Silver nanoparticles as selective ionization probes for analysis of olefins by mass spectrometry

Laser desorption/ionization (LDI) using silver nanoparticles (AgNPs) is shown to selectively ionize olefinic compounds, e.g., cholesterol, 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), and carotenoids. Selective AgNP LDI can be carried out from complex mixtures without the addition of an organic matrix, sample cleanup, or prefractionation. Results presented in this report are the first to demonstrate the selective ionization of specific compounds from a complex mixture using metal nanoparticles.     

On-plate selective enrichment and self-desalting of peptides/proteins for direct MALDI MS analysis

In this paper, a new technique has been proposed to achieve simultaneous peptides/proteins enrichment and wash-free self-desalting on a novel sample support with a circle hydrophobic-hydrophilic-hydrophobic pattern. Upon deposition, the sample solution is first concentrated in a small area by repulsion of the hydrophobic outer layer, and then, the peptides/proteins and coexisting salt contaminants are selectively captured in different regions of the pattern through strong hydrophobic and hydrophilic attractions, respectively. As a result, the detection sensitivity is improved by 2 orders of magnitude better than the use of the traditional MALDI plate, and high-quality mass spectra are obtained even in the presence of NaCl (1 M), NH(4)HCO(3) (100 mM), or urea (1 M). The practical application of this method is further demonstrated by the successful analysis of myoglobin digests with high sequence coverage, demonstrating the great potential in proteomic research.     

Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins

Phosphopeptides are often detected with low efficiency by MALDI MS analysis of peptide mixtures. In an effort to improve the phosphopeptide ion response in MALDI MS, we investigated the effects of adding low concentrations of organic and inorganic acids during peptide sample preparation in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. Phosphoric acid in combination with 2,5-DHB matrix significantly enhanced phosphopeptide ion signals in MALDI mass spectra of crude peptide mixtures derived from the phosphorylated proteins alpha-casein and beta-casein. The beneficial effects of adding up to 1% phosphoric acid to 2,5-DHB were also observed in LC-MALDI-MS analysis of tryptic phosphopeptides of B. subtilis PrkC phosphoprotein. Finally, the mass resolution of MALDI mass spectra of intact proteins was significantly improved by using phosphoric acid in 2,5-DHB matrix.     

GPC separation of polymer samples for MALDI analysis

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is an important technique to characterize the average molecular weights, oligomer repeat units, and end groups of polymer materials. Although MALDI methods have been developed for a broad variety of different synthetic polymers, MALDI continues to struggle with polymer samples having broad polydispersity (PD). We have combined MALDI and gel permeation chromatography (GPC) analyses for broad PD polymer samples with the use of a liquid chromatography (LC) interface. The LC interface uses heated sheath gas and a capillary nozzle to remove most of the mobile phase and deposit the GPC eluants on the precoated matrix on a moving MALDI plate. Our experiments demonstrate that the combination of GPC-LC interface-MALDI can aid in the characterization of broad PD samples, the verification of the presence of low-intensity, high-mass oligomers, and the detection of minor series in polymer samples.     

Coumarin tags for improved analysis of peptides by MALDI-TOF MS and MS/MS. 1. Enhancement in MALDI MS signal intensities

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF MS and MS/MS. The proposed tags, commercially available fluorescent derivatives of coumarin, can be advantageous for peptide analysis in both MS and MS/MS modes. This paper, part 1, will focus on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. Labeling peptides with tags containing the coumarin core was found to enhance the intensities of peptide peaks (in some cases over 40-fold) in MALDI-TOF MS using CHCA and 2,5-DHAP matrixes. The signal enhancement was found to be peptide- and matrix-dependent, being the most pronounced for hydrophilic peptides. No correlation was found between the UV absorptivity of the tags at the excitation wavelengths typical for UV-MALDI and the magnitude of the signal enhancement. Interestingly, peptides labeled with Alexa Fluor 350, a coumarin derivative containing a sulfo group (i.e., bearing strong negative charge), showed a 5-15-fold increase in intensity of MALDI MS signal in the positive ion mode, relative to the underivatized peptides, when 2,5-DHAP was used as the matrix. The Alexa Fluor 350 tag yielded a significantly higher signal relative to that for the CAF tag, likely due to the increased hydrophobicity of the coumarin structure. With 2,5-DHB, a decrease of MALDI MS signal was observed for all coumarin-labeled peptides, again relative to the unlabeled species. These findings support the hypothesis that derivatization with coumarin, a relatively hydrophobic structure, improves incorporation of hydrophilic peptides into hydrophobic MALDI matrixes, such as CHCA and 2,5-DHAP.     

Photoimmobilization of proteins for affinity capture combined with MALDI TOF MS analysis

Affinity capture surfaces can be prepared in a number of ways. A method of obtaining such surfaces through UV-activated immobilization of binding proteins using a benzophenone derivative is reported. Photoimmobilized protein G was used to selectively capture and preconcentrate bovine IgG from a mixture with BSA, and the affinity of photoattached concanavalin A toward ovalbumin was compared with that of commercially available concanavalin A on agarose beads. The results of the capture after tryptic digestion were analyzed by MALDI TOF MS. Immobilized trypsin was also prepared through photoimmobilization and later used to digest hemoglobin. Immobilized enzyme digestion resulted in more partial cleavages than solution-phase digestion. More methionine and tryptophan oxidation was also observed. Photoimmobilization was shown to be a quick and easy way of immobilizing ligands on surfaces.     

Oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for DNA analysis by hybridization

The highly specific molecular recognition properties of oligonucleotides are combined with the unique optical properties of gold nanoparticles for the development of a dry-reagent strip-type biosensor that enables visual detection of double stranded DNA within minutes. The assay does not require instrumentation and avoids the multiple incubation and washing steps performed in most current assays. Gold nanoparticle reporters with oligo(dT) attached to their surface form an integral part of the strip. Biotinylated PCR products (233 bp or 495 bp) are hybridized (5 min) with a poly(dA)-tailed oligo and applied on the strip, which is then immersed in the appropriate buffer. As the buffer migrates upward, it rehydrates the nanoparticles that are linked to the target DNA through poly(dA)/(dT) hybridization. Capture of the hybrids by immobilized streptavidin in the test zone of the strip generates a characteristic red band. A second red band is formed, by hybridization, in the control zone of the strip to indicate proper test performance. The sensor offers at least 8 times higher detectability than ethidium bromide staining of agarose gels and provides confirmation of the amplified fragments. Quantitative data are obtained by densitometric analysis of the bands. As low as 2 fmol of amplified DNA were detectable by the strip sensor. Also, 500 copies of prostate-specific antigen cDNA were detected by combining PCR and the strip sensor. The sensor was used successfully for detection of hepatitis C virus in plasma samples from 20 patients. The strip detected 16 out of 16 positive samples and gave no signal for 4 samples that were negative for the virus. To our knowledge, this is the first dry-reagent system that makes use of oligonucleotide-conjugated gold nanoparticles as probes.     

Single-neuron analysis using CE combined with MALDI MS and radionuclide detection

Capillary electrophoresis (CE) has been combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and radionuclide detection to assay mass-limited biological samples. Nanovial sampling techniques enable injections into the CE capillary from 50 to 150-nL volume samples; after the separation, nanoliter fraction collection combines the CE effluent with a MALDI matrix and minimizes sample spreading, thus allowing both MALDI MS and radionuclide detection on the CE fractions. MALDI MS complements the elution time information of CE by providing accurate molecular mass data, and radionuclide detection provides zeptomole limits of detection with quantitative information. While MALDI MS detects all fully processed peptides at sufficient concentration, culturing the neuron in media containing 35S-Met provides selective radionuclide detection of newly synthesized methionine-containing peptides. The analysis and detection of the expected neuropeptides and hormones in a single 40-microm bag cell neuron from Aplysia californica with CE/MALDI MS/radionuclide detection demonstrates the ability of this hyphenated approach to work with chemically complex mass-limited samples.     

A general method for producing bioaffinity MALDI probes.

A bioaffinity probe based on the idea of immobilizing avidin on the probe surface to extract biotinylated oligosaccharide is described. The probe is produced by taking advantage of the natural affinity of proteins for hydrophobic polymer films. The avidin is immobilized by simply drying the solution on a polymer film surface. This produces a bioaffinity probe that shows enhanced activity for biotin-labeled oligosaccharides. The probe is produced in a matter of minutes but is highly effective for concentrating biotinylated oligosaccharide on the surface. The best matrix for the analysis is DHB, and the best film for the probe is a polyester material commonly used for transparency film. The efficacy of the probe is illustrated with neutral and anionic oligosaccharides. Oligosaccharides derivatized with biotin are retained while those that are unlabeled are washed away. No trace of the unlabeled oligosaccharide is observed in the mass spectrum.     

Heterogeneity within MALDI samples as revealed by mass spectrometric imaging

While matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has revolutionized the manner by which many large molecules are characterized, the highly variable appearance of MALDI mass spectra remains a concern. We have developed MALDI-based imaging as a diagnostic tool for examining the relationships between preparation strategy, sample morphology, and spectral quality. The imaging protocol involves the automated acquisition of mass spectra at 400-1600 positions within a single sample, followed by off-line processing and image display. Several sample types have been characterized, including a simple peptide mixture prepared in dried droplets of 2,5-dihydroxybenzoic acid and in thin films of alpha-cyano-4-hydroxycinnamic acid as well as a complex biological sample consisting of intact peptidergic neurons from the marine mollusk Aplysia californica. Imaging experiments provide a wealth of unbiased information concerning sample defects, spectral reproducibility, mass accuracy, differential analyte distributions, and the validity of internal standards.     

Tetraalkylphosphonium-based ionic liquids for a single-step dye extraction/MALDI MS analysis platform

Room temperature ionic liquids, or RTILs, based on tetraalkylphosphonium (PR(4)(+)) cations were used as the basis of a platform that enables separation of dyes from textiles, extraction of dyes from aqueous solution, and identification of the dyes by MALDI-MS in a single experimental step for forensic purposes. Ionic liquids were formed with PR(4)(+) cations and ferulate (FA), α-cyano-4-hydroxycinnamate (CHCA), and 2,5-dihydroxybenzoate (DHB) anions. The use of tetraalkylphosphonium-based ionic liquids in MALDI-MS allowed detection of small molecule dyes without addition of a traditional solid MALDI matrix.     

Phantom nanoparticles as probes of biomolecular interactions

A new light-scattering-based method to detect molecular interactions at the surface of low-refractive-index nanoparticles was recently proposed. Water-dispersed nanoparticles functionalized with receptors typical of immature bacteria cell walls were used to study the activity of the antibiotic vancomycin. This method subtly depends on the specific properties of the nanoparticles. Here we discuss, by comparative experiments and through theoretical evaluation, the effects of size, refractive index, electric charge, and dilution on the reliability and accuracy of the method. Quite surprisingly, perfect index matching and minimal size (i.e., maximum surface), which is almost attained in one of the colloids here employed, do not represent the ideal conditions. Rather, we show that a nanoparticle radius of 100 nm and a refractive index slightly below that of water yields the best signal/background amplitude. We also show that repulsive interactions can lead to artifacts in the adsorption isotherm, thus indicating that electrostatic stabilization should be kept at a minimum. The close agreement between the interaction strengths, as measured with two different nanoparticle systems, testifies to the reliability of the method.     

Using gold nanoclusters as selective luminescent probes for phosphate-containing metabolites

Glutathione-bound gold nanoclusters (AuNCs@GSH) can emit reddish photoluminescence under illumination of ultraviolet light. The luminescence of the AuNCs@GSH is quenched when chelating with iron ions (AuNCs@GSH-Fe(3+)), presumably resulting from the effective electron transfer between the nanoclusters and iron ions. Nevertheless, we found that the luminescence of the gold nanoclusters can be restored in the presence of phosphate-containing molecules, which suggested the possibility of using AuNCs@GSH-Fe(3+) complexes as the selective luminescent switches for phosphate-containing metabolites. Phosphate-containing metabolites such as adenosine-5'-triphosphate (ATP) and pyrophosphate play an important role in biological systems. In this study, we demonstrated that the luminescence of the AuNCs@GSH-Fe(3+) is switched-on when mixing with ATP and pyrophosphate, which can readily be observed by the naked eye. It results from the high formation constants between phosphates and iron ions. When employing fluorescence spectroscopy as the detection tool, quantitative analysis for phosphate-containing metabolites such as ATP and pyrophosphate can be conducted. The linear range for ATP and pyrophosphate is 50 μM to sub-millimolar, while the limit of detection for ATP and pyrophosphate are ～43 and ～28 μM, respectively. Additionally, we demonstrated that the luminescence of the AuNCs@GSH-Fe(3+) can also be turned on in the presence of phosphate-containing metabolites from cell lysates and blood plasma.     

Disposable hydrophobic surface on MALDI targets for enhancing MS and MS/MS data of peptides

Automated analyses in MALDI MS are complicated by the uneven distribution of analyte over the sample spot, resulting in areas of analyte localization, or "sweet spots". Hence, the ability to concentrate and localize samples is advantageous, especially for automated studies involving low concentrations of analyte. A method for rapidly creating a removable and affordable hydrophobic surface that is free from memory effect is presented. The potential for such compounds to serve as a practical coating for MALDI targets is examined. An example compound with a complete methodology is shown to increase sample homogeneity, peak intensity, and resolution when used for peptide mixtures with CHCA and DHB.     

Complementary use of MALDI and ESI for the HPLC-MS/MS analysis of DNA-binding proteins

Proteins from Escherichia coli were isolated based on their ability to bind DNA and digested in-solution with trypsin; the resulting peptides were separated using HPLC and subsequently analyzed using MALDI TOF/TOF and ESI Q-TOF instruments. Various properties of the peptides observed with the two ionization techniques were compared taking into account the differences between the mass analyzers. This empirical analysis of a data set containing hundreds of peptides and thousands of individual amino acids supports some of the currently held notions regarding the complementary nature of the two ionization processes. Specifically, ESI tends to favor the identification of hydrophobic peptides whereas MALDI tends to lead to the identification of basic and aromatic species. Findings from the present study suggest that ESI and MALDI may be complementary due to the biases of the two ionization techniques for certain classes of amino acids. From a practical standpoint, these biases indicate that, for the present at least, analyses must be performed on both types of instruments in order to gain the most information possible out of a given set of samples in a proteomics study.     

Retrograde labeling of single neurons in conjunction with MALDI high-energy collision-induced dissociation MS/MS analysis for peptide profiling and structural characterization

To reveal the peptide contents of the visually nonidentifiable neurons from a neuronal circuit of interest, we combined retrograde labeling of neurons with mass spectrometric single cell analysis. We used the neuronal circuit involved in the copulation behavior of a freshwater snail, Lymnaea stagnalis, as a model. Central neurons that control this behavior are known to send their axons to the penis nerve and innervate the penis complex. By retrograde filling from the penis nerve with nickel-lysine, these neurons were selectively labeled darkish blue. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric analyses of single stained neurons in the parietal ganglion from different animals reveal consistently the presence of several molecular ion species in the range of 800-1200 Da. From a single neuron, six molecular ion species were further characterized with MALDI time-of-flight/time-of-flight mass spectrometry, which demonstrates that the peptides are derived from a previously reported -FLRFamide precursor.     

Luminescent CdS quantum dots as selective ion probes

Water-soluble luminescent CdS quantum dots (QDs) capped by polyphosphate, L-cysteine, and thioglycerol were synthesized in aqueous solution. The ligands were found to have a profound effect on the luminesence response of CdS QDs to physiologically important metal cations. Polyphosphate-capped CdS QDs were sensitive to nearly all mono- and divalent cations, showing no ion selectivity. Conversely, thioglycerol-capped CdS QDs were sensitive to only copper and iron ions. Similar concentrations of physiologically relevant cations, such as zinc, sodium, potassium, calcium, and magnesium ions did not affect the luminescence of thioglycerol-capped CdS QDs. On the other hand, L-cysteine-capped CdS QDs were sensitive to zinc ions and insensitive to other physiologically important cations, such as copper, calcium, and magnium ions. To demonstrate the detection capability of these new ion probes, L-cysteine and thioglycerol-capped CdS QDs were used to detect zinc and copper ions in physiological buffer samples. The detection limits were 0.8 microM for zinc (II) and 0.1 microM for copper (II) ions. The emission enhancement of the QDs by zinc (II) is attributed to activation of surface states, whereas the effective reduction of copper (II) to copper (I) may explain the emission decrease of the thioglycerol-capped CdS QDs when charged with copper ions. Unlike organic fluorescent dyes, the thioglycerol-capped luminescent CdS QDs discriminate between copper and zinc ions and are therefore suitable for the analysis of copper ions in biological samples in the presence of physiological concentrations of zinc ions. The interference of iron ions with zinc and copper ion detection is attributed to an inner filter effect, which is eliminated by adding fluoride ions to form the colorless complex FeF6(3-). To the best of our knowledge, this is first use of luminescent semiconductor quantum dots as selective ion probes in aqueous samples.     

Gold and silica-coated gold nanoparticles as thermographic labels for DNA detection

The infrared emissivity of Au and silica-coated Au nanoparticles (Au NPs) deposited on indium tin oxide substrates was investigated. NPs were irradiated with laser light at a frequency close to the Au plasmon resonance band, and the blackbody radiation emitted as a result was monitored with an IR camera equipped with an InAs array detector. The differences in temperature before and after laser irradiation were recorded (T-jumps) and were found to be directly proportional to the number of particles present on the slide and to the laser power used in the experiment. Coating Au NPs with silica increased the measured T-jumps 2-5 times, depending on the thickness of the silica shell. This was in agreement with the observation that silica has a much higher IR emissivity than Au. Both Au and silica-coated Au NPs were then tested as labels for thermographic DNA detection. Target DNA concentrations as low as 100 pM were recorded when Au NPs were used as labels and as low as 10 pM when silica-coated Au NPs were used.