Distributed computing strategies for processing of FT-ICR MS imaging datasets for continuous mode data visualization

High-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry imaging enables the spatial mapping and identification of biomolecules from complex surfaces. The need for long time-domain transients, and thus large raw file sizes, results in a large amount of raw data (“big data”) that must be processed efficiently and rapidly. This can be compounded by large-area imaging and/or high spatial resolution imaging. For FT-ICR, data processing and data reduction must not compromise the high mass resolution afforded by the mass spectrometer. The continuous mode “Mosaic Datacube” approach allows high mass resolution visualization (0.001 Da) of mass spectrometry imaging data, but requires additional processing as compared to feature-based processing. We describe the use of distributed computing for processing of FT-ICR MS imaging datasets with generation of continuous mode Mosaic Datacubes for high mass resolution visualization. An eight-fold improvement in processing time is demonstrated using a Dutch nationally available cloud service.

Open image in new window

Graphical abstract ?

     

High mass accuracy and high mass resolving power FT-ICR secondary ion mass spectrometry for biological tissue imaging

Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the sub-micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for elemental formula assignment based on exact mass measurement. We have recently reported a secondary ion mass spectrometer with the combination of a C₆₀ primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy, and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 μm spatial resolution (75 μm primary ion spot size) with mass resolving power (m/Δm _50%) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with m/Δm _50%?>?3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 μm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging.

Fourier transform ion cyclotron resonance mass spectrometry with NanoLC/microelectrospray ionization and matrix-assisted laser desorption/ionization: analytical performance in peptide mass fingerprint analysis

Protein identifications by peptide mass fingerprint analyses with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were performed using microelectrospray ionization coupled to nano liquid chromatography (NanoLC), as well as using matrix-assisted laser desorption/ionization (MALDI). Tryptic digests of bovine serum albumin (BSA), diluted down to femtomole quantities, have been desalted by fast NanoLC under isocratic elution conditions as the high resolving power of FT-ICR MS enables peptides to be separated during the mass analysis stage of the experiment. The high mass accuracy achieved with FT-ICR MS (a few ppm with external calibration) facilitated unambiguous protein identification from protein database searches, even when only a few tryptic peptides of a protein were detected. Statistical confidence in the database search results was further improved by internal calibration due to increased mass accuracy. Matrix-assisted laser desorption/ionization and micro electrospray ionization (ESI) FT-ICR showed good mass accuracies in the low femtomole range, yet a better sensitivity was observed with MALDI. However, in higher femtomole ranges slightly lower mass accuracies were observed with MALDI FT-ICR than with microESI FT-ICR due to scan-to-scan variations of the ion population in the ICR cell. Database search results and protein sequence coverage results from NanoLC FT-ICR MS and MALDI FT-ICR MS, as well as the effect of mass accuracy on protein identification for the peptide mass fingerprint analysis are evaluated.     

精确质量数在单四极杆质谱定性分析农药中的应用

利用MassWorks软件对GC/MS采集的目标农药谱图经过校正后,实现了在单位分辨率质谱上测定6种农药化合物的精确质量数,质量误差小于20mDa;在精确质量数基础上,进一步采用同位素峰形校正检索技术(CLIPs), 实现了对目标农药分子式的准确识别,建立了在单位分辨率质谱上准确测定农药小分子化合物的方法,提升了单位分辨率质谱的定性能力。通过对丙草胺及哒螨灵二种农药的质谱碎片所获得的MassWorks精确质量数及元素组成推导其质谱碎裂机理, 通过对碎片的精确质量数测定可提高对目标物定性的准确性。     

An External Matrix-Assisted Laser Desorption Ionization Source for Flexible FT-ICR Mass Spectrometry Imaging with Internal Calibration on Adjacent Samples

We describe the construction and application of a new MALDI source for FT-ICR mass spectrometry imaging. The source includes a translational X-Y positioning stage with a 10×10 cm range of motion for analysis of large sample areas, a quadrupole for mass selection, and an external octopole ion trap with electrodes for the application of an axial potential gradient for controlled ion ejection. An off-line LC MALDI MS/MS run demonstrates the utility of the new source for data- and position-dependent experiments. A FT-ICR MS imaging experiment of a coronal rat brain section yields ～200 unique peaks from m/z 400-1100 with corresponding mass-selected images. Mass spectra from every pixel are internally calibrated with respect to polymer calibrants collected from an adjacent slide.     

Assigning product ions from complex MS/MS spectra: The importance of mass uncertainty and resolving power

This study offers a unique insight into the mass accuracy and resolving power requirements in MS/MS analyses of complex product ion spectra. In the examples presented here, accurate mass assignments were often difficult because of multiple isobaric interferences and centroid mass shifts. The question then arose whether the resolving power of a medium-resolution quadrupole time-of flight (QqTOF) is sufficient or high-resolution Fourier-transform ion cyclotron resonance (FT-ICR) is required for unambiguous assignments of elemental compositions. For the comparison, two paralytic shellfish poisons (PSP), saxitoxin (STX) and neosaxitoxin (NEO), with molecular weights of 299 and 315 g x mol(-1), respectively, were chosen because of the high peak density in their MS/MS spectra. The assessment of QqTOF collision-induced dissociation spectra and FT-ICR infrared multiphoton dissociation spectra revealed that several intrinsic dissociation pathways leading to isobaric fragment ions could not be resolved with the QqTOF instrument and required FT-ICR to distinguish very close mass differences. The second major source of interferences was M + 1 species originating from coactivated 13C12Cc-1 ion contributions of the protonated molecules of the PSPs. The problem in QqTOF MS results from internal mass calibration when the MH+ ions of analyte and mass calibrant are activated at the same time in the collision or trapping cell. Although FT-ICR MS readily resolved these interfering species, the QqTOF did not provide resolving power >20,000 (full width at half maximum) required to separate most isobaric species. We were able to develop a semi-internal QqTOF calibration technique that activated only the isolated 12C isotope species of the protonated molecules, thus reducing the M + 1 interferences significantly. In terms of overall automated elemental formulas assignment, FT-ICR MS achieved the first formula hit for 100% of the product ions, whereas the QqTOF MS hit rate was only 56 and 65% for STX and NEO product ions, respectively. External mass calibration from commercial FT-ICR and QqTOF instruments gave similar results.     

Characterization of orphan monooxygenases by rapid substrate screening using FT-ICR mass spectrometry

Characterization of orphan enzymes, for which the catalytic functions and actual substrates are still not elucidated, is a significant challenge in the postgenomic era. Here, we describe a general strategy for exploring the catalytic potentials of orphan monooxygenases based on direct infusion analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Eight cytochromes P450 from Bacillus subtilis were recombinantly expressed in Escherichia coli and subjected to a reconstitution system containing appropriate electron transfer components and many potential substrates. The reaction mixtures were directly analyzed using FT-ICR/MS, and substrates of the putative enzymes were readily identified from the mass spectral data. This allowed identification of previously unreported CYP109B1 substrates and the functional assignment of two putative cytochromes P450, CYP107J1 and CYP134A1. The FT-ICR/MS-based approach can be easily applied to large-scale screening with the aid of the extremely high mass resolution and accuracy.     

Towards analytically useful two-dimensional Fourier transform ion cyclotron resonance mass spectrometry

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) achieves high resolution and mass accuracy, allowing the identification of the raw chemical formulae of ions in complex samples. Using ion isolation and fragmentation (MS/MS), we can obtain more structural information, but MS/MS is time- and sample-consuming because each ion must be isolated before fragmentation. In 1987, Pfändler et al. proposed an experiment for 2D FT-ICR MS in order to fragment ions without isolating them and to visualize the fragmentations of complex samples in a single 2D mass spectrum, like 2D NMR spectroscopy. Because of limitations of electronics and computers, few studies have been conducted with this technique. The improvement of modern computers and the use of digital electronics for FT-ICR hardware now make it possible to acquire 2D mass spectra over a broad mass range. The original experiments used in-cell collision-induced dissociation, which caused a loss of resolution. Gas-free fragmentation modes such as infrared multiphoton dissociation and electron capture dissociation allow one to measure high-resolution 2D mass spectra. Consequently, there is renewed interest to develop 2D FT-ICR MS into an efficient analytical method. Improvements introduced in 2D NMR spectroscopy can also be transposed to 2D FT-ICR MS. We describe the history of 2D FT-ICR MS, introduce recent improvements, and present analytical applications to map the fragmentation of peptides. Finally, we provide a glossary which defines a few keywords for the 2D FT-ICR MS field.     

Trapped-ion cell with improved dc potential harmonicity for FT-ICR MS

The trapped-ion cell is a key component critical for optimal performance in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). To extend the performance of FT-ICR MS, we have developed a new cell design that is capable of generating a DC trapping potential which closely approaches that of an ideal Penning trap, i.e., a 3D axial quadrupolar potential distribution. The new cell design was built upon an open cylindrical geometry, supplemented with two pairs of cylindrical compensation segments. Electric potential calculations for trial cell geometries were aimed at minimizing spatial variations of the radial electric field divided by radius. The resulting cell proportions and compensation voltages delivered practically constant effective ion cyclotron frequency that was independent of ion radial and axial positions. Our customized 12 tesla FT-ICR instrument was upgraded with the new cell, and the performance was characterized for a range of ion excitation power and ion populations. Operating the compensated cell at increased postexcitation radii, approximately 0.7 of the cell inner radius, resulted in improved mass measurement accuracy together with increased signal intensity. Under these same operating conditions the noncompensated open cell configuration exhibited peak splitting and reduced signal life time. Mass accuracy tests using 11 calibrants covering a wide m/z range reproducibly produced under 0.05 ppm RMS precision of the internal calibration for reduced ion populations and the optimal excitation radius. Conditions of increased ion population resulted in a twofold improvement in mass accuracy compared with the noncompensated cell, due to the larger achievable excitation radii and correspondingly lower space charge related perturbations of the calibration law.     

Mass calibration of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer including the rise time of the delayed extraction pulse

To utilize fully modern MALDI-TOF and TOF/TOF mass spectrometers with mass resolution exceeding 10,000 and 2 ppm precision of flight time measurements for high mass accuracy, the model of ion motion used in the mass calibration equation must be expanded. The standard three-term equation providing up to 5-10 ppm (rms) mass accuracy with internal standards was modified with an additional term accounting for the finite rise time of the high-voltage extraction pulse. This new four-term calibration equation minimizes the effect of systematic error resulting from the fact that ion velocities are mass dependent due to the rise time of the extraction pulse. Applying this new calibration equation to a mass spectrum obtained in an axial MALDI-TOF MS containing 70 peaks (sodiated PEG), each with a signal-to-noise ratio greater than 100, a mass accuracy of 1.6 ppm (rms) was obtained over the mass range 1.0-4.0 kDa compared with 3.6 ppm (rms) with the standard three-term equation. The physical basis of the effects of the finite extraction pulse rise time on mass calibration is examined for axial MALDI-TOF mass spectrometers, as well as for orthogonal acceleration TOF mass spectrometers.     

Mass spectrometry imaging with high resolution in mass and space (HR<Superscript>2</Superscript> MSI) for reliable investigation of drug compound distributions on the cellular level

Mass spectrometry (MS) imaging is a versatile method to analyze the spatial distribution of analytes in tissue sections. It provides unique features for the analysis of drug compounds in pharmacokinetic studies such as label-free detection and differentiation of compounds and metabolites. We have recently introduced a MS imaging method that combines high mass resolution and high spatial resolution in a single experiment, hence termed HR² MS imaging. In the present study, we applied this method to analyze the spatial distribution of the anti-cancer drugs imatinib and ifosfamide in individual mouse organs. The whole kidney of an animal dosed with imatinib was measured at 35 μm spatial resolution. Imatinib showed a well-defined distribution in the outer stripe of the outer medulla. This area was analyzed in more detail at 10 μm step size, which constitutes a tenfold increase in effective spatial resolution compared to previous studies of drug compounds. In parallel, ion images of phospholipids and heme were used to characterize the histological features of the tissue section and showed excellent agreement with histological staining of the kidney after MS imaging. Ifosfamide was analyzed in mouse kidney at 20 μm step size and was found to be accumulated in the inner medulla region. The identity of imatinib and ifosfamide was confirmed by on-tissue MS/MS measurements. All measurements including mass spectra from 10 μm pixels featured accurate mass (≤2 ppm root mean square) and mass resolving power of R = 30,000. Selected ion images were generated with a bin size of ∆m/z = 0.01 ensuring highly specific information. The ability of the method to cover larger areas was demonstrated by imaging a compound in the intestinal tract of a rat whole-body tissue section at 200 μm step size. The described method represents a major improvement in terms of spatial resolution and specificity for the analysis of drug compounds in tissue sections.     

A high-resolution scanning microprobe matrix-assisted laser desorption/ionization ion source for imaging analysis on an ion trap/Fourier transform ion cyclotron resonance mass spectrometer

A new scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) ion source for high spatial resolution has been developed for linear ion trap and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The source is fully compatible with commercial ion trap flanges (such as the LTQ series, Thermo Fisher Scientific). The source is designed for atmospheric pressure (AP) operation but is also suitable for mid-pressure operation. The AP mode is especially useful for investigating volatile compounds. The source can be interchanged with other ion sources within a minute when operated in the AP mode. Combining high-lateral resolution MALDI imaging with high mass resolution and high mass accuracy mass spectrometry, available in the FT-ICR mode, provides a new quality of analytical information, e.g. from biological samples. First results obtained with the new ion source demonstrate a maximum lateral resolution of 0.6 by 0.5 microm. Depending on the limit of detection of the chosen mass analyzer, however, the size of the focus had to be enlarged to a diameter of up to 8 microm in the FT-ICR mode, in order to create enough ions for detection. Mass spectra acquired for analytical imaging were obtained from single laser pulses per pixel in all the experiments. This mode allows us to investigate biological thin sections with desorption focus diameters in the micrometer range, known to cause complete evaporation of material under the laser focus with a very limited number of laser pulses. As a first example, peptide samples deposited in microstructures were investigated with the new setup. A high quality and validity of the acquired images were obtained in the ion trap mode due to the low limit of detection. High mass resolution and accuracy but poorer image quality were obtained in the ICR mode due to the lower detection sensitivity of the ICR detector.     

Improved mass accuracy for tandem mass spectrometry

With the emergence of top-down proteomics, the ability to achieve high mass measurement accuracy on tandem MS/MS data will be beneficial for protein identification and characterization. (FT-ICR) Fourier transform ion cyclotron resonance mass spectrometers are the ideal instruments to perform these experiments with their ability to provide high resolution and mass accuracy. A major limitation to mass measurement accuracy in FT-ICR instruments arises from the occurrence of space charge effects. These space charge effects shift the cyclotron frequency of the ions, which compromises the mass measurement accuracy. While several methods have been developed that correct these space charge effects, they have limitations when applied to MS/MS experiments. It has already been shown that additional information inherent in electrospray spectra can be used for improved mass measurement accuracy with the use of a computer algorithm called DeCAL (deconvolution of Coulombic affected linearity). This paper highlights a new application of the strategy for improved mass accuracy in tandem mass analysis. The results show a significant improvement in mass measurement accuracy on complex electron capture dissociation spectra of proteins. We also demonstrate how the improvement in mass accuracy can increase the confidence in protein identification from the fragment masses of proteins acquired in MS/MS experiments.     

Mass Recalibration of FT-ICR Mass Spectrometry Imaging Data Using the Average Frequency Shift of Ambient Ions

Achieving and maintaining high mass measurement accuracy (MMA) throughout a mass spectrometry imaging (MSI) experiment is vital to the identification of the observed ions. However, when using FTMS instruments, fluctuations in the total ion abundance at each pixel due to inherent biological variation in the tissue section can introduce space charge effects that systematically shift the observed mass. Herein we apply a recalibration based on the observed cyclotron frequency shift of ions found in the ambient laboratory environment, polydimethylcyclosiloxanes (PDMS). This calibration method is capable of achieving part per billion (ppb) mass accuracy with relatively high precision for an infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI dataset. Comparisons with previously published mass calibration approaches are also presented.

Determination of elemental composition of volatile organic compounds from Chinese rose oil by spectral accuracy and mass accuracy

Elemental composition determination of volatile organic compounds through high mass accuracy and isotope pattern matching could not be routinely achieved with a unit-mass resolution mass spectrometer until the recent development of the comprehensive instrument line-shape calibration technology. Through this unique technology, both m/z values and mass spectral peak shapes are calibrated simultaneously. Of fundamental importance is that calibrated mass spectra have symmetric and mathematically known peak shapes, which makes it possible to deconvolute overlapped monoisotopes and their (13)C-isotope peaks and achieve accurate mass measurements. The key experimental requirements for the measurements are to acquire true raw data in a profile or continuum mode with the acquisition threshold set to zero. A total of 13 ions from Chinese rose oil were analyzed with internal calibration. Most of the ions produced high mass accuracy of better than 5 mDa and high spectral accuracy of better than 99%. These results allow five tested ions to be identified with unique elemental compositions and the other eight ions to be determined as a top match from multiple candidates based on spectral accuracy. One of them, a coeluted component (Nerol) with m/z 154, could not be identified by conventional GC/MS (gas chromatography/mass spectrometry) and library search. Such effective determination for elemental compositions of the volatile organic compounds with a unit-mass resolution quadrupole system is obviously attributed to the significant improvement of mass accuracy. More importantly, high spectral accuracy available through the instrument line-shape calibration enables highly accurate isotope pattern recognition for unknown identification.     

Mass spectrometry of natural organic phosphorus

This article provides a review of the use of modern mass spectrometry (MS) for quantitative and qualitative measurements of organic phosphorus compounds in nature. Included is a brief discussion of recent developments in large molecule mass spectrometry, focusing on time-of-flight (TOF) and ion cyclotron resonance (ICR) mass analysis techniques, as well as electrospray (ESI) and inductively coupled plasma (ICP) ionization. The use of ICP with high-resolution mass spectrometry for quantitative measurements of total phosphorus and as a detector coupled to HPLC and CE for defining organic phosphorus speciation is demonstrated using results from a study of phosphorus cycling in a treatment wetland. Qualitative identifications of individual phosphorus compounds by ultrahigh resolution Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is demonstrated using dissolved organic phosphorus isolated from this same wetland.     

Mapping of Neuropeptides in the Crustacean Stomatogastric Nervous System by Imaging Mass Spectrometry

Considerable effort has been devoted to characterizing the crustacean stomatogastric nervous system (STNS) with great emphasis on comprehensive analysis and mapping distribution of its diverse neuropeptide complement. Previously, immunohistochemistry (IHC) has been applied to this endeavor, yet with identification accuracy and throughput compromised. Therefore, molecular imaging methods are pursued to unequivocally determine the identity and location of the neuropeptides at a high spatial resolution. In this work, we developed a novel, multi-faceted mass spectrometric strategy combining profiling and imaging techniques to characterize and map neuropeptides from the blue crab Callinectes sapidus STNS at the network level. In total, 55 neuropeptides from 10 families were identified from the major ganglia in the C. sapidus STNS for the first time, including the stomatogastric ganglion (STG), the paired commissural ganglia (CoG), the esophageal ganglion (OG), and the connecting nerve stomatogastric nerve (stn) using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) and the MS/MS capability of this technique. In addition, the locations of multiple neuropeptides were documented at a spatial resolution of 25 μm in the STG and upstream nerve using MALDI-TOF/TOF and high-mass-resolution and high-mass-accuracy MALDI-Fourier transform ion cyclotron resonance (FT-ICR) instrument. Furthermore, distributions of neuropeptides in the whole C. sapidus STNS were examined by imaging mass spectrometry (IMS). Different isoforms from the same family were simultaneously and unambiguously mapped, facilitating the functional exploration of neuropeptides present in the crustacean STNS and exemplifying the revolutionary role of this novel platform in neuronal network studies.

傅立叶变换离子回旋共振质谱及其研究进展

本文综述了傅立叶变换离子回旋共振质谱的基本原理及其颇具特色的数据处理方法.以两种典型的离子化方法为代表,介绍了目前主要使用的离子源,并总结了该方法在分析科学,尤其是在大分子分析、气相离子反应动力学研究和复杂体系分析等领域中的广泛应用.     

Multiplex Mass Spectrometric Imaging with Polarity Switching for Concurrent Acquisition of Positive and Negative Ion Images

We have recently developed a multiplex mass spectrometry imaging (MSI) method which incorporates high mass resolution imaging and MS/MS and MS³ imaging of several compounds in a single data acquisition utilizing a hybrid linear ion trap-Orbitrap mass spectrometer (Perdian and Lee, Anal. Chem. 82, 9393–9400, 2010). Here we extend this capability to obtain positive and negative ion MS and MS/MS spectra in a single MS imaging experiment through polarity switching within spiral steps of each raster step. This methodology was demonstrated for the analysis of various lipid class compounds in a section of mouse brain. This allows for simultaneous imaging of compounds that are readily ionized in positive mode (e.g., phosphatidylcholines and sphingomyelins) and those that are readily ionized in negative mode (e.g., sulfatides, phosphatidylinositols and phosphatidylserines). MS/MS imaging was also performed for a few compounds in both positive and negative ion mode within the same experimental set-up. Insufficient stabilization time for the Orbitrap high voltage leads to slight deviations in observed masses, but these deviations are systematic and were easily corrected with a two-point calibration to background ions.

Evaluation of a high-resolution time-of-flight mass spectrometer for the gas chromatographic determination of selected environmental contaminants

A benchtop high-resolution time-of-flight mass spectrometer (TOF MS) was evaluated for the determination of key organic microcontaminants. The major advantage of the TOF MS proved to be the high mass resolution of about 0.002 Da (10 ppm). Consequently, the detectability of polar pesticides, polynuclear aromatic hydrocarbons and polychlorinated biphenyls is excellent, and detection limits are in the order of 1–4 pg injected mass. Best mass spectral resolution was obtained for medium-scale peaks. It is a disadvantage that the calibration range is rather limited, viz. to about two orders of magnitude. The high mass spectral resolution was especially useful to improve the selectivity and sensitivity when analyzing target compounds in complex samples and to prevent false-positive identifications.