Mass Spectrometric Data Acquisition Research Articles

Targeted Data Acquisition for Improved Reproducibility and Robustness of Proteomic Mass Spectrometry Assays

Quantitative mass spectrometry-based proteomic assays often suffer from a lack of robustness and reproducibility. We here describe a targeted mass spectrometric data acquisition strategy for affinity enriched subproteomes-in our case the kinome-that enables a substantially improved reproducibility of detection, and improved quantification via isobaric tags. Inclusion mass lists containing m/z, charge state, and retention time were created based on a set of 80 shotgun-type experiments performed under identical experimental conditions. For each target protein, peptides were selected according to their frequency of observation and isobaric tag for relative and absolute quantitation (iTRAQ) reporter ion quality. Retention times of selected peptides were aligned using similarity driven pairwise alignment strategy yielding <1 min standard deviation for 4 h gradients. Multiple fragmentation of the same peptides resulted in better statistics and more precise reporter ion based quantification without any loss in coverage. Overall, 24% more target proteins were quantified using the targeted data acquisition approach, and precision of quantification improved by >1.5-fold. We also show that a combination of higher energy collisional dissociation (HCD) with collisional induced dissociation (CID) outperformed pulsed-Q-dissociation (PQD) on the OrbitrapXL. With the CID/HCD based targeted data acquisition approach 10% more quantifiable target proteins were identified and a 2-fold increase in quantification precision was achieved. We have observed excellent reproducibility between different instruments, underlining the robustness of the approach.

Bioimaging of metals in brain tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and metallomics

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been developed and established as an emerging technique in the generation of quantitative images of metal distributions in thin tissue sections of brain samples (such as human, rat and mouse brain), with applications in research related to neurodegenerative disorders. A new analytical protocol is described which includes sample preparation by cryo-cutting of thin tissue sections and matrix-matched laboratory standards, mass spectrometric measurements, data acquisition, and quantitative analysis. Specific examples of the bioimaging of metal distributions in normal rodent brains are provided. Differences to the normal were assessed in a Parkinson's disease and a stroke brain model. Furthermore, changes during normal aging were studied. Powerful analytical techniques are also required for the determination and characterization of metal-containing proteins within a large pool of proteins, e.g., after denaturing or non-denaturing electrophoretic separation of proteins in one-dimensional and two-dimensional gels. LA-ICP-MS can be employed to detect metalloproteins in protein bands or spots separated after gel electrophoresis. MALDI-MS can then be used to identify specific metal-containing proteins in these bands or spots. The combination of these techniques is described in the second section.

Nanostructure-initiator mass spectrometry: a protocol for preparing and applying NIMS surfaces for high-sensitivity mass analysis

Nanostructure-initiator mass spectrometry (NIMS) is a new surface-based MS technique that uses a nanostructured surface to trap liquid ('initiator') compounds. Analyte materials adsorbed onto this 'clathrate' surface are subsequently released by laser irradiation for mass analysis. In this protocol, we describe the preparation of NIMS surfaces capable of producing low background and high-sensitivity mass spectrometric measurement using the initiator compound BisF17. Examples of analytes that adsorb to this surface are small molecules, drugs, lipids, carbohydrates and peptides. Typically, NIMS is used to analyze samples ranging from simple analytical standards and proteolytic digests to more complex samples such as tissues, cells and biofluids. Critical experimental considerations of NIMS are described. Specifically, NIMS sensitivity is examined as a function of pre-etch cleaning treatment, etching current density, etching time, initiator composition, sample concentration, sample deposition method and laser fluence. Typically, NIMS surface preparation can be completed in less than 2 h. Subsequent sample preparation requires 1-5 min, depending on sample deposition method. Mass spectrometric data acquisition typically takes 1-30 s per sample.

Quantitative determination of ondansetron in human plasma by enantioselective liquid chromatography-tandem mass spectrometry

A sensitive and enantioselective method was developed and validated for the determination of ondansetron enantiomers in human plasma using enantioselective liquid chromatography-tandem mass spectrometry. The enantiomers of ondansetron were extracted from plasma using ethyl acetate under alkaline conditions. HPLC separation was performed on an ovomucoid column using an isocratic mobile phase of methanol–5 mM ammonium acetate–acetic acid (20:80:0.02, v/v/v) at a flow rate of 0.40 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/ z 294 → 170 for ondansetron enantiomers, and m/ z 285 → 124 for tropisetron (internal standard). The method was linear in the concentration range of 0.10–40 ng/mL for each enantiomer using 200 μL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.10 ng/mL. The intra- and inter-assay precision was 3.7–11.6% and 5.6–12.3% for R-(−)-ondansetron and S-(+)-ondansetron, respectively. The accuracy was 100.4–107.1% for R-(−)-ondansetron and 103.3–104.9% for S-(+)-ondansetron. No chiral inversion was observed during the plasma storage, preparation and analysis. The method was successfully applied to characterize the pharmacokinetic profiles of ondansetron enantiomers in healthy volunteers after an intravenous infusion of 8 mg racemic ondansetron.

Enantioselective determination of azelnidipine in human plasma using liquid chromatography–tandem mass spectrometry

A sensitive and simple method was developed for determination of the enantiomers of azelnidipine, ( R)-(−)-azelnidipine and ( S)-(+)-azelnidipine, in human plasma using chiral liquid chromatography with positive ion atmospheric pressure chemical ionization tandem mass spectrometry. Plasma samples spiked with stable isotope-labeled azelnidipine, [ 2H 6]-azelnidipine, as an internal standard, were processed for analysis using a solid-phase extraction in a 96-well plate format. The azelnidipine enantiomers were separated on a chiral column containing α 1-acid glycoprotein as a chiral selector under isocratic mobile phase conditions. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, monitoring the transitions from m/ z 583 → 167 for ( R)-(−)-azelnidipine and ( S)-(+)-azelnidipine, and from m/ z 589 → 167 for [ 2H 6]-azelnidipine. The standard curve was linear over the studied range (0.05–20 ng/mL), with r 2 > 0.997 using weighted (1/ x 2) quadratic regression, and the chromatographic run time was 5.0 min/injection. The intra- and inter-assay precision (coefficient of variation), calculated from the assay data of the quality control samples, was 1.2–8.2% and 2.4–5.8% for ( R)-(−)-azelnidipine and ( S)-(+)-azelnidipine, respectively. The accuracy was 101.2–117.0% for ( R)-(−)-azelnidipine and 100.0–107.0% for ( S)-(+)-azelnidipine. The overall recoveries for ( R)-(−)-azelnidipine and ( S)-(+)-azelnidipine were 71.4–79.7% and 71.7–84.2%, respectively. The lower limit of quantification for both enantiomers was 0.05 ng/mL using 1.0 mL of plasma. All the analytes showed acceptable short-term, long-term, auto-sampler and stock solution stability. Furthermore, the method described above was used to separately measure the concentrations of the azelnidipine enantiomers in plasma samples collected from healthy subjects who had received a single oral dose of 16 mg of azelnidipine.

An integrated chemical, mass spectrometric and computational strategy for (quantitative) phosphoproteomics: application to Drosophila melanogaster Kc167 cells

Current methods for phosphoproteome analysis have several limitations. First, most methods for phosphopeptide enrichment lack the specificity to truly purify phosphopeptides. Second, fragmentation spectra of phosphopeptides, in particular those of phosphoserine and phosphothreonine containing peptides, are often dominated by the loss of the phosphate group(s) and therefore lack the information required to identify the peptide sequence and the site of phosphorylation, and third, sequence database search engines and statistical models for data validation are not optimized for the specific fragmentation properties of phosphorylated peptides. Consequently, phosphoproteomic data are characterized by large and unknown rates of false positive and false negative phosphorylation sites. Here we present an integrated chemical, mass spectrometric and computational strategy to improve the efficiency, specificity and confidence in the identification of phosphopeptides and their site(s) of phosphorylation. Phosphopeptides were isolated with high specificity through a simple derivatization procedure based on phosphoramidate chemistry. Identification of phosphopeptides, their site(s) of phosphorylation and the corresponding phosphoproteins was achieved by the optimization of the mass spectrometric data acquisition procedure, the computational tools for database searching and the data post processing. The strategy was applied to the mapping of phosphorylation sites of a purified transcription factor, dFOXO and for the global analysis of protein phosphorylation of Drosophila melanogaster Kc167 cells.

Detection of non-covalent protein interactions by 'intensity fading' MALDI-TOF mass spectrometry: applications to proteases and protease inhibitors

Among the main objectives of biomedical and proteomic research is to identify non-covalent interactions involving proteins. Here we provide a detailed protocol to apply matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry for such a purpose using proteases and protease inhibitors in complex biological samples. Our methodology is based on monitoring the reduction in intensity of inhibitors' mass spectrometric signals when their protease target is added to the MALDI sample. The versatility of the protocol permits the target to be added in a soluble form (direct protocol) or immobilized form (indirect protocol). The 'intensity fading' phenomenon is greatly favored when the binding assay is carried out in the sub-micromolar range and the interacting partners occur in mixtures of non-binding compounds. This protocol can be completed in 10 h, taking 20 or 30 min per sample to perform the mass spectrometric data acquisition, depending on whether a soluble or an immobilized target is used.

Proteomic applications of protein quantification by isotope-dilution mass spectrometry

Over the decades, isotope-dilution mass spectrometry (IDMS) has been implemented extensively for accurate quantification of drugs, metabolites and peptides in body fluids and tissues. More recently, it has been extended for quantifying specific proteins in complex mixtures. In this extended methodology, proteins are subjected to endoprotease action and specific resultant peptides are quantified by using synthetic stable isotope-labeled standard (SIS) peptides and IDMS. This article outlines the utilities and applications of quantifying proteins by IDMS, emphasizing its complementary value to global survey-based proteomic studies. The potential of SIS peptides to provide quantitative insights into cell signaling is also highlighted, with specific examples. Finally, we propose several novel mass spectrometric data acquisition strategies for large-scale applications of IDMS and SIS peptides in systems biology and protein biomarker validation studies.

On-site determination of nerve and mustard gases using a field-portable gas chromatograph-mass spectrometer

A field-portable gas chromatograph-mass spectrometer (the Hapsite system) was evaluated for onsite determination of nerve gases and blistering agents. The analytical sequence involved gas sampling for 1 min, microtrap concentration (1 min), apolar capillary gas chromatographic separation (10 min), electron ionization, quadrupole mass spectrometric data acquisition, and personal computer-based data analysis. Vapor containing 1mg/m3 sarin (GB), 1mg/m3 soman (GD), 3mg/m3 tabun (GA), and 0.5mg/m3 mustard gas (HD) was analyzed using the Hapsite system. The chemical warfare agents (CWAs) were detected within 10 min, and identified from their respective mass spectra according to the built-in NIST library database. Being based on the signal levels of the CWA peaks observed in mass chromatograms for m/z 99 (GB), 126 (GD), 70 (GA), and 109 (HD), the detection limits (S/N = 3) were estimated to be 0.2, 0.5, 8, and 0.3μg/m3, respectively. A carryover phenomenon was observed after injecting actual CWA samples, necessitating several blank-air purges of the system. The extent of adsorption was in the order: HD > GA > GD > GB.

Regulation of the Monomer-Dimer Equilibrium in Inducible Nitric-oxide Synthase by Nitric Oxide

The oxygenase domain of inducible nitric-oxide synthase exists as a functional tight homodimer in the presence of the substrate L-arginine and the cofactor tetrahydrobiopterin (H4B). In the absence of H4B, the enzyme is a mixture of monomer and loose dimer. We show that exposure of H4B-free enzyme to NO induces dissociation of the loose dimer into monomers in a reaction that follows single exponential decay kinetics with a lifetime of approximately 300 min. It is followed by a faster autoreduction reaction of the heme iron with a lifetime of approximately 30 min and the concurrent breakage of the proximal iron-thiolate bond, forming a five-coordinate NO-bound ferrous species. Mass spectrometry revealed that the NO-induced monomerization is associated with intramolecular disulfide bond formation between Cys104 and Cys109, located in the zinc-binding motif. The regulatory effect of NO as a dimer inhibitor is discussed in the context of the structure/function relationships of this enzyme.

Increasing Peptide Identification in Tandem Mass Spectrometry Through Automatic Function Switching Optimization

Comprehensive proteomic studies that employ MS directed peptide sequencing are limited by optimal peptide separation and MS and tandem MS data acquisition routines. To identify the optimal parameters for data acquisition, we developed a system that models the automatic function switching behavior of a mass spectrometer using an MS-only dataset. Simulations were conducted to characterize the number and the quality of simulated fragmentation as a function of the data acquisition routines and used to construct operating curves defining tandem mass spectra quality and the number of peptides fragmented. Results demonstrated that one could optimize for quality or quantity, with the number of peptides fragmented decreasing as quality increased. The predicted optimal operating curve indicated that significant improvements can be realized by selecting the appropriate data acquisition parameters. The simulation results were confirmed experimentally by testing 10 LC MS/MS data acquisition parameter sets on an LC-Q-TOF-MS. Database matching of the experimental fragmentation returned peptide scores consistent with the predictions of the model. The results of the simulations of mass spectrometer data acquisition routines reveal an inverse relationship between the quality and the quantity of peptide identifications and predict an optimal operating curve that can be used to select an optimal data acquisition parameter for a given (or any) sample.

Analysis of Peptide and Protein Samples Containing Surfactants by MALDI-MS

Protein samples containing ionic surfactants such as sodium dodecyl sulfate (SDS) can be analyzed by matrix-assisted laser desorption/ionization mass spectrometry, contrary to what is currently accepted. SDS at varying concentrations was added to four polypeptide samples. As expected, a decrease in the signal was observed for increasing concentrations of SDS up to 0.1% (w/v), at which no signal was observed for three of the analytes. However, a recovery of the signals of all the analytes was observed at concentrations of SDS above 0.3%, with useful spectra at concentrations as high as 10%. The SDS concentration at which signal recovery starts is always the same, regardless of the type and the concentration of the analyte. Another two surfactants 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and octylphenolpoly(ethylene glycol ether)10 (Triton X-100) were also tested. The trend for signal variation with CHAPS addition is similar to that for SDS addition, but for Triton X-100, the signal always diminishes with increasing surfactant concentration. For protein samples containing surfactants with anionic groups, addition of more surfactant allows the acquisition of useful mass spectrometric data.

Inter-laboratory note. Laser ablation inductively coupled plasma mass spectrometric transient signal data acquisition and analyte concentration calculation

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) produces complex, time-dependent signals. These require significantly different treatment both during data acquisition and reduction from the more steady-state signals produced by solution sample introduction. This paper discusses, in detail, data acquisition and reduction considerations in LA-ICP-MS analysis. Optimum data acquisition parameters are suggested. Equations are derived for the calculation of sample concentrations and LOD when time-resolved data acquisition is employed, sensitivity calibration is obtained from reference materials with known analyte concentrations and naturally occurring internal standards are used to correct for the multiplicative correction factors of drift, matrix effects and the amount of material ablated and transported to the ICP.

C 15 H 24 Volatile Compounds Unique to Aflatoxigenic Strains of Aspergillus flavus

Headspace volatiles from eight strains of Aspergillus flavus (four aflatoxigenic strains and four nonaflatoxigenic strains), grown for 1, 2, 3, 4, 8, and 10 days in submerged cultures, were collected in Tenax GC traps. The traps were desorbed onto a 50-m gas-liquid chromatography capillary column by heat and gas purge from an external direct injector device. The column was interfaced with a mass spectrometer data acquisition system. Peaks were identified by comparing retention times and mass spectra with those obtained from authentic compounds and by using a computer-assisted mass spectral data base. Aflatoxigenic strains of A. flavus produced several C(15)H(24) compounds (e.g., alpha-gurjunene, trans-caryophyllene, and cadinene) which peaked in 3-day cultures and were not present in earlier (1- and 2-day) or later (8- and 10-day) cultures. None of these volatiles were detected in nonaflatoxigenic strains of A. flavus. There was an apparent correlation between the release of C(15)H(24) volatile compounds and the initiation of aflatoxin biosynthesis, and a correlation between decline of aflatoxin synthesis and the disappearance of the C(15)H(24) compounds unique to aflatoxigenic A. flavus also existed.

A microcomputer system for mass spectrometer control and data acquisition

A microcomputer system has been designed for semi-automatic operation of a solid source mass spectrometer used for geochronological studies. It sequentially steps the magnetic field through pre-selected values, reads the digitized ion currents for a given time and temporarily stores the data which can be transferred to a paper tape or directly to a desk top calculator for further analysis. The unit is relatively inexpensive, made of readily available components and can be adapted to many laboratory automation tasks

An Electronic Peak Selector for Mass Spectrometric Data Acquisition

An Electronic Peak Selector for Mass Spectrometric Data Acquisition