Uncovering matrix effects on lipid analyses in MALDI imaging mass spectrometry experiments

The specific matrix used in matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can have an effect on the molecules ionized from a tissue sample. The sensitivity for distinct classes of biomolecules can vary when employing different MALDI matrices. Here, we compare the intensities of various lipid subclasses measured by Fourier transform ion cyclotron resonance (FT‐ICR) IMS of murine liver tissue when using 9‐aminoacridine (9AA), 5‐chloro‐2‐mercaptobenzothiazole (CMBT), 1,5‐diaminonaphthalene (DAN), 2,5‐Dihydroxyacetophenone (DHA), and 2,5‐dihydroxybenzoic acid (DHB). Principal component analysis and receiver operating characteristic curve analysis revealed significant matrix effects on the relative signal intensities observed for different lipid subclasses and adducts. Comparison of spectral profiles and quantitative assessment of the number and intensity of species from each lipid subclass showed that each matrix produces unique lipid signals. In positive ion mode, matrix application methods played a role in the MALDI analysis for different cationic species. Comparisons of different methods for the application of DHA showed a significant increase in the intensity of sodiated and potassiated analytes when using an aerosol sprayer. In negative ion mode, lipid profiles generated using DAN were significantly different than all other matrices tested. This difference was found to be driven by modification of phosphatidylcholines during ionization that enables them to be detected in negative ion mode. These modified phosphatidylcholines are isomeric with common phosphatidylethanolamines confounding MALDI IMS analysis when using DAN. These results show an experimental basis of MALDI analyses when analyzing lipids from tissue and allow for more informed selection of MALDI matrices when performing lipid IMS experiments.     

Study of synthetic aliphatic copolyamides by time-of-flight matrix assisted laser desorption/ionization mass spectrometry

Synthetic copolyamides based on aliphatic diamines (1,3-propanediamine and 1,4-butanediamine) and dichlorides of aliphatic carboxylic acids (adipic and sebacic acid dichlorides) were investigated using time-of-flight matrix assisted laser desorption/ionization mass spectrometry. Their mass spectra showed peaks for cationized (Na⁺ and K⁺) and protonated (less intense peaks) oligomers with NH₂-NH₂, NH₂-COOH, or COOH-COOH end groups. No cyclic oligomers were detected in the samples. The compositions of oligomers were determined, and the relative reactivities of homologous comonomers in polycondensation were estimated. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1320–1324, July, 2007.     

基质辅助激光解吸电离飞行时间质谱研究2: 四硫 富瓦烯化合物

采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),对四硫富瓦烯化合物进行质谱表征。在所用的实验条件下,样品很容易解吸电离生成单电荷分子离子,得到单同位素分辨的质谱图。26种实际样品的质谱分析结果表明;MALDI-TOF-MS可以比其它质谱方法更有效、更方便地用于此类化合物的质谱分析,解决了此类化合物不易进行质谱鉴定的难题。     

Mass spectrometry imaging of latent fingerprints using titanium oxide development powder as an existing matrix

Recent research has focused on increasing the evidentiary value of latent fingerprints through chemical analysis. Although researchers have optimized the use of organic and metal matrices for matrix‐assisted laser desorption/ionization‐mass spectrometry imaging (MALDI‐MSI) of latent fingerprints, the use of development powders as matrices has not been fully investigated. Carbon forensic powder (CFP), a common nonporous development technique, was shown to be an efficient one‐step matrix; however, a high‐resolution mass spectrometer was required in the low mass range due to carbon clusters. Titanium oxide (TiO₂) is another commonly used development powder, especially for dark nonporous surfaces. Here, forensic TiO₂ powder is utilized as a single‐step development and matrix technique for chemical imaging of latent fingerprints without the requirement of a high‐resolution mass spectrometer. All studied compounds were successfully detected when TiO₂ was used as the matrix in positive mode, although, generally, the overall ion signals were lower than the previously studied CFP. TiO₂ provided quality mass spectrometry (MS) images of endogenous and exogenous latent fingerprint compounds. The subsequent addition of traditional matrices on top of the TiO₂ powder was ineffective for universal detection of latent fingerprint compounds. Forensic TiO₂ development powder works as an efficient single‐step development and matrix technique for MALDI‐MSI analysis of latent fingerprints in positive mode and does not require a high‐resolution mass spectrometer for analysis.     

Operation of an academic open access mass spectrometry facility with particular reference to the analysis of synthetic compounds

Open access mass spectrometry now provides the opportunity to move this spectroscopic method to the beginning of the analytical chain, a place formerly the exclusive province of NMR and TLC. To date this transition has been occurring in industrial settings but there has been less change in the academic environment. This paper provides one blueprint for setting up such a facility, primarily in support of organic synthesis but also for the use of biological scientists. The open access format used at UCI utilizes four instruments: an ESI-TOFMS system used in the flow injection mode, two GC/MS systems (one in EI and one in CI) and a MALDI-TOFMS system. The first three instruments have autosamplers and open access software whereas the MALDI system has a fully automated plate handling interface. This level of automation allows access to the instruments by a user community of more than 100 users, day or night. The decisions made in setting up these instruments were based on a 'keep it simple' philosophy, given the fact that the primary type of data of interest is the molecular mass of the analyte and that data are required for a very wide range of structures.     

Mass spectrometric analysis of low molecular mass polyesters by laser desorption/ionization on porous silicon

A low molecular mass polyester was analyzed by desorption/ionization on porous silicon (DIOS) mass spectrometry. The results were compared with those of matrix-assisted laser desorption ionization (MALDI) mass spectrometry using matrixes of alpha-cyano-4-hydroxycinnamic acid (CHCA) and 10,15,20-tetrakis(pentafluorophenyl)porphyrin (F20TPP). The CHCA matrix was not suitable for characterization of low molecular mass components of the polyester because the matrix-related ions interfered with the component ions. On the other hand, the F20TPP matrix showed no interference because no matrix-related ions appeared below m/z 822. However, the solvent selection for determining optimal conditions of sample preparation was limited, because F20TPP does not dissolve readily in any of the available organic solvents. In the DIOS spectra, the polymer ions were observed at high sensitivity without a contaminating ion. No matrix is needed for DIOS spectra of low molecular mass polyesters, facilitating sample preparation and selectivity of a precursor ion in post-source decay measurements.     

基体辅助激光解吸/电离质谱法对核酸分子的分析测定

基体辅助激光解吸／电离质谱（ＭＡＬＤＩＭＳ）法自８０年代末由Ｋａｒａｓ,Ｈｉｌｅｎｋａｍｐ报道以来,已获得极大的发展．近几年来,其应用范围迅速扩大,各种生物大分子如蛋白质,核酸（ＤＮＡ）,多糖等都已能用ＭＡＬＤＩＭＳ法进行分子量测定．１９９３年,...     

Characterization of some synthetic Ru and Ir complexes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied to the analysis of Ru(OCOCF(3))(2)(CO)(PPh(3))(2), Ru(OCOC(3)F(7))(2)(CO)(PPh(3))(2), Ir(tBuppy)(3) and Ir(ppy)(2)(acac) complexes. A troublesome problem in the MALDI-TOFMS characterization of these metal complexes is the possible replacement of complex ligands by matrix. In this contribution, 10 matrices, ranging from acidic to basic, were investigated: alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), dithranol, 2,4,6-trihydroxyactophenone (THAP), 6-azo-2-thiothymine (ATT), norharman, 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB), 4-nitroaniline (NA) and 2-amino-5-nitrophyridine (ANP). With most of the matrices, including the neutral and basic ones, matrix substitution of ligand could clearly be detected. Based on the experimental results, possible mechanisms of matrix substitution were discussed. It was demonstrated that the ligand exchange process might also occur through the gas-phase reactions initiated by laser shots. Among the matrices tested, DCTB was found to be the best one for the complexes that are prone to ligand exchange by matrix.     

Study of the cyanoacrylate fuming mechanism by matrix‐assisted laser desorption/ionization mass spectrometry

Despite cyanoacrylate fuming being widely used in the forensic science field, its mechanism is not well understood. In this study, matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry is used to study latent fingerprints that have been cyanoacrylate fumed in an attempt to gain insight into the fuming mechanism. In the negative mode mass spectrometry data, four compounds related to the polymerization of cyanoacrylate are identified and their structures are determined from accurate mass and MS/MS. A mechanism is proposed for the formation of these compounds that are regarded as intermediates in the polymerization reaction. In addition, based on the fuming of standard endogenous compounds, we suggest that fatty acids and amino acids are the major catalytic nucleophiles that initiate the polymerization reactions.     

Detection of water-soluble vitamins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using porphyrin matrices

The detection of water-soluble vitamins B(1), B(2), B(6), B(12) and C by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) was attempted by studying 17 porphyrin matrices. Comparative studies of porphyrin matrices, useful mass spectral window, matrix/analyte molar ratio (M/A), ultraviolet-visible absorption characteristics and quantitative results were made. Most porphyrin matrices provide a useful mass spectral window in the low-mass range. The optimal M/A increases with increasing molecular mass of the vitamin. Vitamin B(12) possesses the highest molecular mass and requires a higher M/A. The presence of hydroxyl or carboxyl groups in the porphyrin is an indicator of a useful MALDI matrix. Vitamins B(2) and B(6) were readily ionized upon irradiation with a 337 nm laser without the use of any porphyrin matrix. Improved linearity and sensitivity of the calibration curves were obtained with samples prepared with a constant M/A. The limits of detection and quantitation are at the picomole level. The results indicate that MALDI-TOFMS with porphyrin matrices is a rapid and viable technique for the detection of low molecular mass water-soluble vitamins.     

An approach to analysis of primary amines by a combination of thin‐layer chromatography and matrix‐assisted laser desorption ionization mass spectrometry in conjunction with post‐chromatographic derivatization

On‐spot derivatization has been suggested for the modification of primary amine containing compounds for their analysis by thin‐layer chromatography hyphenated with matrix‐assisted laser desorption ionization mass spectrometry. The proposed approach was based on post‐chromatographic treatment of separated analytes inside the chromatographic zones on the thin‐layer chromatography plates by tris(2,6‐dimethoxyphenyl)methilium reagent. The derivatives, containing permanent positive charge, reveal exceptionally intense peaks of their cationic moieties and high signal/noise ratio in mass spectra recorded directly from the plates. The method was tested on a series of aliphatic, aromatic, and amine‐containing pharmaceuticals.     

以2, 4, 6-三羟基苯乙酮为基体使用基体辅助激光解吸/电离质 谱法测定核酸分 子的研究

报道以2,4,6-三羟基苯乙酮(2,4,6-THAP)为基体用基体辅助激光解吸/电离飞行时间质谱法(MALDI-TOF-MS)测定核酸分子的研究。2,4,6-THAP解吸和电离DNA分子的效率很高,测定DNA分子d(T)~1~0的[M-H]^-分辨率可达1130.0,信噪比为366.0,检出限达5×10^-^1^4mol,可以测定相对分子质量高达14800以上混合碱基组成的DNA分子。为提高基体对样品解吸/电离效率,对纯化DNA样品的方法进行了讨论。     

Miniaturized linear time-of-flight mass spectrometer with pulsed extraction

Improved resolution for a miniaturized instrument is demonstrated at high masses using a pulsed extraction, 3(") linear time-of-flight (TOF) mass analyzer. This illustrates the utility of a small and simple mass spectrometer for biological/medical analyses. Current and future applications suggested by this instrument include rapid mass spectral reading of oligonucleotides that differ in one base (single nucleotide polymorphisms), distinction of biomarker signatures from different species of bacterial spores (biological weapons detection) and point-of-care instruments for proteomics-based diagnostics. We have incorporated a two-stage, pulsed-extraction design that places the focal plane of the ions at the detector channel plate surface. The ions are accelerated to a total energy of 12 keV to enable detection of high-mass proteins in a design that incorporates a floatable flight tube set at the voltage of the front channel plate of the detector. The resultant elimination of post-acceleration at the detector is intended to improve mass resolution by reducing the difference in arrival times between ions and their neutral products. Resolutions of one part in 1200 at m/z 4500 and one part in 600 at m/z 12 000 have been achieved. Proteins with molecular masses up to 66 000 Da, mixtures of oligonucleotides, and biological spores have all been successfully measured, results that increase the potential use of this TOF analyzer.     

Profiling stage-dependent changes of protein expression in Caenorhabditis elegans by mass spectrometric proteome analysis leads to the identification of stage-specific marker proteins

Proteome maps obtained by synchronization of the wild-type Caenorhabditis elegans development reflected stage-dependent molecular differences and revealed dynamic cytoskeletal processes during ontogenesis. Distinct protein spots that may function as molecular markers for the corresponding developmental stages were mass spectrometrically identified. The amount of the Cu(2+)- Zn(2+) superoxide dismutase (CE23550) and an aspartyl proteinase (CE21681) was highest in the first larval stage (L1) and decreased during the ontogenesis from the first larval stage to the adult. Tropomyosin III (CE29059) was prominently present in the first and second larval stage (L1/L2). Abundances of actin 1 or 4 (CE12358 or CE13148) and tropomyosin I (CE28782) were particularly high in multiple spots in the third larval stage (L3). Interestingly, the amount of DIM-1 protein (CE27706), reflected by two spots, was the lowest in this stage. A particular splicing factor (CE31089) was detected only in the fourth larval stage (L4), whereas a spot with high abundance representing the cuticle collagen (CE02272) was only found highly expressed in adult animals (A). In addition, a Ca(2+)-binding protein (CE12368) and one protein spot which has not yet been identified, both reached their maximal spot intensities in the adult stage (A). Moreover, the ASP-1, CCT-5, GPD-1, GPD-2, HSP-6, HSP-16.2, IFB-2, LEC-2, LIN-53, LMN-1, MDH-1, NUD-1, RPA-0, RSP-12, SOD-1, TBB-1, TBB-2, TMY-1, UNC-60, and VIT-2 proteins for which mutants are available and two still unidentified protein spots which were present in all developmental stages, have been reproducibly localized in proteome maps of distinct ontogenesis states.     

Automated normal phase nano high performance liquid chromatography/matrix assisted laser desorption/ionization mass spectrometry for analysis of neutral and acidic glycosphingolipids

The coupling of nano high-performance liquid chromatography (nanoHPLC) with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) via an automatic spotting roboter was developed and adapted for the first time for the analysis of complex mixtures of glycosphingolipids (GSLs). The 2,5-dihydroxybenzoic acid and 6-azo-2-thiothymine matrix systems were adjusted to concurrently meet the requirements for reproducible and homogeneous crystal formation with the liquid chromatography (LC) eluent under the variable LC solvent composition over the course gradient and high ionization efficiency of the GSL species, without the need for recrystallization. Precise adjustment of the automatic spotting parameters in terms of matrix flow rate, on-tip collection time of the matrix/LC eluent solution and the matrix spotting mode, i.e., continuous and discontinuous, was accomplished to collect individually nanoHPLC-separated species within distinct spots and consequently recover by MALDI MS screening all major and minor GSL species in the mixtures. The nanoHPLC/MALDI MS coupling protocol was developed and applied to a mixture of neutral GSLs purified from human erythrocytes and a monosialoganglioside mixture expressed by the murine MDAY-D2 cell line. Additionally, on-line nanoHPLC/MALDI doping with lithium cations of individually separated neutral GSLs was introduced to enhance data interpretation of the GSL MS pattern, while preserving the same level of information and ultimately to enhance structural assignment of components of interest. The method is demonstrated to be highly sensitive, reaching the low femtomole level of detection of individual GSL species and is highlighted as a versatile analytical tool for glycolipidomic studies. Figure Automatic LC/MALDI MS profiling of glycosphingolipids Mostafa Zarei and Stephan Kirsch contributed equally to this work.     

Fabrication of homogenous three‐dimensional biomimetic tissue for mass spectrometry imaging

Reference samples are essential for mass spectrometric method optimization, data quality control, and target analyte quantitation. However, it is highly challenging to prepare an ideal homogeneous, standard‐spiked tissue sample for mass spectrometry imaging (MSI) research. Herein, we present a standard‐spiked 3D biomimetic tissue model fabricated with native cells, homogenate matrix, and biocompatible polymer. Unlike traditional homogenized tissue surrogates or those constructed with “on‐tissue” or “under‐tissue” micropipetting strategies, this simulated tissue shares both structural integrity of cells and homogeneous properties of matrix. As a result, analyte standards could undergo more in‐depth incorporation and has a more comparable native status with a real tissue. Series of tissue sections made from the 3D tissue model were proven to be feasible and useful for the parameter optimization, analyte quantitation, and calibration curve fitting for the air‐flow assisted desorption electrospray ionization MSI. Additionally, by analyzing the quality control model sections, we proposed a median principal component score calibration and demonstrated that this method can normalize instrumental fluctuations to stable levels in a large‐scale untargeted MSI experiments for the reliable metabolomic biomarker discovery. Thus, these results indicated that the standard‐spiked 3D biomimetic tissue has convincing significance in MSI analysis     

Application of high‐resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex

We investigated the application of a high‐resolution Orbitrap mass spectrometer equipped with an electrospray ionization (ESI) source and a matrix‐assisted laser desorption/ionization‐time‐of‐flight (MALDI‐TOF) mass spectrometer to the metabolite profiling of a model small interfering RNA (siRNA) duplex TSR#34 and compared their functions and capabilities. TSR#34 duplex was incubated in human serum in vitro, and the duplex and its metabolites were then purified by ion exchange chromatography in order to remove the biological matrices. The fraction containing the siRNA duplex and its metabolites was collected and desalted and then subjected to high‐performance liquid chromatography (HPLC) equipped with a reversed phase column. The siRNA and its metabolites were separated into single strands by elevated chromatographic temperature and analyzed using the ESI‐Orbitrap or the MALDI‐TOF mass spectrometer. Using this method, the 5' and/or 3' truncated metabolites of each strand were detected in the human serum samples. The ESI‐Orbitrap mass spectrometer enabled differentiation between two possible RNA‐based sequences, a monoisotopic molecular mass difference which was less than 2 Da, with an intrinsic mass resolving power. In‐source decay (ISD) analysis using a MALDI‐TOF mass spectrometer allowed the sequencing of the RNA metabolite with characteristic fragment ions, using 2,4‐dihydroxyacetophenone (2,4‐DHAP) as a matrix. The ESI‐Orbitrap mass spectrometer provided the highest mass accuracy and the benefit of on‐line coupling with HPLC for metabolite profiling. Meanwhile, the MALDI‐TOF mass spectrometer, in combination with 2,4‐DHAP, has the potential for the sequencing of RNA by ISD analysis. The combined use of these methods will be beneficial to characterize the metabolites of therapeutic siRNA compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry

A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.     

Identification of formylglycine in sulfatases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

C(alpha)-Formylglycine, the catalytic amino acid residue in the active site of sulfatases, is generated by post-translational modification of a cysteine or serine residue. We describe a highly sensitive procedure for the detection of C(alpha)-formylglycine-containing peptides in tryptic digests of sulfatase proteins. The protocol is based on the formation of hydrazone derivatives of C(alpha)-formylglycine-containing peptides when using dinitrophenylhydrazine as a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives desorb and ionize with high efficiency and can be detected in the sub-femtomole range. The presence of C(alpha)-formylglycine is indicated by a mass increment of 180.13 u, corresponding to the hydrazone moiety, and also by a unique C-terminal fragment ion, characteristic of sulfatases, that becomes prominent in MALDI post-source decay mass spectra of the hydrazone derivatives.     

Structural characterization of mesquite (Prosopis velutina) gum and its fractions

Structural and physicochemical characteristics of mesquite gum (from Prosopis velutina) were investigated using FT-IR spectroscopic, mass spectrometric and chromatographic methods. Four fractions (F-I, F-IIa, F-IIb and F-III) were isolated by hydrophobic interaction chromatography. The samples were characterized and analyzed for their monosaccharide and oligomers composition by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). L-Arabinose (L-Ara) and D-galactose (D-Gal) were found as the main carbohydrate constituent residues in the polysaccharides from mesquite gum and their ratio (L-Ara/D-Gal) varied within the range 2.54 to 3.06 among the various fractions. Small amounts of D-glucose (D-Glc), D-mannose (D-Man) and D-xylose (D-Xyl) were also detected, particularly in Fractions IIa, IIb and III. Infrared spectroscopy identified polysaccharides and protein in all the samples. Data from mass spectrometry (MALDI-TOF MS) was consistent with the idea that the structure corresponding to the periphereal chains of Fraction I is predominantly a chain of pentoses attached to uronic acid.