Direct Analysis of Triacylglycerols from Crude Lipid Mixtures by Gold Nanoparticle-Assisted Laser Desorption/Ionization Mass Spectrometry

Triacylglycerols (TAGs), essential energy storage lipids, are easily detected by conventional MALDI MS when occurring on their own. However, their signals are easily overwhelmed by other lipids, mainly phosphatidylcholines (PCs) and, therefore, require purification. In order to profile TAGs from crude lipid mixtures without prefractionation, we investigated alternative matrixes that can suppress phospholipid ion signals and enhance cationization of TAGs. We found that an aqueous solution of citrate-capped gold nanoparticles (AuNPs) with a diameter of 12 nm is a superior matrix for the laser desorption/ionization mass spectrometry (LDI MS) of TAGs in crude lipid mixtures. The AuNP matrix effectively suppressed other lipid signals such as phospholipids and also provided 100 times lower detection limit for TAGs than 2,5-dihydroxybenzoic acid (DHB), the best conventional MALDI matrix for TAGs. The AuNP-assisted LDI MS enabled us to obtain detailed TAG profiles including minor species directly from crude beef lipid extracts without phospholipid interference. In addition, we could detect TAGs at a trace level from a total brain lipid extract.

Thin-layer chromatography combined with MALDI-TOF-MS and 31P-NMR to study possible selective bindings of phospholipids to silica gel

High-performance thin-layer chromatography (HPTLC) is a highly established separation method in the field of lipid and (particularly) phospholipid (PL) research. HPTLC is not only used to identify certain lipids in a mixture but also to isolate lipids (preparative TLC). To do this, the lipids are separated and subsequently re-eluted from the silica gel. Unfortunately, it is not yet known whether all PLs are eluted to the same extent or whether some lipids bind selectively to the silica gel. It is also not known whether differences in the fatty acyl compositions affect the affinities to the stationary phase. We have tried to clarify these questions by using a readily available extract from hen egg yolk as a selected example of a lipid mixture. After separation, the complete lanes or selected spots were eluted from the silica gel and investigated by a combination of MALDI-TOF MS and ³¹P NMR spectroscopy. The data obtained were compared with the composition of the total extract (without HPTLC). Although there were significant, solvent-dependent losses in the amount of each lipid, the relative composition of the mixture remained constant; there were also only very slight changes in the fatty acyl compositions of the individual PL classes. Therefore, lipid isolation by TLC may be used without any risk of major sample alterations.

A direct and simple method of coupling matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to thin-layer chromatography (TLC) for the analysis of phospholipids from egg yolk

Although the most important application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is "proteomics," there is growing evidence that this soft ionization method is also useful for phospholipid (PL) analysis. Although all PLs are detectable by MALDI-TOF MS, some lipid classes, particularly those with quaternary amines such as phosphatidylcholines (PCs), are more sensitively detected than others, and these suppress the signals of less sensitively detected PLs when complex mixtures are analyzed. Therefore, a separation of the total organic extract into individual lipid classes is necessary. As MALDI uses a solid sample, the direct evaluation of thin-layer chromatography (TLC) plates is possible. We report here on a method of directly coupling MALDI-TOF MS and TLC that can be easily implemented on commercially available MALDI-TOF devices. A total extract of hen egg yolk is used as a simple PL mixture to demonstrate the capabilities of this method. It will be shown that "clean" spectra without any major contributions from fragmentation products and matrix peaks can be obtained, and that this approach is even sensitive enough to detect the presence of PLs at levels of less than 1% of the total extract.     

Enzyme-Coupled Nanoparticles-Assisted Laser Desorption Ionization Mass Spectrometry for Searching for Low-Mass Inhibitors of Enzymes in Complex Mixtures

In this report, enzyme-coupled magnetic nanoparticles (EMPs) were shown to be an effective affinity-based tool for finding specific interactions between enzymatic targets and the low-mass molecules in complex mixtures using classic MALDI-TOF apparatus. EMPs used in this work act as nonorganic matrix enabling ionization of small molecules without any interference in the low-mass range (enzyme-coupled nanoparticles-assisted laser desorption ionization MS, ENALDI MS) and simultaneously carry the superficial specific binding sites to capture inhibitors present in a studied mixture. We evaluated ENALDI approach in two complementary variations: ‘ion fading’ (IF-ENALDI), based on superficial adsorption of inhibitors and ‘ion hunting’ (IH-ENALDI), based on selective pre-concentration of inhibitors. IF-ENALDI was applied for two sets of enzyme–inhibitor pairs: tyrosinase–glabridin and trypsin–leupeptin and for the real plant sample: Sparrmannia discolor leaf and stem methanol extract. The efficacy of IH-ENALDI was shown for the pair of trypsin–leupeptin. Both ENALDI approaches pose an alternative for bioassay-guided fractionation, the common method for finding inhibitors in the complex mixtures.

Analysis of the Lipidome of Xenografts Using MALDI-IMS and UHPLC-ESI-QTOF

Human tumor xenografts in immunodeficient mice are a very popular model to study the development of cancer and to test new drug candidates. Among the parameters analyzed are the variations in the lipid composition, as they are good indicators of changes in the cellular metabolism. Here, we present a study on the distribution of lipids in xenografts of NCI-H1975 human lung cancer cells, using MALDI imaging mass spectrometry and UHPLC-ESI-QTOF. The identification of lipids directly from the tissue by MALDI was aided by the comparison with identification using ESI ionization in lipid extracts from the same xenografts. Lipids belonging to PCs, PIs, SMs, DAG, TAG, PS, PA, and PG classes were identified and their distribution over the xenograft was determined. Three areas were identified in the xenograft, corresponding to cells in different metabolic stages and to a layer of adipose tissue that covers the xenograft.

Repeat MALDI MS imaging of a single tissue section using multiple matrices and tissue washes

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) techniques are continually being assessed with a view to improving the quality of information obtained from a given sample. A single tissue section will typically only be analyzed once by MALDI MSI and is then either used for histological staining or discarded. In this study, we explore the idea of repeat analysis of a single tissue section by MALDI MSI as a route toward improving sensitivity, structural characterization, and diversity of detected analyte classes. Repeat analysis of a single tissue section from a fresh frozen mouse brain is investigated with both α-cyano-4-hydroxycinnamic acid (CHCA) and para-nitroaniline (PNA). Repeat analysis is then applied to the acquisition of MALDI MSI and MALDI tandem mass spectrometry imaging employing collision induced dissociation (MS/MS imaging employing CID) from a formalin-fixed mouse brain section. Finally, both lipid and protein data are acquired from the same tissue section via repeat analysis utilizing CHCA, sinapinic acid (SA), and a tissue wash step. PNA was found to outperform CHCA as a matrix for repeat analysis; multiple lipids were identified using MS/MS imaging; both lipid and protein images were successfully acquired from a single tissue section.

A simple method to identify ether lipids in spermatozoa samples by MALDI-TOF mass spectrometry

Plasmalogens (alkenylacyl glycerophospholipids) are important lipid constituents of many tissues and cells (e.g., selected spermatozoa). Since the molecular weights of plasmalogens overlap with that of diacyl- or alkyl acyl lipids, sophisticated mass spectrometry (MS; including MS/MS) analysis is normally used for the unequivocal identification of plasmalogens. We will show here that a simple matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (without MS/MS capability) in combination with acidic hydrolysis and subsequent derivatization with 2,4-dinitrophenylhydrazine (DNPH) and/or digestion with phospholipase A₂ (PLA₂) is sufficient to determine the contributions of ether lipids in spermatozoa extracts. As neither diacyl nor alkylacyl lipids are sensitive to acids and do not react with DNPH, the comparison of the mass spectra before and after treatment with acids and/or DNPH addition readily provides unequivocal information about the plasmalogen content. Additionally, the released aldehydes are readily converted into the 2,4-dinitrophenylhydrazones and can be easily identified in the corresponding negative ion mass spectra. Finally, PLA₂ digestion is very useful in confirming the presence of plasmalogens. The suggested method was validated by analyzing roe deer, bovine, boar, and domestic cat spermatozoa extracts and comparing the results with isolated phospholipids.

Thin layer chromatography–spray mass spectrometry: a method for easy identification of synthesis products and UV filters from TLC aluminum foils

A straightforward procedure for direct mass spectrometric (MS) analysis of spots from thin layer chromatography (TLC) plates, without the need of an external ion source, was developed using the aluminum plate backing as spray tip. The spots were cut out shaped as a tip with a 60° angle, mounted in front of the MS orifice, and after addition of a spray solvent spectra were obtained immediately. A high-resolution time-of-flight MS was used since the method is of particular interest for rapid identification or confirmation of spots from TLC plates. The practical benefits of this technique were demonstrated by detection of by-products of organic reactions, by identification of degradation products, and by accurate confirmation of spots when UV filters in sunscreens were analyzed by TLC. Employing the described method TLC spots can be evaluated fast without the need of an external ion source or devices for analyte transfer from TLC to MS, only a basic MS instrument and a high-voltage power supply is required.

Structural Distinction of Diacyl-, Alkylacyl,and Alk-1-Enylacyl Glycerophosphocholines as [M – 15]– Ions by Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization

We describe a linear ion-trap (LIT) multiple-stage (MSⁿ) mass spectrometric approach towards differentiation of alkylacyl, alk-1-enylacyl- and diacyl-glycerophoscholines (PCs) as the [M – 15]^– ions desorbed by electrospray ionization (ESI) in the negative-ion mode. The MS⁴ mass spectra of the [M – 15 – R₂′CH = CO]^– ions originated from the three PC subfamilies are readily distinguishable, resulting in unambiguous distinction of the lipid classes. This method is applied to two alkyl ether rich PC mixtures isolated from murine bone marrow neutrophils and kidney, respectively, to explore its utility in the characterization of complex PC mixture of biological origin, resulting in the realization of the detailed structures of the PC species, including various classes and many minor isobaric isomers.

Mass spectrometry and inflammation—MS methods to study oxidation and enzyme-induced changes of phospholipids

Many diseases such as arthritis or atherosclerosis are accompanied by inflammatory processes. Inflammation is characterized by the infiltration of cells such as neutrophilic granulocytes and (a) the release of phospholipases [particularly phospholipase A₂ (PLA₂)] and (b) the generation of reactive oxygen as well as nitrogen species (ROS and RNS). While PLA₂ leads to defined lyso products (lacking one acyl residue), lipid oxidation is characterized by much more complex product patterns, including lipid peroxides, aldehydes (by double bond cleavage), and many others. Nevertheless, oxidation processes are highly important under in vivo conditions because molecules with regulatory functions are generated by the oxidation of lipids and/or free fatty acids. Therefore, lipid oxidation products as well as lysolipids are increasingly assumed to represent important disease (bio)markers. Consequently, there is also increasing interest in methods to characterize these products qualitatively and quantitatively. Mass spectrometry (MS) seems to be the method of choice to study (phospho)lipids changed under inflammatory conditions: nowadays, soft ionization MS methods are regularly used to study oxidative lipid modifications because of their high sensitivities and the tremendous mass resolutions that are achievable by using modern mass spectrometers. However, experimental care is required to be able to detect all relevant products. Although electrospray ionization (ESI) MS is so far most popular, applications of matrix-assisted laser desorption and ionization (MALDI) MS are continuously increasing. This review aims to summarize the so far available data on MS analyses of oxidized lipids as well as lysolipids. In addition to model systems, special attention will be paid to the monitoring of oxidized lipids and lysolipids under in vivo conditions. It is the aim of this review to provide a critical survey of the advantages and drawbacks of the different MS methods, with the focus on MALDI and ESI.

MASH Suite: A User-Friendly and Versatile Software Interface for High-Resolution Mass Spectrometry Data Interpretation and Visualization

The rapid advancements in mass spectrometry (MS) instrumentation, particularly in Fourier transform (FT) MS, have made the acquisition of high-resolution and high-accuracy mass measurements routine. However, the software tools for the interpretation of high-resolution MS data are underdeveloped. Although several algorithms for the automatic processing of high-resolution MS data are available, there is still an urgent need for a user-friendly interface with functions that allow users to visualize and validate the computational output. Therefore, we have developed MASH Suite, a user-friendly and versatile software interface for processing high-resolution MS data. MASH Suite contains a wide range of features that allow users to easily navigate through data analysis, visualize complex high-resolution MS data, and manually validate automatically processed results. Furthermore, it provides easy, fast, and reliable interpretation of top-down, middle-down, and bottom-up MS data. MASH Suite is convenient, easily operated, and freely available. It can greatly facilitate the comprehensive interpretation and validation of high-resolution MS data with high accuracy and reliability.

Rapid on-site TLC–SERS detection of four antidiabetes drugs used as adulterants in botanical dietary supplements

A novel facile method has been established for rapid on-site detection of antidiabetes chemicals used to adulterate botanical dietary supplements (BDS) for diabetes. Analytes and components of pharmaceutical matrices were separated by thin-layer chromatography (TLC) then surface-enhanced Raman spectroscopy (SERS) was used for qualitative identification of trace substances on the HPTLC plate. Optimization and standardization of the experimental conditions, for example the method used for preparation of silver colloids, the mobile phase, and the concentration of colloidal silver, resulted in a very robust and highly sensitive method which enabled successful detection when the amount of adulteration was as low as 0.001 % (w/w). The method was also highly selective, enabling successful identification of some chemicals in extremely complex herbal matrices. The established TLC–SERS method was used for analysis of real BDS used to treat diabetes, and the results obtained were verified by liquid chromatography–triple quadrupole mass spectrometry (LC–MS–MS). The study showed that TLC–SERS could be used for effective separation and detection of four chemicals used to adulterate BDS, and would have good prospects for on-site qualitative screening of BDS for adulterants.

Minimization of Fragmentation and Aggregation by Laser Desorption Laser Ionization Mass Spectrometry

Measuring average quantities in complex mixtures can be challenging for mass spectrometry, as it requires ionization and detection with nearly equivalent cross-section for all components, minimal matrix effect, and suppressed signal from fragments and aggregates. Fragments and aggregates are particularly troublesome for complex mixtures, where they can be incorrectly assigned as parent ions. Here we study fragmentation and aggregation in six aromatic model compounds as well as petroleum asphaltenes (a naturally occurring complex mixture) using two laser-based ionization techniques: surface assisted laser desorption ionization (SALDI), in which a single laser desorbs and ionizes solid analytes; and laser ionization laser desorption mass spectrometry (L²MS), in which desorption and ionization are separated spatially and temporally with independent lasers. Model compounds studied include molecules commonly used as matrices in single laser ionization techniques such as matrix assisted laser desorption ionization (MALDI). We find significant fragmentation and aggregation in SALDI, such that individual fragment and aggregate peaks are typically more intense than the parent peak. These fragment and aggregate peaks are expected in MALDI experiments employing these compounds as matrices. On the other hand, we observe no aggregation and only minimal fragmentation in L²MS. These results highlight some advantages of L²MS for analysis of complex mixtures such as asphaltenes.

Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins

Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label.

Simultaneous determination of multi-class antibiotic residues in water using carrier-mediated hollow-fiber liquid-phase microextraction coupled with ultra-high performance liquid chromatography tandem mass spectrometry

A method has been developed for carrier-mediated hollow-fiber liquid-phase microextraction (HF-LPME) and enrichment of multiple classes of antibiotics in water samples. Eleven compounds (erythromycin, spiramycin, tilmicosin, sulfathiazole, sulfamethazine, sulfamerazine, oxytetracycline, tetracycline, ciprofloxacin, danofloxacin and enrofloxacin) from four important classes of antibiotics (of the macrolide, sulfonamide, tetracycline and quinolone type) have been simultaneously preconcentrated with one set of HF-LPME conditions, followed by determination by ultra-HPLC combined with electrospray ionization tandem mass spectrometry (UHPLC-MS/MS). Antibiotics can be determined at ng L^?1 levels using this highly sensitive and selective method. Parameters including immersion time, liquid membrane composition, sample pH, acceptor composition and extraction time were optimized to finally give detection limits in the 10?C250?ng?L^?1 range. Good linearity was achieved, with up to 156 times enrichment over the four classes of antibiotics. This multi-residue method enabled the simultaneous enrichment of all 11 multi-class antibiotics from spiked river water samples, with relative recovery between 79 and 118%.

Reliable Determination of Site-Specific In Vivo Protein N-Glycosylation Based on Collision-Induced MS/MS and Chromatographic Retention Time

Site-specific glycopeptide mapping for simultaneous glycan and peptide characterization by MS is difficult because of the heterogeneity and diversity of glycosylation in proteins and the lack of complete fragmentation information for either peptides or glycans with current fragmentation technologies. Indeed, multiple peptide and glycan combinations can readily match the same mass of glycopeptides even with mass errors less than 5 ppm providing considerably ambiguity and analysis of complex mixtures of glycopeptides becomes quite challenging in the case of large proteins. Here we report a novel strategy to reliably determine site-specific N-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- and deglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predicted sites for N-glycosylation. Taking advantage of CID features such as high scan speed and high intensity of fragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information about glycan compositions and peptide sequences, as well as the N-glycosylation site occupancy and relative abundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provide guidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoprotein mixtures.

Multiplex Mass Spectrometric Imaging with Polarity Switching for Concurrent Acquisition of Positive and Negative Ion Images

We have recently developed a multiplex mass spectrometry imaging (MSI) method which incorporates high mass resolution imaging and MS/MS and MS³ imaging of several compounds in a single data acquisition utilizing a hybrid linear ion trap-Orbitrap mass spectrometer (Perdian and Lee, Anal. Chem. 82, 9393–9400, 2010). Here we extend this capability to obtain positive and negative ion MS and MS/MS spectra in a single MS imaging experiment through polarity switching within spiral steps of each raster step. This methodology was demonstrated for the analysis of various lipid class compounds in a section of mouse brain. This allows for simultaneous imaging of compounds that are readily ionized in positive mode (e.g., phosphatidylcholines and sphingomyelins) and those that are readily ionized in negative mode (e.g., sulfatides, phosphatidylinositols and phosphatidylserines). MS/MS imaging was also performed for a few compounds in both positive and negative ion mode within the same experimental set-up. Insufficient stabilization time for the Orbitrap high voltage leads to slight deviations in observed masses, but these deviations are systematic and were easily corrected with a two-point calibration to background ions.

Automated quantitative analysis of lipid accumulation and hydrolysis in living macrophages with label-free imaging

The accumulation of lipids in macrophages is a key factor that promotes the formation of atherosclerotic lesions. Several methods such as biochemical assays and neutral lipid staining have been used for the detection of lipids in cells. However, a method for real-time quantitative assessment of the lipid content in living macrophages has yet to be shown, particularly for its kinetic process with drugs, due to the lack of suitable tools for non-invasive chemical detection. Here we demonstrate label-free real-time monitoring of lipid droplets (LDs) in living macrophages by using coherent anti-Stokes Raman scattering (CARS) microscopy. In addition, we have established an automated image analysis method based on maximum entropy thresholding (MET) to quantify the cellular lipid content. The result of CARS image analysis shows a good correlation (R ²?>?0.9) with the measurement of biochemical assay. Using this method, we monitored the processes of lipid accumulation and hydrolysis in macrophages. We further characterized the effect of a lipid hydrolysis inhibitor (diethylumbelliferyl phosphate, DEUP) and determined the kinetic parameters such as the inhibition constant, K _i. Our work demonstrates that the automated quantitative analysis method is useful for the studies of cellular lipid metabolism and has potential for preclinical high-throughput screening of therapeutic agents related to atherosclerosis and lipid-associated disorders.

Interpretation and Deconvolution of Nanodisc Native Mass Spectra

Nanodiscs are a promising system for studying gas-phase and solution complexes of membrane proteins and lipids. We previously demonstrated that native electrospray ionization allows mass spectral analysis of intact Nanodisc complexes at single lipid resolution. This report details an improved theoretical framework for interpreting and deconvoluting native mass spectra of Nanodisc lipoprotein complexes. In addition to the intrinsic lipid count and charge distributions, Nanodisc mass spectra are significantly shaped by constructive overlap of adjacent charge states at integer multiples of the lipid mass. We describe the mathematical basis for this effect and develop a probability-based algorithm to deconvolute the underlying mass and charge distributions. The probability-based deconvolution algorithm is applied to a series of dimyristoylphosphatidylcholine Nanodisc native mass spectra and used to provide a quantitative picture of the lipid loss in gas-phase fragmentation.