Reflectron Mode Research Articles

Tracking the disulfide rearrangement of heated lactoglobulin by matrix-assisted laser desorption/ionization-in-source decay top-down analysis.

Disulfide bond rearrangement is a common occurrence during protein analysis or treatment. A convenient and rapid method has been developed to investigate heat-induced disulfide rearrangement of lactoglobulin using matrix-assisted laser desorption/ionization-in-source decay (MALDI-ISD) technology. By analyzing heated lactoglobulin in reflectron and linear mode, we demonstrated that cysteines C66 and C160 exist as free residues other than linked ones in some protein isomers. This method provides a straightforward and expeditious way to assess the cysteine status and structural changes of proteins under heat stress.

Stability of MeV primary ion induced secondary ions

We report on the analysis of the onset and the intensity of metastable secondary ions, which are desorbed from the target material as a result of bombarding organic samples with MeV primary ions. The bimodal time-of-flight mass spectrometer, which can analyze secondary ion in linear and reflectron modes, was used for detection and characterization of such ions. The use of the bimodal mass spectrometer for this specific purpose is demonstrated on amino acid arginine, where three main fragments were detected. We have also analyzed the influence of the primary ion beam on the intensity of metastable ion signal. Results from chlorine ion beams with energies between 3 and 10 MeV have exhibited the importance of electronic sputtering on the product/precursor ion peak intensity ratio, which is significantly decreased when using primary ions with higher energy.

Quantitative Assessment of Serine-8 Phosphorylated β-Amyloid Using MALDI-TOF Mass Spectrometry

The study of the molecular mechanisms of the pathogenesis of Alzheimer's disease (AD) is extremely important for identifying potential therapeutic targets as well as early markers. In this regard, the study of the role of post-translational modifications (PTMs) of β-amyloid (Aβ) peptides is of particular relevance. Serine-8 phosphorylated forms (pSer8-Aβ) have been shown to have an increased aggregation capacity and may reflect the severity of amyloidosis. Here, an approach for quantitative assessment of pSer8-Aβ based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is proposed. The relative fraction of pSer8-Aβ was estimated in the total Aβ-pool with a detection limit of 1 fmol for pSer8-Aβ (1-16) and an accuracy of 2% for measurements in the reflectron mode. The sensitivity of the developed method is suitable for determining the proportion of phosphorylated peptides in biological samples.

Fragmentation analysis of O-specific polysaccharide from bacteria Vibrio cholerae O139 by MALDI-TOF and LC/ESI-MS/MS.

Cholera is a life-threatening diarrhoeal disease caused by ingestion of Vibrio cholerae. There are at least 200 serogroups of V. cholerae but only two of them are causing epidemics - O1 and O139 serogroups. Fragmentation analysis of O-antigen, also known as O-specific polysaccharide (OSP), from lipopolysaccharide (LPS) is important to obtain new information about its structure, such as fragmentation patterns and fragment structures. In the present study, OSP and core (OSPc) structure from V. cholerae O139 was studied using matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) and direct injection electrospray ionization (ESI)-MS methods. MALDI-TOF analysis was performed in positive-ion reflectron mode, while ESI-MS was performed in negative ionization mode. ESI-MS analysis was followed by ESI-MS/MS analysis. Using this analytical approach, we managed to obtain two possible fragmentation pathways of OSP from V. cholerae O139. Mutual sign of these two pathways is shortening the length of the oligosaccharide by neutral loss of monosaccharide residues. Additionally, liquid chromatography-MS analysis was performed to separate depicted molecular forms of OSPc.

Identification of Snake Venoms According to their Protein Content Using the MALDI-TOF-MS Method

Snakebites cause mortality and morbidity in tropical regions. Bites by Viperidae and Elapidae family snakes are common in Turkey. Snake venom characterization and anti-venom production are important in the diagnosis and treatment of snakebites. Our objective was to develop a method for analyzing all venoms by identifying basic protein structures in venom using a MALDI-TOF-MS and to create a venom library. Five Viperidae family and one from the Elapidae family venoms, were identified. Although the proportions vary depending on the species and subspecies, the identification procedure involved determining the protein structures of PLA2, VEGF, svMP, CRISP, svSP, PIII-MP, and LAAO, common to all venoms. Six matrices (CHCA, DHAP, DHB, SA, S-DHB and THAP,) with the MALDI-TOF-MS and six snake venoms were studied separately. On the other hand, peptide profiles were studied in the mass range of 1000-10000 Da using reflectron mode. It was found that biomarkers peptides were 4398, 4026, 6395, 3800 and 6672 Da for ML, MX, VAA, VBB, VAM and WA respectively. The highest relative abundance values of the protein signals were mostly obtained with both the CHCA and the DHAP matrices. Some targeted protein structures were not observed in studies involving the S-DHB and the THAP matrices, and those were detected exhibited relatively low abundance.

Properties of Polyhexamethylene Guanidine (PHMG) Associated with Fatal Lung Injury in Korea.

The use of humidifier disinfectant (HD) has been determined to be associated with lung injuries (HDLI) in Korea. Although HD brands containing polyhexamethylene guanidine (PHMG) oligomers have been found to cause more HDLI compared to brands containing other disinfectants, the physicochemical properties of PHMG have been poorly defined. We aimed to quantify the PHMG dissolved in HD brands, characterize the number-average (Mn) and weight-average (Mw) molecular masses, and identify the polymerization degree of PHMG. Analysis of the PHMG oligomers was performed using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) operated in positive-ion reflectron mode. Eight brands of HD containing PHMG were identified. The PHMG concentrations in these brands ranged from 160 to 37,200 ppm (mean = 3100.9 ppm). Concentration was a significant variable among and within HD brands. The degree of PHMG oligomerization fell within the range of two to four. The averages of Mn and Mw were 517.2 g/mol (range: 422–613 g/mol) and 537.3 g/mol (range: 441.0–678.0 g/mol), respectively. Based on the average molecular weight and the degree of polymerization, the PHMG examined here could be regarded as oligomers, which may be associated with the highest proportion of HDLI being caused by PHMG.

The interaction of 355nm laser light on Adenine and Uracil

SynopsisDissociation and ionization of adenine and uracil were investigated in the multiphoton absorption re-gime using laser pulse at 355nm in conjunction with TOF mass spectrometry in reflectron mode (R-TOF). In contrast of previous investigations where a notorious amount of low mass ions was detected arising from both molecules.

NorHarmane containing ionic liquid matrices for low molecular weight MALDI-MS carbohydrate analysis: The perfect couple with α-cyano-4-hydroxycinnamic acid.

Cinnamic acid derivatives, particularly α-cyano-4-hydroxycinnamic acid (E-α-cyano-4-hydroxycinnamic acid or (E)-2-cyano-3-(4-hydroxyphenyl)prop-2-enoate; CHCA), have been extensively used especially for protein and peptide analysis. Together with the introduction of ionic liquid MALDI matrix (ILM) started the study of applications of IL prepared with CHCA and a counter organic base (ie, aliphatic amines) in which CHCA moiety is the chromophore responsible of UV-laser absorption. Despite the extensive studies of norharmane (9H-pyrido[3,4-b]indole; nHo) applications as matrix and its peculiar basic properties in the ground and electronic excited state, nHo containing ILM was never tested in MALDI-MS experiments. This pyrido-indole compound was introduced as MALDI matrix 22years ago for different applications including low molecular weight (LMW) carbohydrates (neutral, acidic, and basic carbohydrates). These facts encouraged us to use it as a base, for the first time, for ILM preparation. As a rational design of new IL MALDI matrices, E-α-cyanocinnamic acid.nHo and E-cinnamic acid.nHo were prepared and their properties as matrices studied. Their performance was compared with that of (a) the corresponding IL prepared with butylamine as basic component, (b) the corresponding crystalline E-α-cyanocinnamic and E-cinnamic acid, and (c) the classical crystalline matrices (2,5-dihydroxybenzoic acid, DHB; nHo) used in the analysis of neutral/sulfated carbohydrates. The IL DHB.nHo was tested, too. Herein, we demonstrate the outstanding performance for the IL CHCA.nHo for LMW carbohydrate in positive and negative ion mode (linear and reflectron modes). Sulfated oligosaccharides were detected in negative ion mode, and although the dissociation of sulfate groups was not completely suppressed the relative intensity (RI) of [M-Na]- peak was quite high. Additionally, to better understand the quite different performance of each IL tested as matrix, the physical and morphological properties in solid state were studied (optical image; MS image).

Aniline derivative/DHB/alkali metal matrices for reflectron mode MALDI-TOF and TOF/TOF MS analysis of unmodified sialylated oligosaccharides and glycopeptides

Sialylglycoconjugates play biologically important roles in nature. The labile nature of the sialic acid residue in matrix-assisted laser desorption/ionization–mass spectrometry (MALDI-MS) analysis demands additional modification processes of the carboxylate group to avoid in-source or post-source decay. This paper reports a versatile and high-sensitivity matrix system, aniline derivative/2,5-dihydroxybenzoic acid/alkali metal salt, for MALDI-MS analysis of sialyloligosaccharides and sialylglycoconjugates without any modifications. A simple adduct ion signal was obtained from the femtomole range of the analytes tested with the new matrix system by MALDI-MS in reflectron mode. Besides, the laser power-dependent in-source decay and post-source decay pattern of the sialylated analytes with this matrix system allows a pseudo MS3 strategy for in-depth sequencing studies of the analytes.

Unprecedented Ionization Processes in Mass Spectrometry Provide Missing Link between ESI and MALDI.

In the field of mass spectrometry, producing intact, highly-charged protein ions from surfaces is a conundrum with significant potential payoff in application areas ranging from biomedical to clinical research. Here, we report on the ability to form intact, highly-charged protein ions on high vacuum time-of-flight mass spectrometers in the linear and reflectron modes achievable using experimental conditions that allow effective matrix removal from both the sample surfaces and from the charged clusters formed by the laser ablation event. The charge states are the highest reported on high vacuum mass spectrometers, yet they remain at only around a third of the highest charge obtained using laser ablation with a suitable matrix at atmospheric pressure. Other than physical instrument modifications, the key to forming abundant and stable highly-charged ions appears to be the volatility of the matrix used. Cumulative results suggest mechanistic links between the ionization process reported here and traditional ionization methods of electrospray ionization and matrix-assisted laser desorption/ionization.

Intraspecific venom variation in southern African scorpion species of the genera Parabuthus, Uroplectes and Opistophthalmus (Scorpiones: Buthidae, Scorpionidae)

Scorpion venoms comprise cocktails of proteins, peptides, and other molecules used for immobilizing prey and deterring predators. The composition and efficacy of scorpion venoms appears to be taxon-specific due to a coevolutionary arms race with prey and predators that adapt at the molecular level. The taxon-specific components of scorpion venoms can be used as barcodes for species identification if the amount of intraspecific variation is low and the analytical method is fast, inexpensive and reliable. The present study assessed the extent of intraspecific variation in newly regenerated venom collected in the field from geographically separated populations of four southern African scorpion species: three buthids, Parabuthus granulatus (Ehrenberg, 1831), Uroplectes otjimbinguensis (Karsch, 1879), and Uroplectes planimanus (Karsch, 1879), and one scorpionid, Opistophthalmus carinatus (Peters, 1861). Although ion signal patterns were generally similar among venom samples of conspecific individuals from different populations, MALDI-TOF mass spectra in the mass range m/z 700–10,000 revealed only a few ion signals that were identical suggesting that species identification based on simple venom mass fingerprints (MFPs) will be more reliable if databases contain data from multiple populations. In general, hierarchical cluster analysis (HCA) of the ion signals in mass spectra was more reliable for species identification than counts of mass-identical substances in MFPs. The statistical approach revealed conclusive information about intraspecific diversity. In combination with a comprehensive database of MALDI-TOF mass spectra in reflectron mode, HCA may offer a method for rapid species identification based on venom MFPs.

Enhancing ganglioside species detection for MALDI-TOF imaging mass spectrometry in negative reflectron mode

Enhanced ganglioside species detection was achieved for matrix assisted laser desorption ionization time-of-flight imaging mass spectrometry (MALDI-TOF IMS) in negative reflectron mode using a novel sample preparation protocol that involves washing the tissue in ammonium salt solutions followed by spray depositing ammonium salts and waiting 24h after sublimation of 1,5-diaminonaphthalene (DAN) before data acquisition. Application of this novel method to normal adult mouse brains led to more than 10-fold increase in total ion intensity in the ganglioside mass range and an increase in the number of detected sialylated species from 3 to 15, with no apparent delocalization observed at 20μm spatial resolution, making it a powerful technique with the potential to provide greater information about gangliosides in numerous biological contexts.

Production and Characterization of a Polyphenol Enriched Date Palm Extract from California Date Palm

Date palm (Phoenix dactylifera) is an important food cultivated in the Coachella Valley in Southern California. Medjool and deglet noor dates are the two most popular varieties and both pack an impressive list of nutrients, including vitamins, minerals, lipids and proteins. Dates are also rich in phenolic compounds including phenolic acid, flavonoids, tannins, anthocyanins and carotenoids. These compounds are associated with a variety of beneficial health effects. However, very few studies have identified the molecular mechanisms underlying these observed effects. Therefore, the aim of the present study was to produce and characterize a date palm extract (DPE) made from California dates that can then be used in subsequent in vitro and in vivo studies to elucidate the mechanisms by which dates confer beneficial health effects. California dates were harvested during the 2014 and 2015 harvest seasons and freeze‐dried date extract was produced from equal part medjool and deglet noor dates by means of liquid extraction and solid phase separation. The extracts were characterized using High Performance Liquid Chromatography (HPLC) and Matrix‐Assisted Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry (MALDI‐TOF MS).HPLC chromatograms show that the predominant polyphenols present within the extracts have spectral characteristics indicative of hydroxycinnamic acids; specifically, the UV‐Visible spectra has a maximum absorbance in the range of 320–330nm. The extracts also have spectral characteristics indicative of proanthocyanidins (PAC). Specifically, the UV‐Visible spectra have an absorbance at 280nm and resolve as a poorly eluting ‘hump’ rather than well‐defined individual peaks. The qualitative and quantitative presence of PAC in the date extract was detected by butanol‐HCl and by separation of the extract on a Sephadex LH20 column prior to HPLC and MALDI‐TOF MS analysis. This separation provides an enriched PAC fraction, which helps with the subsequent characterization of PACs, providing sufficient separation of compounds to allow for the detection of PAC oligomers from 3–7 degrees of polymerization by MALDI‐TOF MS. HPLC chromatograms of the PAC fraction at 280 nm show two broad and unresolved humps, due to the structural heterogeneity of the PAC oligomers. Furthermore, positive reflectron mode MALDI‐TOF MS exhibited a series of compounds which appear to be oligomeric in nature with repeatable extension units. We propose that this mass may correspond to suberin compounds, specifically an ester of ferulic acid with long‐chain w ‐ hydroxyfatty acids (C16, C18, C20). The pattern of MALDI peaks suggests the presence of a series of w ‐ hydroxyfatty acids varying in degree of unsaturation.This study shows that date palm fruits are a good source of phenolic compounds mainly hydroxycinnamic acids, PAC and lipophilic polyphenols, and that this extract can be used for further in vitro and in vivo analysis.Support or Funding InformationFunded by California Date Commission

Laser Desorption/Ionization-Time of Flight (LDI-TOF) and Matrix-Assisted Laser Desorption/Ionization - Time of Flight (MALDI – TOF) mass spectrometry of an Algerian asphaltene

Both LDI-TOF and MALDI-TOF Mass spectroscopy experiments of an Algerian asphaltene derived from a deposit were performed. LDI mass experiments were conducted for both linear and reflectron modes under laser wavelength/attenuation variation. The different LDI-Mass spectra show that mass distribution depends on experimental condition for masses below 1000amu and that the average molecular weight is around 650 for the polar fraction and beyond 1000amu for non-polar ones. The use of different matrices as CHCA, HABA and Dithranol changes slightly the aspect of the spectra.

Analysis of the molecular weight distribution of vacuum residues and their molecular distillation fractions by laser desorption ionization mass spectrometry

Average molecular weight analysis of vacuum residues using laser desorption ionization (LDI) can be a difficult task due to the significant influence of the experimental parameters and gas-phase reactions. In this paper, laser desorption/ionization time-of-flight mass spectrometry in reflectron mode (LDI TOF MS) was used to analyze vacuum residues and their molecular distillations (MD) fractions obtained at distillation cuts of 510–603°C, 510–645°C and 510–687°C. Those samples associated with the lowest distillation temperature presented the narrowest molecular weight distributions and lowest average molecular weight (Mw) indicating lower complexity. American Petroleum Institute gravity, or API gravity (API°), is a measure of the weight of liquid petroleum compared to water, as expected a correlation between Mw and API° was determined, where lower API° correlated with higher molecular weight. When using higher laser energies mass spectra were acquired with a spacing of 24Da between the peaks, indicating the production of carbon clusters or “fullerenes”. This suggests that asphaltenes could be the precursors of the clusters that extend over 2500Da in reflectron mode. Under appropriate experimental conditions, it was possible to produce repeatable molecular distribution for all the samples. Likewise, mass spectrometric data can be used in Principal Component Analysis (PCA) and Partial Least Square (PLS) analysis to discriminate and to predict density of the samples with low percentage of errors.

Comparison of A-type Proanthocyanidins in Cranberry and Peanut Skin Extracts Using Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Cranberry products have long been used to treat urinary tract infections. It is believed that the A-type proanthocyanidins in cranberries contribute to this function. Peanut is one of the other, few food sources that primarily contain A-type proanthocyanidins. The skin on the outside of the peanut kernels (testa), which is treated as an agriculture waste product, contains high levels of A-type proanthocyanidins. In this study, an HPLC diol column separation method and MALDI-TOF MS were used to characterize and compare the proanthocyanidin compositions of peanut skins and cranberries. MALDI-TOF MS in linear mode was able to detect a group of proanthocyanidins with DP (degree of polymerization) 10 in peanut skin extract, but was only able to detect DP 8 in cranberry extract. The reflectron mode showed clusters of clear narrow peaks at DP 7 in peanut skin extract, while the highest DP resolved for cranberry extract was only 3 in reflectron mode. This might be due to the low response intensity of the cranberry samples with the current cleanup method and the matrix. Based on the resolved peaks in reflectron mode, pPeanut skins and cranberries have similar proanthocyanidins composition; they contain both A-type and B-type proanthocyanidins, with the A-type being predominant. This result may inspire future studies on the comparison of biological functions between peanut skins and cranberries and further comparison of their polymeric proanthocyanidin composition.

Photofragmentation of Formic Acid

SynopsisDissociation and ionization of formic acid was investigated by multiphotonic absorption regime using laser pulse at 355nm in conjunction with TOF mass spectrometry in reflectron mode (R-TOF). From the different type of detected fragment ions is inferred the formation of some small clusters of formic acid.

Laser desorption ionization time-of-flight mass spectrometry of Ge-As-Te chalcogenides.

Ge(x)As(y)Te(z) glasses have a broad window of optical transparency and high refractive index which make them promising for applications in the infrared region. The aim of this work is to reveal structural motifs which could be present during the fabrication of Ge-As-Te thin films by plasma deposition techniques; such knowledge is important for the optimization of thin film growth. Mass spectra were acquired using a laser desorption ionization time-of-flight (LDI-TOF) mass spectrometer equipped with a nitrogen laser (337 nm) coupled with a quadrupole ion trap, and recorded in positive and negative ion reflectron modes. XRD, SEM (EDX) and Raman spectroscopy were also used for the characterization of Ge-As-Te bulk or powdered samples. Bulk Ge(x)As(y)Te(z) samples (x = 10-20%, y = 20-60%, z = 30-70%) were synthesized. LDI-MS of Ge-As-Te powders provided evidence for the formation of both positively and negatively charged clusters, whose stoichiometry was determined as Te(n)(+/-) (n = 1-4), Te(5)(+), binary AsTe(n)(+/-) (n = 1-3), GeTe(n)(+/-) (n = 1-3), As(2)Te(+/-), As(2)Te(3)(+), As(3)Te(+), AsTe(4) (+), Ge(2)H(6) (+/-), ternary GeAsTe(+), GeAsTe(2)(+/-), GeAsH5 (+), GeAsH(6)(+), GeAsH(12)(+), and tertiary GeAsTeH(5)(-), GeAsTeH(8)(-), GeAsTe(2)H(3) (+/-). The local structure of Gex Asy Tez materials is at least partly different from that of species identified in plasma by mass spectrometry, as deduced from Raman scattering spectroscopy analysis. However, LDI-TOFMS was found to be a suitable technique for the partial structure characterization of Ge-As-Te bulk samples and especially for the identification of the structural motifs present in the plasma during the preparation of the corresponding thin films.

Effective methods for the measurement of CsI cluster ions using MALDI‐MS with suitable solvent combinations and additives

The original version of this paper, published in Journal of Mass Spectrometry, 2014, 49, 1199–1202, DOI 10.1002/jms.3446, contains the following errors. Page 1201, figure 2 caption, ‘DCTB acetone solution’ should be changed to ‘DCTB ethanol solution’. Page 1201, right column, line 3, The phrase ‘in the linear mode’ is not necessary because Figure 2 shows the results of data in both linear and reflectron modes. Page 1202, left column, line 19, The phrase “in the linear mode” is not necessary because Figure S3 shows the results of data in both linear and reflectron modes. Supporting Information, Table S2, The value for sucrose given in the (CsI)nI- column should be “6622”, not ‘6882’ as shown. This error was caused by miscounting the number of " n" in (CsI)nI.

GUMBOS matrices of variable hydrophobicity for matrix-assisted laser desorption/ionization mass spectrometry.

Detection of hydrophobic peptides remains a major obstacle for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This stems from the fact that most matrices for MALDI are hydrophilic and therefore have low affinities for hydrophobic peptides. Herein, 1-aminopyrene (AP) and AP-derived group of uniform materials based on organic salts (GUMBOS) as novel matrices for MALDI-MS analyses of peptides were investigated for hydrophobic and hydrophilic peptides. A number of solid-phase AP-based GUMBOS are synthesized with variable hydrophobicity simply by changing the counterions. Structures were confirmed by use of (1)H NMR and electrospray ionization mass spectrometry (ESI-MS). 1-Octanol/water partition coefficients (Ko/w) were used to measure the hydrophobicity of the matrices. A dried-droplet method was used for sample preparation. All spectra were obtained using a MALDI-TOF mass spectrometer in positive ion reflectron mode. A series of AP-based GUMBOS was synthesized including [AP][chloride] ([AP][Cl]), [AP][ascorbate] ([AP][Asc]) and [AP][bis(trifluoromethane)sulfonimide] ([AP][NTf2]). The relative hydrophobicities of these compounds and α-cyano-4-hydroxycinnamic acid (CHCA, a common MALDI matrix) indicated that AP-based GUMBOS can be tuned to be much more hydrophobic than CHCA. A clear trend is observed between the signal intensities of hydrophobic peptides and hydrophobicity of the matrix. MALDI matrices of GUMBOS with tunable hydrophobicities are easily obtained simply by varying the counterion. We have found that hydrophobic matrix materials are very effective for MALDI determination of hydrophobic peptides and, similarly, the more hydrophilic peptides displayed greater intensity in the more hydrophilic matrix.