonly observed with other ionization methods such as CI, FD, and FAB, but the other fragment ions have not been observed in other mass spectra with one exception on desorption CI. Ions b and d (e.g., m/z 464 and 328, respectively, for PC 16:0, 16:0) contain one fatty acyl residue and the other ion containing the phosphorylcholine moiety appears at m/z 196 for PC. Thus the masses of the MH+ ion and these fragment ions provide useful structural information even in the case of a mixture. The ion b (e.g., m/z 488 of PC 18:0, 18:2) observed during an early period of heating was formed mainly by the loss of one acyl group at sn-1 of the glycerol backbone and thus may be used to differentiate the positional specificities of the constituent fatty acids. The temperature of the sample, however, should be controlled precisely, because it has a significant effect on the mass spectrum. The present method (LI) also provided useful information for a mixture of PC and PE.     

Peptide and protein imaging mass spectrometry in cancer research

MALDI mass spectrometry is able to acquire protein profiles directly from tissue that can describe the levels of hundreds of distinct proteins. MALDI imaging MS can simultaneously reveal how each of these proteins varies in heterogeneous tissues. Numerous studies have now demonstrated how MALDI imaging MS can generate different protein profiles from the different cell types in a tumor, which can act as biomarker profiles or enable specific candidate protein biomarkers to be identified.     

Ammonia desorption chemical ionization mass spectrometry of phospholipids

Ammonia desorption chemical ionization mass spectra (NH₃-DCIMS) of phosphatidyl-sulfocholines, a mixture of a homologous series of phosphatidylsulfocholines (PSC) and a mixture of a PSC phosphatidylcholine (PC), were measured by a flash heating method involving introduction of 1 μg of the samples on a tungsten wire, quickly heated, into the ion source of a Finnigan 4000/INCOS quadrupole instrument. It was possible to observe mass spectra during several seconds.The first spectra recorded of the PSCs gave essentially only the quasi-molecular [M + 18]⁺ peaks and the ‘A’ (see text) peaks. Spectra of a mixture of three homologous PSCs clearly showed three pairs of the above peaks. In contrast spectra of the PCs gave principally the [M + 1]⁺ and the ‘A’ peaks after 2s so that one can distinguish the diagnostic peaks in a mixture of a PC and a PSC.These results show that ammonia desorption chemical ionization by fast heating is a suitable technique for the charaterization of some head groups of phospholipids as well as a promising method for the analysis of phospholipid mixtures.     

Structural analysis of choline phospholipids by fast atom bombardment mass spectrometry and tandem mass spectrometry

The structures of intact choline phospholipids were determined by positive and negative ion mode fast atom bombardment mass spectrometry, tandem mass spectrometry, and B2/E and B/E constant linked scan mass spectrometry. The molecular weight of the choline lipid could be clearly determined by the appearance of [M + H]+ or [M + Na]+ in the positive ion mode and triplet ions, e.g., [M - 15]-, [M - 60]-, and [M - 86]-, in the negative ion mode. The structures of the triplet ions were assigned to [M - CH3]-, [M - HN(CH3)3]-, and [M - CH2 = CHN(CH3)3]-, respectively, by the MS/MS of each triplet ion, and the origin of the triplet ions was found as the matrix-ion adduct to the target molecule by using the B2/E linked scan technique. The polar group could be identified by the existence of ions indicating glycerophosphocholine and its cleavage products and by the presence of the triplet ions in the negative ion mode. Positional determination of the distribution of constituent fatty acyl groups was carried out by comparing the intensity of deacylated ions from positions 1 and 2 in the positive ion mode and of the ions produced by MS/MS of the triplet ions. From the mass number of the [RCOO]- ion which appeared in the negative ion mode, the molecular weight and degree of unsaturation of the fatty acyl group were determined. The position of double bond(s) in the acyl group was determined from the MS/MS of the [RCOO]- ion.     

Salamander retina phospholipids and their localization by MALDI imaging mass spectrometry at cellular size resolution

Salamander large cells facilitated identification and localization of lipids by MALDI imaging mass spectrometry. Salamander retina lipid extract showed similarity with rodent retina lipid extract in phospholipid content and composition. Like rodent retina section, distinct layer distributions of phospholipids were observed in the salamander retina section. Phosphatidylcholines (PCs) composing saturated and monounsaturated fatty acids (PC 32:0, PC 32:1, and PC 34:1) were detected mainly in the outer and inner plexiform layers (OPL and IPL), whereas PCs containing polyunsaturated fatty acids (PC 36:4, PC 38:6, and PC 40:6) composed the inner segment (IS) and outer segment (OS). The presence of PCs containing polyunsaturated fatty acids in the OS layer implied that these phospholipids form flexible lipid bilayers, which facilitate phototransduction process occurring in the rhodopsin rich OS layer. Distinct distributions and relative signal intensities of phospholipids also indicated their relative abundance in a particular cell or a cell part. Using salamander large cells, a single cell level localization and identification of biomolecules could be achieved by MALDI imaging mass spectrometry.     

Epigenetic modifications of metastasis suppressor genes in colon cancer metastasis

Colon and rectal cancer (colorectal cancer, CRC) is the third most common cancer worldwide. Deaths from CRC account for around 8% of all cancer deaths, making it the fourth most common cause of death from cancer. The high mortality rate of colon cancer is mainly attributable to its metastasis. Efforts have been made to identify metastasis suppressor genes, which encode proteins responsible for inhibiting the metastasis but not suppressing the growth of primary tumors. Studies on metastasis suppressor genes demonstrated that epigenetic modifications, such as DNA promoter methylation and histone modification, play crucial roles in regulating the expression of many metastasis suppressor genes, which indicates the association between aberrant epigenetic alterations and cancer metastasis. This review will focus on the recent findings regarding metastasis suppressors regulated by epigenetic modifications, particularly DNA methylation and histone modification, in CRC metastasis. Also discussed will be recent progress on the suppression of CRC metastasis by genistein, a soy isoflavone, with a focus on epigenetic mechanisms.     

Visualizing spatial lipid distribution in porcine lens by MALDI imaging high-resolution mass spectrometry

The intraocular lens contains high levels of both cholesterol and sphingolipids, which are believed to be functionally important for normal lens physiology. The aim of this study was to explore the spatial distribution of sphingolipids in the ocular lens using mass spectrometry imaging (MSI). Matrix-assisted laser desorption/ionization (MALDI) imaging with ultra high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was used to visualize the lipid spatial distribution. Equatorially-cryosectioned, 12 μm thick slices of tissue were thaw-mounted to an indium-tin oxide (ITO) glass slide by soft-landing to an ethanol layer. This procedure maintained the tissue integrity. After the automated MALDI matrix deposition, the entire lens section was examined by MALDI MSI in a 150 μm raster. We obtained spatial- and concentration-dependent distributions of seven lens sphingomyelins (SM) and two ceramide-1-phosphates (CerP), which are important lipid second messengers. Glycosylated sphingolipids or sphingolipid breakdown products were not observed. Owing to ultra high resolution MS, all lipids were identified with high confidence, and distinct distribution patterns for each of them are presented. The distribution patterns of SMs provide an understanding of the physiological functioning of these lipids in clear lenses and offer a novel pathophysiological means for understanding diseases of the lens.     

Emerging technologies in mass spectrometry imaging

Mass spectrometry imaging (MSI) as an analytical tool for bio-molecular and bio-medical research targets accurate compound localization and identification. In terms of dedicated instrumentation, this translates into the demand for more detail in the image dimension (spatial resolution) and in the spectral dimension (mass resolution and accuracy), preferably combined in one instrument. At the same time, large area biological tissue samples require fast acquisition schemes, instrument automation and a robust data infrastructure. This review discusses the analytical capabilities of an "ideal" MSI instrument for bio-molecular and bio-medical molecular imaging. The analytical attributes of such an ideal system are contrasted with technological and methodological challenges in MSI. In particular, innovative instrumentation for high spatial resolution imaging in combination with high sample throughput is discussed. Detector technology that targets various shortcomings of conventional imaging detector systems is highlighted. The benefits of accurate mass analysis, high mass resolving power, additional separation strategies and multimodal three-dimensional data reconstruction algorithms are discussed to provide the reader with an insight in the current technological advances and the potential of MSI for bio-medical research.     

Different localization patterns of anthocyanin species in the pericarp of black rice revealed by imaging mass spectrometry

Black rice (Oryza sativa L. Japonica) contains high levels of anthocyanins in the pericarp and is considered an effective health-promoting food. Several studies have identified the molecular species of anthocyanins in black rice, but information about the localization of each anthocyanin species is limited because methodologies for investigating the localization such as determining specific antibodies to anthocyanin, have not yet been developed Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a suitable tool for investigating the localization of metabolites. In this study, we identified 7 species of anthocyanin monoglycosides and 2 species of anthocyanin diglycosides in crude extracts from black rice by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. We also analyzed black rice sections by MALDI-IMS and found 2 additional species of anthocyanin pentosides and revealed different localization patterns of anthocyanin species composed of different sugar moieties. Anthocyanin species composed of a pentose moiety (cyanidin-3-O-pentoside and petunidin-3-O-pentoside) were localized in the entire pericarp, whereas anthocyanin species composed of a hexose moiety (cyanidin-3-O-hexoside and peonidin-3-O-hexoside) were focally localized in the dorsal pericarp. These results indicate that anthocyanin species composed of different sugar moieties exhibit different localization patterns in the pericarp of black rice. This is the first detailed investigation into the localization of molecular species of anthocyanins by MALDI-IMS.     

Drug localization in different lung cancer phenotypes by MALDI mass spectrometry imaging

Lung cancer is a common cause of cancer mortality in the world, largely due to the risk factor of tobacco smoking. The drug therapy at the molecular level includes targeting the epidermal growth factor receptor (EGFR) tyrosine kinase activity by using inhibitors, such as erlotinib (Tarceva) and gefitinib (Iressa). The heterogeneity of disease phenotypes and the somatic mutations presented in patient populations have a great impact on the efficacy of treatments using targeted personalized medicine. In this study, we report on basic physical and chemical properties of erlotinib and gefitinib in three different lung cancer tumor phenotypes, using MALDI instrumentation in imaging mode, providing spatial localization of drugs without chemical labeling. Erlotinib and gefitinib were analyzed in i) planocellular lung carcinoma, ii) adenocarcinoma and iii) large cell lung carcinoma following their deposition on the tissue surfaces by piezo-dispensing, using a controlled procedure. The importance of high-resolution sampling was crucial in order to accurately localize the EGFR tyrosine kinase inhibitors deposited in heterogeneous cancer tissue compartments. This is the first report on personalized drug characterization with localizations at a lateral resolution of 30μm, which allowed us to map these compounds at attomolar concentrations within the lung tumor tissue microenvironments.     

A fermented grain food mixture, AOB, inhibits liver metastasis in the metastasis model of rat colon cancer

Natural antioxidants have been shown to be rich sources of microchemicals with the potential to prevent human cancers. We examined whether dietary supplement of a fermented grain food mixture (AOB), which has been shown to have a strong antioxidative effect, may protect against colon cancer metastasis. At day 5 of AOB (6.5%) supplementation in a basal diet, the rat colon cancer cells (RCN-H4) were injected beneath the capsule of the spleen and one min later rats were splenectomized. All rats without AOB supplementation had multiple liver metastases. However, the number and the size of liver tumors were reduced by about 80% in AOB group. Combined use of cisplatin and AOB enhanced an anti-metastatic effect. The inhibition of liver metastasis by AOB was caused by the regulation of cell cycle. This product may be used as an adjuvant in the therapy of malignant neoplasia.     

Shotgun analysis of phospholipids from mouse liver and brain by nanoflow liquid chromatography/tandem mass spectrometry

It was demonstrated that a shotgun approach can be utilized for the characterization of phospholipids (PLs) extracted from mouse liver and brain by using nanoflow reversed phase liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). In this study, a dual scan method was introduced for the high throughput analysis of complex PL mixtures. Two consecutive LC-ESI-MS-MS runs were made in positive ion mode (for phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs)) first followed by analysis in negative ion mode (for phosphatidylserine (PSs) and phosphatidylinositol (PIs)) using the same binary gradient elution with and without adding formic acid, respectively. The separation of the PLs was carried out using a home made pull tip capillary column (C18) with an end frit. The MS analysis of the eluted PL molecules was performed with a precursor scan followed by a data dependent MS-MS scan. The developed dual scan method was tested with the extracts of PCs and PIs mixtures from soybean, PEs from Escherichia coli, and PSs from bovine brain. It was further applied for the characterization of intact PL samples that were extracted from both mouse liver and mouse brain in the laboratory, and resulted in the identification of 90 and 80 PL species, respectively.     

Visualization of the cell-selective distribution of PUFA-containing phosphatidylcholines in mouse brain by imaging mass spectrometry

Previous studies have shown that MALDI-imaging mass spectrometry (IMS) can be used to visualize the distribution of various biomolecules, especially lipids, in the cells and tissues. In this study, we report the cell-selective distribution of PUFA-containing glycerophospholipids (GPLs) in the mouse brain. We established a practical experimental procedure for the IMS of GPLs. We demonstrated that optimization of the composition of the matrix solution and spectrum normalization to the total ion current (TIC) is critical. Using our procedure, we simultaneously differentiated and visualized the localizations of specific molecular species of GPLs in mouse brain sections. The results showed that PUFA-containing phosphatidylcholines (PCs) were distributed in a cell-selective manner: arachidonic acid- and docosahexaenoic acid-containing PCs were seen in the hippocampal neurons and cerebellar Purkinje cells, respectively. Furthermore, these characteristic localizations of PUFA-PCs were formed during neuronal maturation. The phenomenon of brain cell-selective production of specific PUFA-GPLs will help elucidate the potential physiological functions of PUFAs in specific brain regions.     

Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry

Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.