Negative mode nanostructure-initiator mass spectrometry for detection of phosphorylated metabolites

The chemical complexity of the metabolome requires the development of new detection methods to enlarge the range of compounds detectable in a biological sample. Recently, a novel matrix-free laser desorption/ionization method called nanostructure-initiator mass spectrometry (NIMS) [Northen et al., Nature 449(7165):1033–1036, 2007] was reported. Here we investigate NIMS in negative ion mode for the detection of endogenous metabolites, namely small phosphorylated molecules. 3-Aminopropyldimethylethoxysilane was found to be suitable as initiator for the analytes studied and a limit of detection in the tens of femtomoles was reached. The detection of different endogenous cell metabolites in a yeast cell extract is demonstrated.     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the discrimination of food-borne microorganisms

A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify Escherichia coli O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.     

Rapid mass spectrometric analysis for ascorbate and related organic acids in small volumes of plasma for use in pediatric subjects

A gas chromatographic–mass spectrometric isotope dilution method was developed for analysis of ascorbate on 10 μl samples of plasma. This assay was reproducible (standard deviation of less than 4%) and gave values for plasma ascorbate content within 8% of our previously published gas chromatographic–mass spectrometric method. Non-specific sample preparation allowed other analytes to be determined on the same sample by adjusting data acquisition parameters and adding the appropriate internal standard. Analysis on 28 subjects fell within the expected range for plasma ascorbate 68±29 μm (11.9±5.0 μg/ml) and established a normal range for plasma threonate of 28.1±2.4 μm (3.8±0.4 μg/ml).     

Integration of surface plasmon resonance with mass spectrometry: automated ligand fishing and sample preparation for MALDI MS using a Biacore 3000 biosensor.

Integrating surface plasmon resonance analysis with mass spectrometry allows detection and characterization of molecular interactions to be complemented with identification of interaction partners. We have developed a procedure for Biacore 3000 that automatically performs all steps from ligand fishing and recovery to sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry including on-target digestion. In the model system used in this study a signal transduction protein, calmodulin, was selectively captured from brain extract by one of its interaction partners immobilized on a sensor chip. The bound material was eluted, deposited directly onto a MALDI target, and analyzed by mass spectrometry both as an intact protein and after on-target tryptic digestion. The procedure with direct deposition of recovered material on the MALDI target reduces sample losses and, in combination with automatic sample processing, increases the throughput of surface plasmon resonance mass spectrometry analysis.     

Microbead device for isolating biotinylated oligonucleotides for use in mass spectrometric analysis

We describe a prototypical device for isolating biotinylated oligonucleotides for use in mass spectrometric analysis. It consists of monomeric avidin-coated microbeads trapped in a pipette tip and has been used for genotyping single nucleotide polymorphisms (SNPs) with the previously developed solid phase capture-single base extension (SPC-SBE) method. The device reduces processing time for genotyping by SPC-SBE and allows direct spotting of sample for rapid analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, it allows simultaneous processing of multiple samples and can be reused after regeneration of beads with no carryover effects. These results indicate that the microbead device is a low-cost tool that enhances sample cleanup prior to MS for SNP genotyping.     

Determination of Tirofiban in human serum by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method with a rapid and simple sample preparation was developed and validated for the determination of Tirofiban in biological fluids. Tirofiban in serum samples was extracted and cleaned up by using an automated solid phase extraction method. An external calibration was used. The mass spectrometer was operated in the multiple reaction monitoring mode (MRM). A good linear response over the range of 2-200ng/ml was demonstrated. The accuracy for Tirofiban ranged from 94.8 to 110.8% within-day and from 103.0 to 104.7% between-day. The lower limit of quantification was 2ng/ml. This method is suitable for pharmacokinetic studies.     

Liquid chromatographic–tandem mass spectrometric method for the determination of the neuraminidase inhibitor zanamivir (GG167) in human serum

An LC–MS–MS method for the analysis of the neuraminidase inhibitor, zanamivir, in human serum is described. Zanamivir was extracted from protein precipitated human serum samples using Isolute SCX solid-phase extraction cartridges and analysed using reversed-phase chromatography with TurboIonSpray atmospheric pressure ionisation followed by mass spectrometric detection. The method uses a stable isotope internal standard, is highly specific and sensitive for a compound of this type and has been used for the analysis of human serum and urine samples from clinical studies. The method was extended to the analysis of serum and plasma samples from pre-clinical studies involving the rat, ferret and cell culture media. The method has been shown to be robust and valid over a concentration range of 10–5000 ng/ml using a 0.2-ml sample volume. The main advantages of this method compared to earlier procedures are primarily specificity, sensitivity, ease of sample preparation, small sample volume and short analysis time (ca. 5 min).     

Liquid ultraviolet matrix-assisted laser desorption/ionization -- mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet -- matrix-assisted laser desorption/ionisation -- mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. The low-femtomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydroxybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and low-mass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.     

Modular mass spectrometric tool for analysis of composition and phosphorylation of protein complexes

The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC) and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples.     

Characterizing peptides in individual mammalian cells using mass spectrometry

Cell-to-cell chemical signaling plays multiple roles in coordinating the activity of the functional elements of an organism, with these elements ranging from a three-neuron reflex circuit to the entire animal. In recent years, single-cell mass spectrometry (MS) has enabled the discovery of cell-to-cell signaling molecules from the nervous system of a number of invertebrates. We describe a protocol for analyzing individual cells from rat pituitary using matrix-assisted laser desorption/ionization MS. Each step in the sample preparation process, including cell stabilization, isolation, sample preparation, signal acquisition and data interpretation, is detailed here. Although we employ this method to investigate peptides in individual pituitary cells, it can be adapted to other cell types and even subcellular sections from a range of animals. This protocol allows one to obtain 20-30 individual cell samples and acquire mass spectra from them in a single day.     

Proteomic analysis of novel marine bacteria using MALDI and ESI mass spectrometry.

The objective of this study was to develop a mass spectrometric protocol to search for proteins related to phototrophy in marine bacteria. The genes that produce proteins involved in conversion of light into energy have been detected by cloning-sequencing from some of these bacteria, but it was previously unknown if these proteins were actually expressed. Attaining this study's goal was complicated by the fact that the samples consisted of miniscule cell pellets, which yielded small amounts of very complex mixtures of proteins. Sample preparation and analysis were tailored to optimize the probability of detecting the proteins of interest. It has been reported that using both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) to analyze a mixture of peptides leads to the identification of more peptides that either technique alone. In order to exploit this complementarity between ESI and MALDI for proteomic analysis, samples were analyzed using both ionization techniques. With correct choices in sample preparation and ionization process, biologically relevant proteins can be identified out of small samples containing whole proteomes.     

Selective metabolite and peptide capture/mass detection using fluorous affinity tags

A new and general methodology is described for the targeted enrichment and subsequent direct mass spectrometric characterization of sample subsets bearing various chemical functionalities from highly complex mixtures of biological origin. Specifically, sample components containing a chemical moiety of interest are first selectively labeled with perfluoroalkyl groups, and the entire sample is then applied to a perfluoroalkyl-silylated porous silicon (pSi) surface. Due to the unique hydrophobic and lipophobic nature of the perfluorinated tags, unlabeled sample components are readily removed using simple surface washes, and the enriched sample fraction can then directly be analyzed by desorption/ionization on silicon mass spectrometry (DIOS-MS). Importantly, this fluorous-based enrichment methodology provides a single platform that is equally applicable to both peptide as well as small molecule focused applications. The utility of this technique is demonstrated by the enrichment and mass spectrometric analysis of both various peptide subsets from protein digests as well as amino acids from serum.     

Purification of tryptic peptides for mass spectrometry using polyvinylidene fluoride membrane.

A simple procedure for the purification of tryptic peptides, prior to mass spectrometric analysis, using polyvinylidene fluoride membrane (PVDF) is described. The sensitivity of mass spectrometric analysis is such that minor impurities in tryptic peptide digests suppress the signal obtained. However, we obtained useful signal, from a sample that did not yield any spectra earlier, by purifying the sample using PVDF membrane. For this, the tryptic peptide digest was first spotted on the membrane which was then air-dried and washed. Further, the membrane was extracted with trifluoroacetic acid (TFA) and acetonitrile and subjected to mass spectrometric analysis. This procedure enabled us to identify a cross-reactive D1 antigen on the neutrophil surface that bound antibodies that targeted 60 kD Ro autoantigen in systemic lupus erythematosus, an autoimmune disorder.     

The application of hybrid mass spectrometry/mass spectrometry and high-resolution mass spectrometry to the analysis of fish samples for polychlorinated dibenzo-p-dioxins and dibenzofurans

A hybrid mass spectrometer operated in low-resolution selected decomposition monitoring (SDM) mode was used for the analysis of whole fish samples for the 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and poly-chlorinated dibenzofurans (PCDD/Fs). These fish samples were previously analyzed for PCDD/Fs using a high-resolution mass spectrometer following EPA protocols. The hybrid tandem mass spectrometric method using loss of COCl gave similar quantification results to those obtained by high-resolution mass spectrometry and eliminated the interferences attributed to polychlorinated biphenyls that were encountered in the high-resolution mass spectrometric analysis. Comparison of the two methods shows that the high-resolution mass spectrometric method surpasses the tandem mass spectrometric method in other analytical figures of merit, such as detection limit, linearity and reproducibility.     

MALDI imaging of lipids after matrix sublimation/deposition

Mass spectrometric techniques have been developed to record mass spectra of biomolecules including lipids as they naturally exist within tissues and thereby permit the generation of images displaying the distribution of specific lipids in tissues, organs, and intact animals. These techniques are based on matrix-assisted laser desorption/ionization (MALDI) that requires matrix application onto the tissue surface prior to analysis. One technique of application that has recently shown significant advantages for lipid analysis is sublimation of matrix followed by vapor deposition directly onto the tissue. Explanations for enhanced sensitivity realized by sublimation/deposition related to sample temperature after a laser pulse and matrix crystal size are presented. Specific examples of sublimation/deposition in lipid imaging of various organs including brain, ocular tissue, and kidney are presented.     

Accurate determination of the molecular weight of the major surface layer protein isolated from Clostridium thermosaccharolyticum by time-of-flight mass spectrometry.

Matrix-assisted laser desorption with concomitant ionization, in combination with a linear time-of-flight mass spectrometer, was used to analyze underivatized and hard-to-solubilize surface layer proteins and glycoproteins by depositing them on top of a microcrystalline layer of the matrix alpha-cyano-4-hydroxycinnamic acid. Use of this special sample preparation technique allowed the first successful desorption-ionization of intact surface layer proteins and accurate determination of their molecular weights by mass spectrometry. The molecular mass of the monomeric subunit of the major surface layer protein isolated from Clostridium thermosaccharolyticum E207-71 was determined to be 75,621 +/- 81 Da. The obtainable mass accuracy of the technique is conservatively considered to be within +/- 0.2%. This result deviates from that given by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by approximately 7.4 kDa because this method is strongly affected and biased by the three-dimensional structure of this type of surface protein. With the apparent advantages of unsurpassed mass accuracy, low dependence on the physicochemical properties of the surface layer proteins, and high sensitivity, it can be concluded that a linear time-of-flight instrument combined with UV matrix-assisted laser desorption with concomitant ionization is better suited for molecular weight determination than is gel electrophoresis.     

Antigenic surveillance of the influenza virus by mass spectrometry

The use of a mass spectrometric-based immunoassay to survey the antigenic identity of a type A influenza strain is described. Antigenic surveillance of the influenza virus remains a critical step in the identification of new viral strains and the subsequent use of such strains or synthetic constructs in vaccine preparations. The immunoassay is shown to be capable of detecting a determinant of a single hemagglutinin antigen, responsible for the initial stages of infection, in a mixture of viral proteins with high sensitivity and specificity. The determinant comprises residues 207-225 of the hemagglutinin HA1 chain of the type A strain which occupies a highly accessible region of beta-sheet atop the antigen. Peptide determinants are identified without the need to immobilize antibody or isolate antibody-peptide complexes which are shown to be preserved during mass spectrometric analysis. This immunoassay achieves optimal sensitivity (femtomole level) with minimal sample handling and is amenable to high sample throughput and automation. Determinants are identified by a direct comparison of the matrix-assisted laser desorption ionization mass spectra obtained for an antibody reaction mixture and unreacted control. The sequence and antigenic identity of a component antigen can be rapidly identified by searching protein databases with the mass spectral data in conjunction with tandem mass spectrometric sequencing.     

Determination of cefaclor in human plasma by a sensitive and specific liquid chromatographic-tandem mass spectrometric method

A sensitive and specific liquid chromatographic-tandem mass spectrometric method is described for the determination of cefaclor in human plasma. The plasma samples were treated by two sample preparation procedures, i.e. protein precipitation (PPT) and solid-phase extraction (SPE). The pretreated samples were analyzed on a C(18) HPLC column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization (ESI) was employed as the ionization source. The analyte and internal standard ampicillin (for PPT) or cefetamet (for SPE) were detected by use of selected reaction monitoring (SRM) mode. The lower limit of quantitation obtained as a result of the PPT procedure was 100 ng/ml. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 12% for cefaclor. The accuracy as determined from QC samples was within +/-3% for the analyte. The SPE procedure could provide the lower limit of quantitation of 2 ng/ml. The precision and accuracy were measured to be below 7.1% and between -3.6% and 1.1%, respectively, for all QC samples. The method was applied for the evaluation of the pharmacokinetic profiles of cefaclor sustained-release formulation.     

Combined experimental and statistical strategy for mass spectrometry based serum protein profiling for diagnosis of breast cancer: a case-control study

Serum protein profiling by mass spectrometry is a promising method for early detection of cancer. We have implemented a combined strategy based on matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and statistical data analysis for serum protein profiling and applied it in a well-described breast cancer case-control study. A rigorous sample collection protocol ensured high quality specimen and reduced bias from preanalytical factors. Preoperative serum samples obtained from 48 breast cancer patients and 28 controls were used to generate MALDI MS protein profiles. A total of nine mass spectrometric protein profiles were obtained for each serum sample. A total of 533 common peaks were defined and represented a 'reference protein profile'. Among these 533 common peaks, we identified 72 peaks exhibiting statistically significant intensity differences ( p < 0.01) between cases and controls. A diagnostic rule based on these 72 mass values was constructed and exhibited a cross-validated sensitivity and specificity of approximately 85% for the detection of breast cancer. With this method, it was possible to distinguish early stage cancers from controls without major loss of sensitivity and specificity. We conclude that optimized serum sample handling and mass spectrometry data acquisition strategies in combination with statistical analysis provide a viable platform for serum protein profiling in cancer diagnosis.     

Determination of scopolamine in human serum by gas chromatography-ion trap tandem mass spectrometry

The objective of this study was to develop a very sensitive and selective method for the determination of scopolamine in serum with a rapid and simple sample preparation. A capillary column gas chromatographic-ion trap tandem mass spectrometric technique has been applied. Scopolamine and the internal standard mexiletine were extracted from serum samples and cleaned up by using a single step liquid-liquid extraction. Derivatization was carried out using 2,2,2-trifluoro-N-methyl-N-trimethylsilylacetamide. The mass spectrometer was operated with positive ions in the selected reaction mode with chemical ionisation using methane. The sum of peak height of two daughter ions was used for quantification. The detection limit was 50 pg/ml in serum.