Multisample preparation methods for the solvent-free MALDI-MS analysis of synthetic polymers

A limitation of any current approach using solvent-free MALDI mass spectrometry is that only one sample at a time can be prepared and transferred to the MALDI-plate. For this reason, multiple-sample preparation approaches for solvent-free MALDI MS analysis of synthetic polymers were developed that are simple and practical. One approach multiplexed sample preparation by simultaneously preparing multiple samples. With this approach, as many as 384 samples could be prepared by addition of analyte, matrix, salt, and 1-mm metal beads to each well of a 384-well disposable bacti plate, capping the plate with the lid and homogenizing all samples simultaneously using a common laboratory vortex device. Besides the time savings achieved by a single vortex step for multiple samples, an additional advantage of this method relative to previously reported solvent-free preparation methods is that the mixing volume per sample is reduced, which allows a reduction in the amount of analyte required. This method, however, still requires the transfer of each homogenized sample to the MALDI plate for subsequent analysis. Here we report a novel approach that combines multiple simultaneous solvent-free sample preparation with automatic sample transfer to the MALDI target plate. This approach reduces the possibility of cross-contamination, the amount of sample and matrix consumed for an analysis, and the time required for preparation of multiple samples. These methods were shown to provide high-quality mass spectra for various synthetic polymer standards with M(n) values to 10 kDa. The methods are efficient in that small sample amounts are required, the sample/salt/matrix ratio is not critical, and the time necessary to achieve sufficient homogenization of multiple samples is less than 5 min.     

Imaging the morphology of solvent-free prepared MALDI samples

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is an important technique to characterize many different materials, including synthetic polymers. MALDI mass spectral data can be used to determine the polymer average molecular weights, repeat units, and end groups. The development of solvent-free sample preparation methods has enabled MALDI to analyze insoluble materials and, interestingly, can provide higher-quality mass spectral data. Although the utility of solvent-free sample preparation for MALDI has been demonstrated, the reasons for its success are only now being discovered. In this study, we use microscopy tools to image samples prepared using solvent-free methods to examine the morphology of these samples. The samples are prepared using a simple vortex method. Our results show that the average particle size of typical MALDI matrices is reduced from their original tens to hundreds of micrometers to hundreds of nanometers. This size reduction of the matrix occurs in one minute using the vortex method. We also observe remarkably smooth and homogeneous sample morphologies for the laser to interrogate, especially considering the relatively crude methods used to prepare our samples.     

Determining the time needed for the vortex method for preparing solvent-free MALDI samples of low molecular mass polymers

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is an important technique to characterize many different materials, including synthetic polymers. MALDI mass spectral data is used to determine the polymer average molecular weights, repeat units, and end groups. The development of the vortex method of solvent-free sample preparation showed that remarkably short mixing times could prepare samples that yielded high quality MALDI mass spectra. In this paper, we use microscopy images and MALDI mass spectra to evaluate the mixing time required by the vortex method to produce mass spectra for low molecular mass polymer samples. Our results show that mixing times of as little as 10 s can generate homogeneous thin films that produce high quality mass spectra with S/N ∼ 100. In addition, ultrashort mixing times of only 2 s still produce samples with mostly smooth morphology and mass spectra with S/N ∼ 10.     

Application and evaluation of solvent-free matrix-assisted laser desorption/ionization mass spectrometry for the analysis of derivatized fullerenes

A variety of derivatized fullerenes have been studied by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Of particular emphasis has been the evaluation of a recently introduced solvent-free sample/target preparation method. Solvent-free MALDI is particularly valuable in overcoming adverse solvent-related effects, such as insolubility and/or degradation of the sample. The method was applied to fullerene derivatives susceptible to decomposition under insufficiently "soft" MALDI conditions. Analytes included the hydrofullerene: C(60)H(36), fluorofullerenes: C(60)F(x) where x = 18, 36, 46, 48 and C(70)F(x) where x = 54, 56, methano-bridged amphiphilic ligand adducts to C(60) and the [4 + 2] cycloadduct of tetracene to C(60). The new solvent-free sample preparation is established as an exceedingly valuable addition to the repertoire of preparation protocols within MALDI. The MALDI mass spectra were of very high quality throughout, providing a testimony that "soft" MALDI conditions could be achieved. Using the [4 + 2] cycloadduct of tetracene to C(60) as the model analyte for direct comparison with solvent-based MALDI, the solvent-free approach led to less fragmentation and more abundant analyte ions. Applying solvent-free sample preparation, different matrix compounds have been examined for use in the MALDI of derivatized fullerenes, including sulfur, tetracyanoquinodimethane (TCNQ), 9-nitroanthracene (9-NA) and trans-2-[3-(4-tert-butylphenyl)-2-methyl-2- propenylidene]malononitrile (DCTB). DCTB was confirmed as the best performing matrix, reducing unwanted decomposition and suppression effects.     

New aspects in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a universal solvent-free sample preparation

A method of solvent-free sample preparation is shown to be of universal applicability for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Results obtained were compared with those of traditional solvent-based sample preparation for MALDI-MS in order to demonstrate their similarities with respect to accuracy, sensitivity and resolution for polymers such as polystyrene and poly(methyl methacrylate) in a mass range from 2 to 100 kDa. The results revealed that there is fundamentally no difference in the quality of the obtained mass spectra, and we conclude that the mechanism of desorption and ionization remains unchanged. However, the solvent-free sample preparation turned out to have some advantages over the traditional method in certain cases: quick and easy applicability is shown for polyetherimide avoiding time-consuming optimization procedures. In particular, industrial pigments that are insoluble in common solvents were characterized without interfering signals from fragments. The method even showed improvements with respect to reproducibility and mass discrimination effects in comparison to traditional sample preparation. Additionally, this contribution provides new insight regarding the analyte/matrix preorganization for the desorption step which now appears to be independent of crystallinity.     

Reproducibility of serum protein profiling by systematic assessment using solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

Protein profiling of human serum by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is potentially a new diagnostic tool for early detection of human diseases, including cancer. Sample preparation is a key issue in MALDI MS and the analysis of complex samples such as serum requires optimized, reproducible methods for handling and deposition of protein samples. Data acquisition in MALDI MS is also a critical issue, since heterogeneity of sample deposits leads to attenuation of ion signals in MALDI MS. In order to improve the robustness and reproducibility of MALDI MS for serum protein profiling we investigated a range of sample preparation techniques and developed a statistical method based on repeated analyses for evaluation of protein-profiling performance of MALDI MS. Two different solid-phase extraction (SPE) methods were investigated, namely custom-made microcolumns and commercially available magnetic beads. Using these two methods, nineteen different sample preparation methods for serum profiling by MALDI MS were systematically tested with regard to matrix selection, stationary phase, selectivity, and reproducibility. Microcolumns were tested with regard to chromatographic properties; reversed phase (C8, C18, SDB-XC), ion-exchange (anion, weak cation, mixed-phase (SDB-RPS)) and magnetic beads were tested with regard to chromatographic properties; reversed phase (C8) or affinity chromatography (Cu-IMAC). The reproducibility of each sample preparation method was determined by enumeration and analysis of protein signals that were detected in at least six out of nine spectra obtained by three triplicate analyses of one serum sample.A candidate for best overall performance as evaluated by the number of peaks generated and the reproducibility of mass spectra was found among the tested methods. Up to 418 reproducible peaks were detected in one cancer serum sample. These protein peaks can be part of a possible diagnostic profile, suggesting that this sample preparation method and data acquisition approach is suitable for large-scale analysis of serum samples for protein profiling.     

A comparison of EDI with solvent-free MALDI and LDI for the analysis of organic pigments

To evaluate the applicability of EDI to material analysis as a new ionization method, a comparison of EDI with solvent-free matrix-assisted laser desorption ionization (MALDI) and laser desorption ionization (LDI) was made for the analysis of organic pigments, e.g. Pigment Yellow 93, Pigment Yellow 180, and Pigment Green 36, as test samples, which are poorly soluble in standard solvents. In EDI, the samples were prepared in two ways: deposition of suspended samples in appropriate solvents and dried on the substrate, and the direct deposition of the powder samples on the substrate. No matrices were used. Both sample preparation methods gave similar mass spectra. Equally strong signals of [M + H](+) and [M - H](-) ions were observed with some fragment ions for azo pigments in the respective positive or negative mode of operation. For the powder sample of the phthalocyanine pigment PG36, M(+*) and [M + H](+) in the positive mode and M(-*) in the negative mode of operation were observed as major ions. Positive-mode, solvent-free MALDI gave M(+), [M + H](+) and [M + Na](+) and negative mode gave [M - H](-) depending on the sample preparation. As solvent-free MALDI, EDI was also found to be an easy-to-operate, versatile method for the samples as received.     

Solvent-free MALDI-MS for the analysis of biological samples via a mini-ball mill approach

An efficient, low sample load mini-ball mill (MBM) sample preparation procedure was developed for solvent-free MALDI analysis of peptides and proteins. Picomole sample amounts can be handled conveniently, with 30 s grinding times being sufficient. Matrix purity and molar analyte/matrix ratios are not as critical as with methods employing solvent. Ammonium salt is employed for protonation of the peptide and suppression of sodiation. This strategy allows for peptide mapping and other biochemical manipulations to be performed prior to MBM sample preparation and mass analysis. The analysis of bovine serum albumin (66 kDa) yielded good results, indicating that higher molecular weight proteins are accessible. A semi-solvent-free strategy by the MBM sample preparation method is also described.     

Characterization of low molecular weight hydrocarbon oligomers by laser desorption/ionization time-of-flight mass spectrometry using a solvent-free sample preparation method.

A new solvent-free sample preparation method using silver trifluoroacetate (AgTFA) was developed for the analysis of low molecular weight paraffins and microcrystalline waxes by laser desorption/ionization time-of-flight mass spectrometry (LDI-TOFMS). Experiments show that spectral quality can be enhanced by dispersing AgTFA directly in liquid paraffins without the use of additional solvents. This preparation mixture is applied directly to the MALDI probe. Solid waxes could be examined by melting prior to analysis. The method also provides sufficiently reproducible spectra that peak area ratios between mono- and bicyclic alkane peaks indicated variations in the cycloalkane content of paraffin samples. Dehydrogenation of hydrocarbons observed during the desorption/ionization process was studied by analysis of alkane standards.     

Independent assessment of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sample preparation quality: A novel statistical approach for quality scoring

Preparation of samples according to an optimized method is crucial for accurate determination of polymer sample characteristics by Matrix-Assisted Laser Desorption Ionization (MALDI) analysis. Sample preparation conditions such as matrix choice, cationization agent, deposition technique or even the deposition volume should be chosen to suit the sample of interest. Many sample preparation protocols have been developed and employed, yet finding the optimal sample preparation protocol remains a challenge. Because an objective comparison between the results of diverse protocols is not possible, “gut-feeling” or “good enough” is often decisive in the search for an optimum. This implies that sub-optimal protocols are used, leading to a loss of mass spectral information quality. To address this problem a novel analytical strategy based on MALDI imaging and statistical data processing was developed in which eight parameters were formulated to objectively quantify the quality of sample deposition and optimal MALDI matrix composition and finally sum up to an overall quality score of the sample deposition. These parameters can be established in a fully automated way using commercially available mass spectrometry imaging instruments without any hardware adjustments. With the newly developed analytical strategy the highest quality MALDI spots were selected, resulting in more reproducible and more valuable spectra for PEG in a variety of matrices. Moreover, our method enables an objective comparison of sample preparation protocols for any analyte and opens up new fields of investigation by presenting MALDI performance data in a clear and concise way.     

MALDI-TOF MS stability study of model poly(p-phenylene terephthalamide)s

In the present study, we address the possibility of matrix-assisted laser desorption/ionization (MALDI)–time-of-flight MS analysis-induced chain fragmentation in poly(p-phenylene terephthalamide) (PPD-T) by considering two possible sources: (1) grinding-induced fragmentation resulting from the evaporation–grinding MALDI sample preparation method (E-G method) and (2) in-source/metastable fragmentation induced by the MALDI laser. An analysis of variance (ANOVA) statistical study found, with a high probability, that obtaining MALDI spectra with the effective laser area as large as possible (the “fanned-out” setting) did not cause any chain fragmentation due to the E-G MALDI sample preparation method, even when three additional grinding steps were used. However, the effect of laser fluence was less clear. A significant effect of laser fluence was observed for lower mass oligomers (<1,400 Da), but there was essentially no effect for higher mass species up to our limit of ANOVA measurement (∼2,300 Da). Plausible explanations are presented to explain these observations. The most likely scenario is that “unexpected” end-group modifications occur during PPD-T synthesis, producing small quantities of low mass species, which are amplified by the MALDI-EG extraction procedure.     

Evaluation of an on-target sample preparation system for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in conjunction with normal-flow peptide high-performance liquid chromatography for peptide mass fingerprint analyses

Large-scale mass spectrometry (MS)-based proteomic analyses require high-throughput sample preparation techniques due to the increasing numbers of samples that make up a typical proteomics experiment. Moreover, extensive sample pre-treatment steps are necessary prior to MS acquisition for even the most rapid and robust MS-based proteomics methodology, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS followed by peptide mass fingerprinting (PMF) analysis. These include sample purification and fractionation, removal of digestion buffers or solvents, and spotting of sample with matrix onto the MALDI target. These multiple steps of time-consuming sample handling can result in high overall analysis costs and the likelihood of sample contamination and loss. In order to overcome some of these limitations in sample processing, we have investigated the use of a novel, simple, inexpensive 96-well elastomeric array that affixes to a MALDI target to create an on-target 96-well plate that accommodates a high solution volume (ca. 200 microL), thereby enabling the on-target processing of samples for MALDI-TOFMS. We explored several factors that influence MALDI sample preparation: type of matrix, solution volume, solution organic composition, solution drying rates and matrix/analyte co-crystallization methods. We also investigated the use of the 96-well elastomeric device for coupling MALDI-TOFMS analysis directly to high flow rate (1 mL/min) reversed-phase (rp)-HPLC. By developing an optimized, robust sample preparation protocol, we were able to obtain mass spectra with a high signal-to-noise ratio from peptide standards present at the 50-fmol level in large starting volumes of solution. PMF analyses were possible from 1-pmol and 500-fmol protein-digest standards. Coupling the device to high-flow HPLC (750 microL/min) yielded a robust and semi-automated means to obtain enhanced MALDI-TOFMS data at 500 ng of protein digest. These methodologies developed for this simple, on-target, elastomeric device show promise for streamlining the sample preparation process from HPLC to MALDI-MS.     

Measuring salty samples without adducts with MALDI MS

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been used for the discovery of hundreds of novel cell to cell signaling peptides. Beyond its advantages of sensitivity and minimal sample preparation requirements, MALDI MS is attractive for biological analyses as high quality mass spectra may be obtained directly from specific locations within prepared tissue sections. However, due to the large quantity of salts present in physiological tissues, these mass spectra often contain many adducts of cationic salts such as sodium and potassium, in addition to the molecular ion [M + H]⁺. To reduce the presence of cation adducts in MALDI mass spectra obtained directly from tissues, we present a methodology that uses a slow condensation procedure to enable the formation of distinct regions of matrix/analyte crystals and cation (salt) crystals. Secondary ion mass spectrometric imaging suggests that the salts and MALDI matrix undergo a mutually exclusive crystallization process that results in the separation of the salts and matrix in the sample.     

Conductive carbon tape as a sample platform for microwave-based MALDI MS detection of proteins and phosphoproteins

In this study, we developed a novel microwave-assisted protein preparation and digestion method for matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry analysis and identification of proteins that involves using conductive carbon tape as a sample platform for sample preparation (reduction and alkylation) and digestion under microwave heating and as a plate for MALDI analysis. This method allows for the enzymatic digestion products of proteins to be directly analyzed by MALDI mass spectrometry and results in a marked reduction in sample loss. Our protocol requires only a small volume (1 μL) of reaction solvent, which increases the frequency of enzyme-to-protein contact, thereby resulting in more efficient digestion of sample than conventional in-solution digestion methods. To test this protocol, we used magnetic iron (II, III) oxide nanoparticles as concentrating probes to enrich phosphopeptides from a mixture of peptides in enzymatically digested protein samples. We found that the one-pot on-tape-based protein preparation and digestion under microwave heating combined with the on-tape-based enrichment method not only dramatically reduced the time required for phosphopeptides analysis but also allowed for the simultaneous identification of phosphoproteins. The advantages of our protocol include ease of use, high digestion efficiency, high specificity, and rapid (15 min) identification of proteins and enrichment of phosphopeptides in a mixture of enzymatically digested protein samples.     

The fragmentation of ethoxylated surfactants by AP-MALDI-QIT

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has become an important technique to characterize the chemical structure of industrial polymer materials. MALDI methods have been developed to address a broad variety of different polymer materials containing different chemistries. One of the key aspects of the typical MALDI experiment is the generation of intact ions. The development of Atmospheric Pressure (AP) MALDI quadrupole ion trap (QIT) instruments has opened another channel to obtaining MS/MS experiments for polymer samples. These experiments provide a new method to obtain chemical structure information from MALDI experiments. Collision-Induced Dissociation (CID) provides an improved MALDI MS/MS experiment that can be done on readily available mass spectrometers. AP MALDI QIT techniques have been successfully applied to a variety of synthetic polymers. This work explores the applicability of AP MALDI QIT methods to relatively low molecular weight ethoxylated surfactants. In these experiments we show the CID fragmentation mass spectra on some ethoxylated surfactants, and demonstrate the existence of analyte matrix clusters.     

固相微萃取技术在形态分析中的应用进展

形态分析比传统的元素分析能提供更为丰富的信息，成为当今分析化学领域前沿课题之一，而固相微萃取（SPME)是近十年来发展起来的新型分离富集技术，简便快速、无污染、易于和其它技术联用．近几年来才开始将固相微萃取应用到形态分析，二者结合对形态分析的发展具有促进作用，本文就固相微萃取技术在元素有机化合物形态分析中的应用进行了评述．     

The effects of molecular weight distribution and sample preparation on matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of petroleum macromolecules

To date there have been no systematic, quantitative investigations of the effect of sample preparation on the matrix-assisted laser desorption/ionization time-of-flight (MALDI) mass spectrometry response for polydisperse systems. To this end, the interrelationships between sample preparation, analyte molecular weight distribution (MWD) and solubility, and signal response were investigated for mixtures of alkylated polycyclic aromatic hydrocarbon (PAH) oligomers, the constituents of petroleum pitch that serve as precursors for advanced carbon materials. These PAH oligomers served as a useful analyte system for study, as their solvent solubilities decrease significantly with each increasing oligomeric unit. Molecular weight standards consisting of relatively pure dimer and trimer cuts of the starting M-50 petroleum pitch were produced using a dense-gas/supercritical extraction (DGE/SCE) technique and were then used to produce oligomeric mixtures of well-defined composition for study. Both traditional, solvent-based and newer, solvent-free sample preparation methods were evaluated, and their effects on both homogeneity and signal response were determined. While solvent-free sample preparation methods produced homogeneous samples and reproducible results regardless of the MWD of the analyte, solvent-based samples that contained more than one oligomeric cut produced non-homogeneous samples and poor reproducibilities. The differing solubilities of dimer, trimer, and tetramer oligomers in a given solvent (e.g., CS(2) or toluene) were found to be the cause of the inhomogeneities observed in solvent-based sample preparation. A quantitative analysis study performed with dimer/trimer mixtures over a wide range of compositions via solvent-free sample preparation indicates that linear, reproducible calibration curves can be generated and used to calculate the molecular composition of unknown dimer/trimer mixtures with confidence.     

Reverse thin layer method for enhanced ion yield of oligosaccharides in matrix‐assisted laser desorption/ionization

A sample preparation method that is suitable for sensitive detection of underivatized oligosaccharides by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) has been investigated. As compared with the conventional dried‐droplet or ethanol (EtOH) recrystallization method, superior mass spectra in terms of ion yield and signal‐to‐noise (s/n) ratio were obtained when methanol (MeOH) was used as a solvent for the mixture of matrix and oligosaccharides. Based on these results, a new sample preparation method, named the ‘reverse thin layer method’, was developed. This method comprises two steps: first, complete drying of the oligosaccharide solution on the MALDI target plate; and second, deposition of the matrix dissolved in a small amount of MeOH. Using this method, a relatively homogeneous matrix crystal was generated and higher yields of both positive and negative ions were obtained from oligosaccharides compared with conventional methods. Notably, the method can be applied to various matrices including both solid and liquid matrices. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of Hanford-Related Organics Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry

Matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI/TOF-MS) was used for the analysis of low-molecular phosphate compounds found in Hanford tank wastes. The mass spectra of these compounds indicate protonated peaks as well as sodium adducts. Analytical methods presently utilized for the analysis of the phosphate-related organics are both time consuming and labor intensive. A promising alternative is MALDI/TOFMS. The MALDI process produces both positive and negative ions directly and very little sample is required. In addition, there is limited sample preparation and minimal hazardous waste production.     

Experimental factors affecting the quality and reproducibility of MALDI TOF mass spectra obtained from whole bacteria cells

Numerous experimental factors are shown to significantly influence the spectra obtained when bacteria are analyzed by MALDI TOF/MS. Detailed investigation of the instrument parameters and sample preparation are all shown to influence the spectra. Of these, the preanalysis sample preparation steps incorporate the most important elements influencing the quality and reproducibility of the spectra. Some of the most important sample preparation factors include the method employed for sterilization, the type of matrix, the matrix solvent and concentration of cells in the matrix, as well as the type and concentration of acid added to the matrix. The effects of these parameters, as well as other aspects of sample preparation and the effects of several instrumental parameters on spectra are presented. Optimization and control of all experimental variables leads to a stable protocol for analysis of bacteria. The protocol employs a Nd:Yag laser and describes both sample handling and instrument conditions which consistently yield reproducible MALDI TOF mass spectra with greater than 25 peaks from both gram-positive and gram-negative bacteria.