Rapid characterization of polyalcohols by silylation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry method for rapidly enumerating hydroxyl groups in analytes is described, and applied to some common polyalcohols (erythritol, mannitol and xylitol). Polyalcohols were derivatized with trimethylsilylimidazole (TMSI) either separately or as mixtures, and were analyzed, without chromatographic separation or purification. The mass spectra revealed consecutive peaks that are separated by 72 m/z units as a consequence of displacement of one hydroxyl hydrogen atom by one TMS group. The number of observed peaks was used to confirm the number of hydroxyl groups in each analyte.     

Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used successfully to detect phosphorylation sites in proteins. Applications may be limited by the low response of phosphopeptides compared to nonphosphorylated peptides in MALDI MS. The addition of ammonium salts to the matrix/analyte solution substantially enhances the signal for phosphopeptides. In examples shown for equimolar mixtures, the phosphorylated peptide peaks become the largest peaks in the spectrum upon ammonium ion addition. This can allow for the identification of phosphopeptides in an unfractionated proteolytic digestion mixture. Sufficient numbers of protonated phosphopeptides can be generated such that they can be subjected to postsource decay analysis, in order to confirm the number of phosphate groups present. The approach works well with the common MALDI matrices such as α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid, and with ammonium salts such as diammonium citrate and ammonium acetate.     

A comparative study of a liquid and a solid matrix in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and collision cross section measurements

We present experimental matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) results comparing a liquid (glycerol/K(4)[Fe(CN)(6)]) and a solid matrix (2,5-dihydroxybenzoic acid, DHB) with respect to analyte signal stability and initial ion velocity. For applications requiring stable production of analyte ions over a long period of time, the liquid matrix is superior to the solid matrix. The stable analyte ion signal obtained from a liquid matrix allowed the measurement of collision cross sections of small poly(ethylene glycol) (PEG(n)) adduct ions in the flight tube with good resolution. The initial velocity of these adduct ions was measured. It was found that analyte molecules from the liquid matrix have initial ion velocities significantly smaller than those from the solid matrix. MALDI-TOF measurements for large molecules using a liquid matrix are therefore likely to result in smaller systematic errors in mass calibrations due to initial ion velocity.     

Small molecule matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a polymer matrix

We have employed a light-absorbing electrically conductive polymer as a matrix to determine the molecular mass of small organic molecules using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method, which is in contrast to the usual MALDI strategy for matrix selection in which a small molecule matrix is used with a high molecular mass analyte, addresses the problem of matrix interference which limits the usefulness of MALDI-TOF for small molecule analysis. Use of negative ion mode offers advantages for this application. Using this approach, we have obtained clean molecular ion mass spectra of small organic molecules in the mass range 100-300 Da.     

Online Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry with In situ Mixing

Online matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is reported with in situ matrix/analyte aerosol mixing. The analyte and matrix were dissolved in separate solvents and pneumatically atomized into particles. Continuous flows of analyte particles and matrix droplets in nitrogen were mixed in a tee. The resulting particles were sampled using a nozzle, focused into a beam with an aerodynamic lens system, and vertically deposited continuously on a movable target. The matrix/analyte mixture was desorbed/ionized using a 266 nanometers pulsed laser at an incident angle of 45°. The nascent ions were analyzed using reflectron TOF MS. The performance of online MALDI-TOF MS was evaluated by the analysis of palmityl palmitate with the lithium salt of 2, 4-dihydroxybenzoic acid as the matrix. Strong and stable MALDI signals of palmityl palmitate were obtained. The matrix solvent mixture and the analyte concentration were optimized and the results demonstrate the development of an alternative for online MALDI analysis.     

2,5-Dihydroxyacetophenone: a matrix for highly sensitive matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of proteins using manual and automated preparation techniques

2,5-Dihydroxyacetophenone (DHAP) is presented as a matrix which enables highly sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of peptides, proteins and glycoproteins on AnchorChip targets. Depending on the protein, lower fmol amounts can be detected due to the increased homogeneity and concentration of the crystallization of the analyte/matrix mixture on the anchors. Best results could be generated in the mass range of 8-100 kDa. All sample/matrix preparation steps starting from mixing of DHAP matrix solution with sample solution to the transfer of the mixture to the MALDI-TOF target can be performed manually or automatically allowing low- and high-throughput analyses.     

Matrix-assisted laser desorption ionization mass spectrometry with 2-(4-hydroxyphenylazo)benzoic acid matrix

A novel matrix substance, 2-(4-hydroxyphenylazo) benzoic acid, or HABA, has been found to be very advantageous for matrix-assisted ultraviolet laser desorption ionization mass spectrometry. This compound has been successfully used for the desorption of peptides, proteins, and glycoproteins up to approximately 250 kDa. For these materials, the most abundant analyte-related peaks correspond to [M + H]⁺ ions and multiply protonated molecules. Comparisons with sinapic acid, 2,5-dihydroxybenzoic acid, and α-cyano-4-hydroxycinnamic acid indicate that the new matrix provides comparable sensitivity for peptides and smaller proteins but results in better sensitivity for larger proteins and glycoproteins in protein mixtures. Other matrices discriminate against the higher mass components in these cases. Somewhat reduced mass resolution has been found for smaller proteins, but for larger proteins and glycoproteins the best mass resolution can often be obtained with the new matrix. For other classes of compounds that form ions predominantly via cation attachment, at least as good sensitivity and even better resolution have been obtained. Derivatized glycolipids and synthetic polymers have been studied in detail. For the analysis of many synthetic polymers, the best performance in terms of sensitivity and mass resolution has been observed with HABA matrix. Mass resolution was higher for cation adducts than for the protonated peptide molecules in the same mass range. The new matrix exhibits greatly extended (in time) analyte ion production and reproducibility. Owing to the uniform sample surface with this matrix, barely any spatial variation of the ion signal could be observed. In addition, many hundreds of single-shot mass spectra could be accumulated from the same spot, even for larger proteins.     

MALDI-TOF MS detection of dilute, volume-limited peptide samples with physiological salt levels

This paper presents a highly efficient sample preparation technique for matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The purpose of the research is to use a conventional MALDI support to directly and conveniently detect sub-nM levels of peptides from volume-limited samples with physiological salt levels. In this new method, highly uniform matrix-nitrocellulose spots with a 500 microm diameter were conveniently generated by direct contact of a capillary tip to a stainless steel MALDI plate. An array of 50 microspots can be blotted from 1 microL matrix-nitrocellulose solution within 1 min. It was found that the addition of high concentration nitrocellulose to the alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution is critical for the formation of microspots. Samples are deposited on top of those microspots and incubated for 3 min. The CHCA-nitrocellulose surface shows a significant peptide binding capability for sub-nM levels of peptide. Restricting the matrix spot diameter to 500 microm gives an analyte enrichment effect because the peptides are confined to a small solid-phase surface area. Selective peptide binding is seen even with >0.15 M salt levels. Loading small aliquots of samples with multiple applications allows low level peptide detection down to 100 pM. Push-pull perfusates collected from the rat striatum were successfully analyzed with the microspot method.     

A novel strategy for MALDI-TOF MS analysis of small molecules

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) does not work efficiently on small molecules (usually with molecular weight below 500 Da) because of the interference of matrix-related peaks in low m/z region. The previous methods developed for this problem focused on reducing the peaks caused by the traditional matrices. Here, we report a novel strategy to analyze small molecules in a high and interference-free mass range by using metal-phthalocyanines (MPcs) as matrices which should be capable of forming matrix-analyte adducts. The mass of the target analyte was calculated by subtracting the mass of MPc from the mass of the MPc-analyte adduct. MPcs were also detectable and could serve as internal standards. Various MPcs with aromatic or aliphatic groups and different metal centers were then synthesized and explored. Aluminum-phthalocyanines (AlPcs), gallium-phthalocyanines (GaPcs), and indium-phthalocyanines (InPcs) were efficient matrices to form MPc-analyte adducts in either the positive or negative ion mode. The detection limits varied from 17 to 75 fmol, depending on analyte types. The mechanism of adducts formation was also proposed. Collectively, our strategy provides a novel and efficient way to analyze small molecules by MALDI-TOF MS.     

Lipid fingerprinting of gram-positive lactobacilli by intact--matrix-assisted laser desorption/ionization mass spectrometry using a proton sponge based matrix

A method of direct lipid analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) in intact membranes, without prior extraction/separation steps, is described. Here, we demonstrate the efficacy of a strong base, 1,8-bis(dimethylamino)naphthalene (DMAN; proton sponge), as a novel matrix for MALDI-time-of-flight (TOF) MS analysis of whole cell bacteria. Initially, individual acidic low-molecular-weight analytes such as standard free fatty acids and phospholipids were analyzed using DMAN as matrix. Clear negative-mode MALDI-TOF MS spectra of all analytes show only deprotonated analyte signals at a low picomole limit of detection with the complete absence of matrix-related signals. These results indicate that DMAN represents a suitable matrix for MALDI-TOF MS analysis of mixtures of complex lipids as the intact membranes of microorganisms. DMAN was successfully applied to the analysis of Lactobacillus sanfranciscensis and L. plantarum microorganisms. Different components were sensitively detected in a single spot, including 16:0, 18:2, 18:3, and 21:0 free acids, glycolipids, phosphatidylglycerols (PGs) and cardiolipins. This method might be of general application, offering the advantage of quickly gaining information about lipid components of other gram-positive bacterial membranes.     

Removal of sodium and potassium adducts using a matrix additive during matrix-associated laser desorption/ionization time-of-flight mass spectrometric analysis of peptides

Monovalent cations often associate with peptides and proteins under mass spectrometry (MS) conditions, resulting in a discernable, but often misleading, adduct cluster pattern. These adduct cluster peaks reduce the signal intensity of specific peptide species by splitting the ion population into multiple mass peaks, suppressing the ionization of neighboring low-abundance peaks, and interfering with identification of post-translational modifications. Further, monovalent contaminants tend to form a distribution of matrix cluster peaks in matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) spectra causing interference and suppression in the mass range below 1400 Da. The most common method for reduction or elimination of adduct clusters is solid-phase extraction via a pipette tip or spin column, which often leads to loss of low-abundance peptide components. In this study we describe the use of a commercially available surfactant blend that markedly reduces the adduction of monovalent cations during peptide analysis by MALDI-TOFMS.     

Sub part-per-million mass accuracy by using stepwise-external calibration in fourier transform ion cyclotron resonance mass spectrometry

A new external calibration procedure for FT-ICR mass spectrometry is presented, stepwise-external calibration. This method is demonstrated for MALDI analysis of peptide mixtures, but is applicable to any ionization method. For this procedure, the masses of analyte peaks are first accurately measured at a low trapping potential (0.63 V) using external calibration. These accurately determined (< 1 ppm accuracy) analyte peaks are used as internal calibrant points for a second mass spectrum that is acquired for the same sample at a higher trapping potential (1.0 V). The second mass spectrum has a approximately 10-fold improvement in detection dynamic range compared with the first spectrum acquired at a low trapping potential. A calibration equation that accounts for local and global space charge is shown to provide mass accuracy with external calibration that is nearly identical to that of internal calibration, without the drawbacks of experimental complexity or reduction of abundance dynamic range. For the 609 mass peaks measured using stepwise-external calibration method, the root-mean-square error is 0.9 ppm. The errors appear to have a Gaussian distribution; 99.3% of the mass errors are shown to lie within three times the sample standard deviation (2.6 ppm) of their true value.     

Use of matrix clusters and trypsin autolysis fragments as mass calibrants in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Trypsin autolysis fragments and matrix clusters are often observed as intense peaks in mass spectra of protein digests. It is demonstrated that these can be exploited to improve the mass calibration of a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometer. Interpretation of some of the autolysis masses is complicated by the existence of disulfide bonds. Surprisingly large matrix clusters are often visible for alpha-cyano-4-hydroxy-cinnamic acid. The fractional part of their masses differentiates them from protein digestion fragments.     

Ruggedness testing of quantitative atmospheric pressure ionization mass spectrometry methods: the effect of co-injected matrix on matrix effects

A number of techniques have been suggested to date for assessing matrix effects on quantitative atmospheric pressure ionization mass spectrometry (API-LC/MS) methods. A newly designed experiment has the aim of efficiently simulating the quantitative behavior of an LC/MS method as a function of the amount of co-injected matrix extract. Two sets of mixtures were prepared in different formats to study matrix effects as a function of analyte or matrix amount. Chromatographic conditions were varied as well, to alter the separation between analyte and co-extractants, and thereby provide different matrix effect conditions for testing the same mixtures. Graphical presentation of the results was used to gain insight into the matrix effect phenomenon. The results suggest that ruggedness for API-LC/MS methods may be defined as the absence of significant variation in results as a function of the amount of co-injected matrix. That is, a non-rugged API-LC/MS method may give consistent results only if a fixed amount of matrix is co-injected on a specific instrument. The results also point to the existence of a specific matrix concentration for the onset of matrix effects, below which these effects are not significant. These issues are important to the US FDA Center for Veterinary Medicine, which has regulatory authority for methods used to monitor for drug residues in food tissues from animals. The ruggedness testing technique suggested here may be an important factor in determining that a method is ready for multi-laboratory testing on multiple instruments.     

Thin-Layer Matrix Sublimation with Vapor-Sorption Induced Co-Crystallization for Sensitive and Reproducible SAMDI-TOF MS Analysis of Protein Biosensors

Coupling immunoassays on self-assembled monolayers (SAMs) to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) provides improved assay selectivity compared with traditional photometric detection techniques. We show that thin-layer-transfer (TLT) of ??-cyano-4-hydroxycinnaminic acid (CHCA) MALDI matrix via vacuum sublimation followed by organic solvent-based vapor-sorption induced co-crystallization (VIC) results in unique matrix/analyte co-crystallization tendencies that optimizes assay reproducibility and sensitivity. Unique matrix crystal morphologies resulted from VIC solvent vapors, indicating nucleation and crystal growth characteristics depend upon VIC parameters. We observed that CHCA microcrystals generated by methanol VIC resulted in >10× better sensitivity, increased analyte charging, and improved precision compared with dried droplet measurements. The uniformity of matrix/analyte co-crystallization across planar immunoassays directed at intact proteins yielded low spectral variation for single shot replicates (18.5?% relative standard deviation, RSD) and signal averaged spectra (<10?% RSD). We envision that TLT and VIC for MALDI-TOF will enable high-throughput, reproducible array-based immunoassays for protein molecular diagnostic assays in diverse biochemical and clinical applications.     

A label-free method based on MALDI-TOF mass spectrometry for the absolute quantitation of troponin T in mouse cardiac tissue

A label-free absolute quantitation method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed. This methodology was applied to mouse heart in order to quantify cardiac troponin T (cTnT), which is considered to be a sensitive marker of heart damage. The cTnT was extracted, isolated by reversed-phase high-performance liquid chromatography, digested, and analyzed by MALDI-TOF MS. The MS-based quantitation was performed using matrix-matched calibration curves (due to a matrix effect) of two synthetic peptides, one cTnT-specific peptide and one internal standard peptide, respectively. Recoveries at three spiking levels ranged from 87–96%, with relative standard deviations of below 10%. The method detection limit and the method quantitation limit, expressed as the amount of cTnT for the amount of total sarcomeric protein extract, were 0.03 mg g⁻¹ and 0.15 mg g⁻¹, respectively. This method appears to be accurate and generally suitable for improving absolute protein quantitation.     

Mechanism of modification by palladium in graphite furnace atomic absorption spectrometry

The effects of palladium and mixtures containing palladium on the absorbance characteristics of lead, thallium, cadmium, selenium, manganese and cobalt are described. These data, together with results of scanning electron microscopy showing the distribution of palladium on the graphite surface, indicate that palladium has a physical mechanism of analyte modification. During furnace heating, the analyte dissolves in molten palladium and may combine with it chemically. However, the rate limiting step leading to atomization appears to be diffusion of the analyte from palladium. The addition of magnesium, molybdenum or powdered carbon increases the speed of diffusion by causing palladium to form smaller droplets, and hence produces sharper absorbance peaks. Palladium becomes less effective as the atomization temperature increases, because the rate of diffusion is higher. This accounts for palladium having only a small stabilizing effect on less volatile elements such as manganese and cobalt. The addition of ascorbic acid to palladium has no significant effect on its modifying properties in a dilute nitric acid matrix. Results of kinetic studies on the atomization of gold are consistent with analyte diffusion out of palladium as the rate-limiting step leading to atomization.     

Silica nanoparticles pre-spotted onto target plate for laser desorption/ionization mass spectrometry analyses of peptides

We report on the simple deposition of Stöber silica nanoparticles (SiO₂ NPs) on conventional MALDI target plate for high throughput laser desorption/ionization mass spectrometry (LDI-MS) analyses of peptide mixtures with sensitivity in the femtomolar range. This low-cost easily prepared material allowed straightforward LDI experiments by deposition of the studied samples directly onto a pre-spotted MALDI plate. This analytical strategy can be performed in any laboratory equipped with a MALDI-TOF instrument. All key benefits of organic matrix-free technologies were satisfied while maintaining a high level of detection performances (sensitivity and reproducibility/repeatability). In particular, sample preparation was simple and detection in the low mass range was not hampered by matrix ions. Imaging studies were undertaken to query sample dispersion into the inert SiO₂ NPs and to help into the search of the best experimental conditions producing homogeneous analyte distribution within the deposit. In contrast to commercial disposable LDI targets designed for single use and requiring an adaptor such as NALDI™, the proposed SiO₂ NPs pre-spotting on a MALDI target plate allowed very easily switching between MALDI and LDI experiments. They can be conducted either simultaneously (positions with an organic matrix or SiO₂ NPs) or in the row (support prepared in advance, stored and washed after use). The overall cost and versatility of the methodology made it very attractive to MALDI users in many domains (peptidomics, proteomics, metabolomics).     

Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry

A simple reversed-phase nano-column purification and sample preparation technique is described, which markedly improves the mass spectrometric analysis of complex and contaminated peptide mixtures by matrix-assisted laser desorption/ionization (MALDI). The method is simple, fast and utilizes only low-cost disposables. After loading the sample on the column and a subsequent washing step, the analyte molecules are eluted with 50-100 nl of matrix solution directly on to the MALDI/MS target. The washing step ensures removal of a wide range of contaminants. The small bed volume of the column allows efficient sample concentration and the elution process yields very small sample spots. This simplifies the analysis and minimizes discrimination effects due to sample heterogeneity, because the desorption/ionization laser simultaneously irradiates a large portion of the sample. Taken together, these features of the method significantly improve the sensitivity for MALDI/MS analysis of contaminated peptide samples compared with the commonly used sample preparation procedures. This is demonstrated with in-gel tryptic digests of proteins from human brain that were separated by 2D gel electrophoresis. Furthermore, it is shown that with this method 2,5-dihydroxybenzoic acid (DHB) acts as an efficient matrix for peptide mapping. Both detection sensitivity and sequence coverage are comparable to those obtained with the currently preferred matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). The higher stability of peptide ions generated with DHB compared with CHCA is advantageous when analyzing fragile sample molecules. Therefore, the method described here is also of interest for the use of Fourier transform ion cyclotron resonance (FT-ICR) or ion-trap mass analyzers.