Polylysine-coated diamond nanocrystals for MALDI-TOF mass analysis of DNA oligonucleotides

A protocol based on aminated diamond nanocrystals has been developed to isolate, concentrate, purify, and digest DNA oligonucleotides in one microcentrifuge tube for matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. It is shown that use of diamond nanocrystals as a solid-phase extraction support not only permits concentration of oligonucleotides in highly diluted solutions but also facilitates separation of oligonucleotides from proteins in heavily contaminated solutions. Enzymatic digestions can be conducted on particle, and additionally, the digests can be easily recovered from the solution for base sequencing. In this method, the aminated diamond nanocrystals ( approximately 100 nm in diameter) were prepared by noncovalent coating of carboxylated/oxidized diamonds with poly(L-lysines) (PL), which form stable complexes with DNA oligonucleotides. While the complexes are sufficiently stable to sustain repeated washing with deionized water, the DNA molecules can be readily eluted after incubation of the diamond adducts in aqueous ammonium hydroxide at elevated temperatures. No preseparation of PL or diamond nanocrystals is required for subsequent MALDI-TOF mass analysis.     

用于MALDI-TOF MS分析小分子化合物的新型基质材料研究进展

基质辅助激光解吸/电离-飞行时间质谱(MALDI-TOF MS)是一种新型的软电离生物质谱技术,其对小分子化合物的分析一直是研究热点。目前,已经有多种新型基质材料被开发,主要分为纳米材料基质、有机小分子基质和有机聚合物基质。新型基质材料的提出,解决了传统基质的强背景离子干扰的问题,但仍处于研究阶段。综述了不同新型基质材料的优点、性质及适用对象,并对新型基质材料的未来发展趋势进行了展望。     

Manipulation of temperature to improve solubility of hydrophobic proteins and cocrystallization with matrix for analysis by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) requires cocrystallization of analyte with a large excess of matrix, which must be mutually soluble in a solvent that encourages crystal growth upon evaporation. MALDI-MS of hydrophobic proteins can be difficult, because they tend to aggregate in polar solutions. High concentrations of denaturants and salts are often employed to combat protein aggregation, but this can result in signal suppression. By using various organic cosolvent systems and matrixes at different protein:matrix ratios, we were able to use MALDI-TOFMS to detect four bacterially expressed hydrophobic proteins comprising alanine-rich mutants of the basic region/leucine zipper protein (bZIP) GCN4. By manipulating sample temperature, we were able to maintain protein solubility. Protein aggregation was suppressed when mixing the protein and matrix solutions at 4 degrees C prior to warming to 37 degrees C, following the temperature-leap technique described by Xie and Wetlaufer (Protein Sci. 1996, 5, 517-523), who used this method to renature bovine carbonic anhydrase II. Manipulation of temperature encouraged our hydrophobic proteins to adopt conformations leading to the nonaggregating state, and solubility was maintained even when the concentration of denaturant was reduced from 4 M to 400 mM. The temperature-leap tactic was critical for maintaining protein solubility, preventing signal suppression normally seen with higher concentrations of salts, allowing for generation of superior spectra, and should prove applicable to other systems prone to aggregation.     

On the asymptotic analysis of surface-stress-driven thin-layer flow

It has been demonstrated experimentally that thin liquid layers may be applied to a solid surface or substrate if a temperature gradient is applied which results in a surface tension gradient and surface traction. Two related problems are considered here by means of the long-wave or lubrication theory. In the first problem, an improved estimate of the applied liquid coating thickness for a liquid being drawn from a bath is found through asymptotic and numerical matching. Secondly, the theory is extended to consider substrates that are not perfectly wetted but exhibit a finite equilibrium contact angle for the coating liquid. This extension incorporates the substrate energetics using a disjoining pressure functional. Unsteady flows are calculated on a substrate of nonuniform wettability. The finite contact angle value required to stop stress-driven flow is predicted and the resulting steady profiles are compared with experimental results for several values of the applied stress.     

Fast screening of low molecular weight compounds by thin-layer chromatography and "on-spot" MALDI-TOF mass spectrometry

Fast screening of low-MW compounds is performed by thin-layer chromatography (TLC) followed by direct on-spot matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification with nearly "matrix-free" mass spectra using an UV-absorbing ionic liquid matrix. Owing to minimal background ions from the proton donor triethylamine/alpha-cyano-4-hydroxycinnamic acid ionic liquid matrix, three arborescidine alkaloids, the anesthesics levobupivacaine and mepivacaine, and the antibiotic tetracycline were readily characterized most frequently by the MS detection of their protonated molecules. The technique is fast and sensitive, requires little sample preparation and manipulation, and is therefore suitable for fast screening with TLC separation and MS identification of low-MW compounds, with potential applications in areas such as phytochemistry, synthetic chemistry, and product manufacturing quality monitoring.     

Optimization of sample preparation for peptide sequencing by MALDI-TOF photofragment mass spectrometry

This paper describes the optimization of sample preparation for MALDI 193-nm photofragment ion time-of-flight mass spectrometry to sequence small to medium-sized peptides from peptide mixtures. We show that matrix additives, such as fructose and phenylbutyric acid have a dramatic effect on the abundance of fragment ions observed in the post-source decay spectra. A dried-droplet MALDI matrix consisting of 1:1 alpha-cyano-4-hydroxycinnamic acid/fructose proves to be an excellent matrix for photodissociation because [M + H]+ ions are formed with low internal energies, and the photofragment ion spectrum contains high abundances of sequence-informative ions. The addition of fructose appears to improve overall sample homogeneity and durability, as compared to conventional alpha-cyano-4-hydroxycinnamic acid dried-droplet preparations. MALDI-TOF photodissociation is then used to selectively sequence the peptides bradykinin (RPPGFSPFR), des-Arg9 bradykinin (RPPGFSPF), and substance P-amide (RPKPQQFFGLM-NH2) from a mixture of five peptides.     

MALDI-TOF mass spectrometric method for detection of hybridized DNA oligomers

Two new approaches for nucleic acid hybridizations by MALDI-TOF mass spectrometry are described. Hybridization using genomic DNA without polymerase chain reaction was demonstrated. Total genomic DNA of bacteriophages bound to charge-modified nylon membranes was identified by the hybridization of species-specific oligonucleotide probes. lambda-Phage DNA and M13 were used for the test with good success. Since MALDI-TOF mass spectrometry can be used to measure the molecular weights of different probes, mass spectrometry can be used for the detection of hybridizations with multiple probes. We demonstrate that multiple-probe hybridization can be resolved by mass spectrometry. Six probes with different mass tag were used for hybridization on a single spot. MALDI-TOF mass spectrometry was successfully used to measure these probes simultaneously. This provides a simple nonradioactive method for multiplex hybridization analysis. It has the potential to drastically increase the speed for microarray hybridization analysis in the future.     

MALDI-TOF mass spectrometry analysis of amphipol-trapped membrane proteins

Amphipols (APols) are amphipathic polymers with the ability to substitute detergents to keep membrane proteins (MPs) soluble and functional in aqueous solutions. APols also protect MPs against denaturation. Here, we have examined the ability of APol-trapped MPs to be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For that purpose, we have used ionic and nonionic APols and as model proteins (i) the transmembrane domain of Escherichia coli outer membrane protein A, a β-barrel, eubacterial MP, (ii) Halobacterium salinarum bacteriorhodopsin, an α-helical archaebacterial MP with a single cofactor, and (iii, iv) two eukaryotic MP complexes comprising multiple subunits and many cofactors, cytochrome b(6)f from the chloroplast of the green alga Chlamydomonas reinhardtii and cytochrome bc(1) from beef heart mitochondria. We show that these MP/APol complexes can be readily analyzed by MALDI-TOF-MS; most of the subunits and some lipids and cofactors were identified. APols alone, even ionic ones, had no deleterious effects on MS signals and were not detected in mass spectra. Thus, the combination of MP stabilization by APols and MS analyses provides an interesting new approach to investigating supramolecular interactions in biological membranes.     

High-throughput SEM preparation of proteinaceaous extracellular matrix

Adhesives of aquatic organisms are of high scientific interest with a view to biomimicry. The visualization of their fine-structure, however, is difficult due to the fragility of the extracellular matrix (ECM). Care must be taken in its preparation for high-resolution SEM. Relating matrix structures to substrate properties demands a high throughput of samples for reliable comparisons. In order to acquaint ourselves with a suitable method we found an easy way to manage the critical steps of preparation for SEM. We show that thin layers of proteinaceous matrix can be satisfactorily prepared by combining freeze-drying and sputtering in a conventional sputter coater after one-step fixation (2.5% glutaraldehyde). The results were superior to CPD.     

Toward prediction: using chemometrics for the optimization of sample preparation in MALDI-TOF MS of synthetic polymers

In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for the study of synthetic polymers although its mechanism is still not understood in detail. Sample preparation plays the key role in obtaining reliable MALDI mass spectra, in particular, the proper choice of matrix, cationization reagent, and solvent. There is still no general sample preparation protocol for MALDI analysis of synthetic polymers. For known synthetic polymers, such as polystyrenes and other frequently investigated polymers, application tables in review articles might be a guide for selecting a MALDI matrix, cationization reagent, and solvent. For unknown polymers (polymers which were not analyzed by MALDI-TOF MS before but whose structures are in part known from the manufacturing process and from NMR analysis as well), the selection of matrix and solvent is based upon the polarity-similarity principle. Chemometric methods provide a useful tool for the investigation of sample preparation because huge data sets can be evaluated in short time, that is, for extracting relevant information and for classification of samples, as well. Furthermore, chemometrics provide a suitable way for the selection of a proper matrix, cationization reagent, and solvent. In this paper, a prediction model is presented using the partial least-squares (PLS) regression. By applying the model, the suitability of appropriate (nontested) combinations (matrix, cationization reagent, solvent) can be predicted for a certain synthetic polymer based upon the investigation of a few combinations. This model may help find suitable combinations in a short time and serve as a starting point for the investigation of unknown polymers. Results are exemplary presented for polystyrene PS2850.     

Cyclic subspace regression with analysis of the hat matrix

Cyclic subspace regression (CSR) is a new approach to the complex multivariate calibration problem. The simple algorithm produces solutions for principal component regression (PCR), partial least squares (PLS), least squares (LS), and other related intermediate regressions. This paper describes further analysis of CSR and shows that by using hat matrices, CSR regression vectors are formed from a summation of weighted eigenvectors where weights are determined from the hat matrix, singular values, and sample space eigenvectors. Examination of CSR weights for PCR and PLS further documents differences and similarities and provides information to assist in determining prediction rank for PCR and PLS. By redefining CSR in terms of weighted eigenvectors, it can be shown when PLS and PCR produce essentially the same results where minor differences stem from overfitting by PLS. Additionally, weights derived from the hat matrix show when PCR and PLS generate different results and why. Equations are shown for the sample space that reveal PLS to be a method based on oblique projections while PCR uses orthogonal projections. The optimal intermediate CSR model can be identified as well. A near infrared data set is studied and illustrates principles involved.     

A method for the analysis of low-mass molecules by MALDI-TOF mass spectrometry

We report a novel method termed matrix suppressed laser desorption/ionization to improve the analysis of low-mass molecules by MALDI-TOF mass spectrometry. In this method, the surfactant of cetrimonium bromide (CTAB) is added to the conventional matrix of alpha-cyano-4-hydroxycinnamic acid solution to prepare the MALDI samples. During the MALDI process, the presence of CTAB could substantially or even completely suppress the matrix-related ion background. As a result, very clean mass spectra can be routinely obtained in the low-mass range. In addition, the presence of CTAB can significantly improve the mass resolution of low-mass molecules. It is seen that high-quality spectra were routinely obtained at a matrix/CTAB ratio of 1000:1. This method has been successfully used to analyze a variety of low-mass molecules.     

Instrumental parameters in the MALDI-TOF mass spectrometric analysis of quaternary protein structures

Several former studies have shown that MALDI-TOF-MS can be applied successfully to investigate the quaternary structure of proteins. Whereas most of these reports were focused on MALDI sample preparation, there is little information about the influence of instrumental parameters on the desorption/ionization and gas-phase behavior of protein subunit assemblies. Therefore, in addition of giving short examples of the quaternary structure analysis of a microheterogeneous glycoprotein, a metalloenzyme, and a heme-binding enzyme by MALDI-TOF-MS, we report a systematic study of the effect of some instrumental parameters on the analysis of chicken egg white avidin. From these tested parameters, only the laser pulse energy was found to influence the relative abundance of the intact assembly as well as the formation of nonspecific cluster ions significantly. This finding suggests that the disruption of the noncovalent interactions during the desorption/ionization process takes place at a very short time interval after the laser ablation, whereas those assemblies that survive this step are rather stable afterward in the gas phase. In addition, we present clear evidence that protein cluster ions are not preformed during sample preparation but originate from nonspecific assemblage during desorption/ionization.     

The limitations of MALDI-TOF mass spectrometry in the analysis of wide polydisperse polymers

Average molecular weight determination of polymers with polydispersities greater than 1.2 is an ongoing challenge in the field of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Mass discrimination effects observed in the analysis of these polymers have been attributed to sample preparation, desorption/ionization, and instrumental factors. In an effort to separate these factors, we studied poly(methyl methacrylate) (PMMA) standards using two different ion detection systems installed on the same time-of-flight mass analyzer. Equimass blends of narrow PMMA standards were used to simulate a polymer with a wide polydispersity. MALDI-MS analysis was also performed on a PMMA standard with a polydispersity of 1.7. All samples were analyzed by size exclusion chromatography for comparison. Although sample preparation and ionization/desorption factors were found to influence the spectral appearance of the MMA distributions, we demonstrate that, under similar sample preparation and instrument conditions, different ion detection systems produce different results for synthetic polymer blends. The differences in the detector responses for the blends and wide polydisperse standard arise from several factors related to the ion detection system: (1) detection mechanisms, (2) saturation effects, and (3) signal-to-noise limitations.     

In situ preparation of reinforced polyimide nanocomposites with the noncovalently dispersed and matrix compatible MWCNTs

High performance MWCNT-reinforced polyimide nanocomposites were prepared through in situ polymerization route. 2,6-Diaminoanthraquinone (2,6-DAAQ) was selected to serve as both a diamine comonomer, and a noncovalent dispersant of MWCNTs through π–π interaction. The good dispersion of MWCNTs in both solution and polymer matrix, and high compatibility derived from the structural similarity have been proved to be particularly advantageous for the interfacial adhesion and load transfer from the polymer matrix to MWCNTs. The nanocomposites with 0.50–0.75 wt% MWCNTs had the most excellent thermal stability, thermomechanical and tensile properties, and the electrical conductivity also achieved a sharp increase at such low content. The load transfer efficiency was calculated based on both theoretical model and microscopical size measurement, and the results were rather reasonable within accepted error range.     

Improving the performance of MALDI-TOF in oligonucleotide analysis using a new SDIFA technology

A new technology termed SDIFA is developed to improve the mass resolution of linear matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in oligonucleotide analysis. Unlike the currently used delayed extraction method, SDIFA allows electrical isolation of the sample holder from ion extraction/acceleration and selectively samples part of the desorbed ions, thereby reducing the initial velocity distribution and improving resolution. In addition, a method was introduced to improve the space focusing of TOF. Isotope-limited mass resolution was obtained for oligonucleotides of up to 62 mer, and the true instrumental resolution reaches to 1,800 at 19.2 kDa. It was also demonstrated that excellent resolution was obtained across a large mass range using a single setting of acquisition parameters. This feature allows unambiguous identification of multiple A/T heterozygote samples in a mass range of 5,200-7,800 Da. Moreover, compared with DE, the performance of SDIFA was more stable, reproducible, and less dependent on the experimental conditions including the laser power, sample spots, sample substrates, delayed time, and extraction field strength. This enhanced ease of data acquisition is the key to automated spectrum acquisition.     

Rapid and direct sequencing of double-stranded DNA using exonuclease III and MALDI-TOF MS

Application of MALDI-TOF MS to direct sequencing of dsDNA substrates is demonstrated using a strategy that employs exonuclease III digestion of a target sequence. Experimental conditions for exonuclease III have been optimized for this application, including addition of essential divalent metal ion cofactors. A short cation-exchange column was designed to provide efficient sample cleanup and overcome major problems arising from salt interference.     

Suppression of matrix clusters and enhancement of peptide signals in MALDI-TOF mass spectrometry using nitrilotriacetic acid

Matrix clusters and their alkali metal ion adducts suppress peptide signals in the 500-1400 Da range and compromise MALDI-TOF mass spectrometric peptide mass fingerprinting and protein identification. Addition of 7 mM nitrilotriacetic acid to the matrix solution significantly reduced matrix clusters and increased signal-to-noise ratio of peptide signals approximately 5 to 20-fold. As a result, reliability in the identification of femtomole amounts of proteins based on peptide mass fingerprinting and database search was significantly enhanced, leading to a higher score and sequence coverage.