Post-Trapping Derivatization of Radical-Derived EPR-Silent Adducts: Application to Free Radical Detection by HPLC/UV in Chemical, Biochemical, and Biological Systems and Comparison with EPR Spectroscopy

Free radicals are conventionally detected by electron paramagnetic resonance (EPR) spectroscopy after being trapped as spin adducts. Albeit this technique has demonstrated utmost efficacy in studying free radicals, its application to biological settings is intrinsically hampered by the inevitable bioreduction of radical-derived paramagnetic adducts. Herein, we describe a reliable technique to detect and quantify free radical metabolites, wherein reduced alkyl- and phenyl-5,5-dimethyl-1-pyrroline N-oxide (DMPO) adducts are converted into ultrastable N-naphthoate esters. To mimic the ubiquitous in vivo microenvironment, bioreductants, exogenous thiols, and sodium borohydride were studied. Nitroxyl reduction was confirmed using EPR and triphenyltetrazolium chloride. The formation of the N-naphthoyloxy derivatives was established by liquid chromatography/mass spectrometry (LC/MS). The derivatives were chromatographed using a binary eluent. HPLC and internal standards were synthesized using Grignard addition. The labeled DMPO adduct is (1) fluorescent, (2) stable as opposed to nitroxyl radical adducts, (3) biologically relevant, and (4) excellently chromatographed. Applications encompassed chemical, biochemical, and biological model systems generating C-centered radicals. Different levels of phenyl radicals produced in situ from whole blood were successfully determined. The method is readily applicable to the detection of hydroxyl radical. Analogously, DMPO, the spin trap, could be detected with extreme sensitivity suitable for in vivo applications. The developed method proved to be a viable alternative to EPR, where for the first time the reductive loss of paramagnetic signals of DMPO-trapped free radicals is transformed into fluorescence emission. We believe the proposed methodology could represent a valuable tool to probe free radical metabolites in vivo using DMPO, the least toxic spin trap.     

Top-down identification and quantification of stable isotope labeled proteins from Aspergillus flavus using online nano-flow reversed-phase liquid chromatography coupled to a LTQ-FTICR mass spectrometer

Online liquid chromatography-mass spectrometric (LC-MS) analysis of intact proteins (i.e., top-down proteomics) is a growing area of research in the mass spectrometry community. A major advantage of top-down MS characterization of proteins is that the information of the intact protein is retained over the vastly more common bottom-up approach that uses protease-generated peptides to search genomic databases for protein identification. Concurrent to the emergence of top-down MS characterization of proteins has been the development and implementation of the stable isotope labeling of amino acids in cell culture (SILAC) method for relative quantification of proteins by LC-MS. Herein we describe the qualitative and quantitative top-down characterization of proteins derived from SILAC-labeled Aspergillus flavus using nanoflow reversed-phase liquid chromatography directly coupled to a linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FTICR-MS). A. flavus is a toxic filamentous fungus that significantly impacts the agricultural economy and human health. SILAC labeling improved the confidence of protein identification, and we observed 1318 unique protein masses corresponding to 659 SILAC pairs, of which 22 were confidently identified. However, we have observed some limiting issues with regard to protein quantification using top-down MS/MS analyses of SILAC-labeled proteins. The role of SILAC labeling in the presence of competing endogenously produced amino acid residues and its impact on quantification of intact species are discussed in detail.     

High-throughput screening and characterization of reactive metabolites using polarity switching of hybrid triple quadrupole linear ion trap mass spectrometry

A highly sensitive and efficient method has been developed for detection and characterization of glutathione (gamma-glutamyl-cysteinylglycine, GSH)-trapped reactive metabolites using a negative precursor ion (PI) as the survey scan to trigger the acquisition of positive enhanced product ion (EPI) spectra on a triple quadrupole linear ion trap mass spectrometer. The negative precursor ion scan step was carried out monitoring the anion at m/z 272, corresponding to deprotonated gamma-glutamyl-dehydroalanyl-glycine originating from the glutathionyl moiety. Because of the uniqueness and abundance of the anion at m/z 272, this single survey scan exhibited broad utility in the detection of unknown GSH conjugates. Further structural characterization was achieved by analyzing positive MS2 spectra that featured rich fragments without mass cutoff and were acquired in the same liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. The effectiveness and reliability of this approach was evaluated using a number of model compounds in human liver microsomal incubations, including acetaminophen, clozapine, diclofenac, imipramine, meclofenamic acid, and ticlopidine. As a result, the PI-EPI approach revealed the presence of known adducts and, in many instances, identified additional conjugates that had not been reported previously. In comparison to the widely used neutral loss (NL) scanning analysis, this approach provided superior sensitivity and selectivity for different types of GSH conjugates. More importantly, the PI-EPI approach is suitable for high-throughput screening of reactive metabolites in the drug discovery process.     

Combined liquid chromatography/electron paramagnetic resonance spectrometry/electrospray ionization mass spectrometry for radical identification.

Electron paramagnetic resonance (EPR) spectrometry and mass spectrometry (MS) have been coupled together on-line with liquid chromatography (LC)/UV detection. These combined techniques have been applied to the determination of spin-trapped radical adducts, including phenyl, 2-, 3-, and 4-chlorophenyl, and 2-bromophenyl radicals trapped with alpha-(1-oxo-4-pyridyl)-N-tert-butylnitrone (4-POBN), and phenyl radicals trapped with 2-methyl-2-nitrosopropane (MNP), alpha-phenyl-N-tert-butylnitrone (PBN), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Oxidized and reduced forms of the radical adducts were also detected by the on-line LC/EPR/MS system.     

Combined top-down and bottom-up mass spectrometric approach to characterization of biomarkers for renal disease

Here we describe a mass spectrometry (MS) approach for biomarker discovery and structural characterization, based on both top-down and bottom-up analyses. Capillary electrophoresis (CE) coupled to electrospray ionization (ESI) time-of-flight (TOF) MS serves to separate and mass-measure the thousands of polypeptides contained in human urine. Statistical analysis of the differences between healthy control samples and patients with focal-segmental glomerulosclerosis, membranous glomerulonephritis, minimal change disease, IgA nephropathy, and diabetic nephropathy validates multiple biomarkers for the control and each of the diseases. To identify those biomarkers, we employ preparative CE, enabling direct infusion ESI MS analysis, followed by sample manipulation and reanalysis where necessary. We show how tandem Fourier transform ion cyclotron resonance (FT-ICR) MS identifies these sometimes large (>8 kDa) biomarkers. Critically, we maintain connectivity between the CE TOF MS data and the ICR data used for biomarker identification.     

Biotinylated analogue of the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide for the detection of low-abundance protein radicals by mass spectrometry

Protein radicals are implicated in oxidative stress and are associated with a wide range of diseases and disorders. In the present work, we describe the specific application of a newly synthesized nitrone spin trap, Bio-SS-DMPO, for the detection of these highly reactive species by mass spectrometry (MS). Bio-SS-DMPO is a biotinylated analogue of the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) that allows for specific capture of the protein(s)/peptide(s) labeled by the spin-trap on a (strept)avidin-bound solid matrix. The disulfide bond in the linker arm joining biotin to DMPO can be cleaved to release captured spin-adduct peptide from the solid matrix. This (strept)avidin-based affinity purification reduces the complexity of the samples prior to MS analyses, thereby facilitating the location of the sites of spin trap addition. In addition, the biotin moiety on the spin-trap can efficiently be probed with (strept)avidin-conjugated reporter. This offers an effective means to visualize the presence of DMPO-adducted proteins in intact cells.     

Top-down proteomics on a chromatographic time scale using linear ion trap fourier transform hybrid mass spectrometers

Proteomics has grown significantly with the aid of new technologies that consistently are becoming more streamlined. While processing of proteins from a whole cell lysate is typically done in a bottom-up fashion utilizing MS/MS of peptides from enzymatically digested proteins, top-down proteomics is becoming a viable alternative that until recently has been limited largely to offline analysis by tandem mass spectrometry. Here we describe a method for high-resolution tandem mass spectrometery of intact proteins on a chromatographic time scale. In a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run, we have identified 22 yeast proteins with molecular weights from 14 to 35 kDa. Using anion exchange chromatography to fractionate a whole cell lysate before online LC-MS/MS, we have detected 231 metabolically labeled (14N/15N) protein pairs from Saccharomyces cerevisiae. Thirty-nine additional proteins were identified and characterized from LC-MS/MS of selected anion exchange fractions. Automated localization of multiple acetylations on Histone H4 was also accomplished on an LC time scale from a complex protein mixture. To our knowledge, this is the first demonstration of top-down proteomics (i.e., many identifications) on linear ion trap Fourier transform (LTQ FT) systems using high-resolution MS/MS data obtained on a chromatographic time scale.     

Nano-LC FTICR tandem mass spectrometry for top-down proteomics: routine baseline unit mass resolution of whole cell lysate proteins up to 72 kDa

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.     

Charge determination of product ions formed from collision-induced dissociation of multiply protonated molecules via ion/molecule reactions.

The use of ion/molecule reactions involving multiply protonated ions derived from electrospray for the determination of the charges of product ions formed from collision-induced dissociation is described. The experiments are carried out with a quadrupole ion trap capable of multiple stages of mass spectrometry. The approach is illustrated with proton transfer from a product ion from quadruply protonated melittin, and from a product ion from the (M + 20H)20+ ion from horse myoglobin, to 1,6-diaminohexane. The major product ion from quadruply protonated bovine insulin is used to illustrate the use of a clustering reaction with 1,6-diaminohexane. The ion trap is shown to be a particularly useful tool for employing both collisional activation and low-energy ion/molecule reactions in the same experiment to determine product ion charge.     

Whole protein dissociation in a quadrupole ion trap: identification of an a priori unknown modified protein

A protein mixture derived from a whole cell lysate fraction of Saccharomyces cerevisiae, which contains roughly 19 proteins, has been analyzed to identify an a priori unknown modified protein using a quadrupole ion trap tandem mass spectrometer. Collection of the experimental data was facilitated by collision-induced dissociation and ion/ion proton-transfer reactions in multistage mass spectrometry procedures. Ion/ion reactions were used to manipulate charge states of both parent ions and product ions for the purpose of concentrating charge into the parent ion of interest and to reduce the product ion charge states for determination of product ion mass and abundance. The identification of the protein was achieved by matching the uninterpreted product ion spectrum against protein sequence databases with varying degrees of annotation, coupled with a scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential sites. The protein was identified to be an acetylated yeast heat shock protein, HS12_Yeast (11.6 kDa), with the initiating methionine residue removed. This constitutes the first example of the identification of an a priori unknown protein that is not present in an annotated protein database using a "top-down" approach with a quadrupole ion trap. This example illustrates the utility of relatively low cost instrumentation with modest mass analysis characteristics for the identification of modified proteins without recourse to enzymatic digestion. It also illustrates how experimental data can be used interactively with protein databases when the modified protein of interest is not initially present in the database.     

CE-microreactor-CE-MS/MS for protein analysis

We present a proof-of-principle for a fully automated bottom-up approach to protein characterization. Proteins are first separated by capillary electrophoresis. A pepsin microreactor is incorporated into the distal end of this capillary. Peptides formed in the reactor are transferred to a second capillary, where they are separated by capillary electrophoresis and characterized by mass spectrometry. While peptides generated from one digestion are being separated in the second capillary, the next protein fraction undergoes digestion in the microreactor. The migration time in the first dimension capillary is characteristic of the protein while migration time in the second dimension is characteristic of the peptide. Spot capacity for the two-dimensional separation is 590. A MS/MS analysis of a mixture of cytochrome c and myoglobin generated Mascot MOWSE scores of 107 for cytochrome c and 58 for myoglobin. The sequence coverages were 48% and 22%, respectively.     

Structural analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry

Improving qualitative and quantitative characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, we describe the performance of ETD-based top-down mass spectrometry for structural analysis of intact 150 kDa monoclonal antibodies, immunoglobulins G (IgGs). Simultaneous mass analysis of intact IgGs as well as a complex mixture of ETD product ions at sufficiently high resolution and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resolution ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissociation. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the experiment sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.     

FTMS structure elucidation of natural products: application to muraymycin antibiotics using ESI multi-CHEF SORI-CID FTMS(n), the top-down/bottom-up approach,and HPLC ESI capillary-skimmer CID FTMS

The molecular formulas for the structures and substructures of muraymycin antibiotics A1 (C52H90N14O19, MW 1214) and B1 (C49H83N11O18, MW 1113) were determined using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The muraymycin A1 and B1 structures were elucidated by utilizing capillary-skimmer fragmentation with up to five stages of mass spectrometry (MS5). Multi-CHEF, a multiple ion isolation method, was used at each stage of MS(n) to isolate a parent ion and up to four reference ions, for exact-mass calibration. The parent ions were fragmented by SORI-CID and the product ions internally calibrated with average absolute mass errors less than 1 ppm at each stage in the fragmentation processes. Using the top-down/bottom-up approach, the molecular formulas for the antibiotics were determined by summing the elemental formulas of the neutral losses, obtained by measuring the mass differences (<500 Da) between the genetically related sequential parent ion masses in the MS(n) spectra, with the unique elemental formula of the lowest parent ion mass (<500 Da). The structures of 12 additional compounds in the muraymycin complex were elucidated using HPLC ESI capillary-skimmer CID FTMS by correlating their fragmentation patterns with those of muraymycins A1 and B1. Sequential neutral losses of an aminosugar, a valine, a uridine, and an ester fatty acid from the muraymycin parent ions provided diagnostic fragments for characterization.     

Intramolecular isobaric fragmentation: a curiosity of accurate mass analysis of sulfadimethoxine in pond water

Liquid chromatography with time-of-flight mass spectrometry (TOF-MS) and quadrupole-time-of-flight (Q-TOF) mass spectrometry/mass spectrometry (MS/MS) were used for the accurate mass analysis of sulfadimethoxine in pond water of a fish hatchery. Sulfadimethoxine is the most important sulfa antimicrobial used in aquaculture to treat bacterial disease in a wide variety of fish. Because correct identification is essential to environmental monitoring of antimicrobial pharmaceuticals, accurate mass analyses (TOF and Q-TOF-MS/MS) were compared to nominal mass measurement (quadrupole ion trap). It was known that all six members of the sulfa antimicrobial family gave a common 6-sulfanilamido ion at a nominal mass of m/z 156; thus, this ion was the focus of TOF confirmation (exact mass 156.0119 u) along with the protonated molecule (exact mass 311.0814 u). In the process of accurate mass confirmation of the 156 m/z fragment ion, a second isobaric ion (exact mass m/z 156.0773), was discovered at the same nominal mass, which was not differentiated by quadrupole ion trap. The structure was assigned as 2-4-dimethoxypyridine and is exactly the other protonated half of the sulfadimethoxine molecule. This discovery led to the subsequent use of Q-TOF-MS/MS and high-resolution identification of five other important ion fragments for the identification of sulfadimethoxine in pond water at environmental concentrations. The caveats of using low-resolution mass spectrometry without MS/MS for environmental monitoring are discussed in the light of high profile monitoring of sulfa antimicrobial pharmaceuticals in the aquatic environment.     

Enzyme-DNA biocolloids for DNA adduct and reactive metabolite detection by chromatography-mass spectrometry

Silica microbead bioreactors (0.5 microm diameter) coated with DNA and enzymes were fabricated to measure reactive metabolite and DNA-adduct formation rates relevant to genotoxicity screening. Cytochrome (cyt) P450 2E1, cyt P450(cam), and myoglobin (Mb) were incorporated into thin films with DNA using the electrostatic layer-by-layer (LbL) method. The utility of these biocolloids was demonstrated by oxidation of guaiacol, styrene, and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Enzyme turnover rates for formation of reactive metabolites were monitored using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC-MS). Capillary LC-MS/MS was employed to determine DNA nucleobase adducts after catalyzing the reactive metabolite formation with DNA-enzyme biocolloids and then using neutral thermal hydrolysis on the biocolloids. Dramatic improvements in surface area to volume ratio over similar films on macroscopic surfaces opens new avenues for genotoxicity screening and enabled the first use of pure cyt P450 enzymes in enzyme-DNA films to produce DNA adducts. The method makes possible identification and formation rate measurements of major and minor DNA adducts as well as the metabolites themselves in <5 min of reaction time using relevant human liver enzymes.     

Application of metal-coded affinity tags (MeCAT): absolute protein quantification with top-down and bottom-up workflows by metal-coded tagging

As the quantification of peptides and proteins extends from comparative analyses to the determination of actual amounts, methodologies for absolute protein quantification are desirable. Metal-coded affinity tags (MeCAT) are chemical labels for peptides and proteins with a lanthanide-bearing chelator as a core. This modification of analytes with non-naturally occurring heteroelements adds the analytical possibilities of inductively coupled plasma mass spectrometry (ICPMS) to quantitative proteomics. We here present the absolute quantification of recombinantly expressed aprotinin out of its host cell protein background using two independent MeCAT methodologies. A bottom-up strategy employs labeling of primary amino groups on peptide level. Synthetic peptides with a MeCAT label which are externally quantified by flow injection analysis (FIA)-ICPMS serve as internal standard in nanoHPLC-ESI-MS/MS. In the top-down approach, protein is labeled on cysteine residues and separated by two-dimensional gel electrophoresis. Flow injection analysis of dissolved gel spots by ICPMS yields the individual protein amount via its lanthanide label content. The enzymatic determination of the fusion protein via its β-galactosidase activity found 8.3 and 9.8 ng/μg (nanogram fusion protein per microgram sample) for batches 1 and 2, respectively. Using MeCAT values of 4.0 and 5.4 ng/μg are obtained for top-down analysis, while 14.5 and 15.9 ng/μg were found in the bottom-up analysis.     

On the utility of isotopic fine structure mass spectrometry in protein identification

Modern mass spectrometry (MS)-based protein identification and characterization relies upon accurate mass measurements of the (13)C isotopic distributions of the enzymatically produced peptides. Interestingly, obtaining peptide elemental composition information from its isotopic fine structure mass spectrum to increase the confidence in peptide and protein identification has not yet been developed into a bottom-up proteomics-grade analytical approach. Here, we discuss the possible utility and limitations of the isotopic fine structure MS for peptide and protein identification. First, we in silico identify the peptides from the E. coli tryptic digest and show the increased confidence in peptide identification by consideration of the isotopic fine structures of these peptides as a function of mass and abundance accuracies. In the following, we demonstrate that the state-of-the-art high magnetic field Fourier transform ion cyclotron resonance (FT-ICR) MS allows a routine acquisition of the isotopic fine structure information of a number of isobaric peptide pairs, including a pair of peptides originating from E. coli. Finally, we address the practical limitation of the isotopic fine structure MS implementation in the time-constraint experiments by applying an advanced signal processing technique, filter diagonalization method, to the experimental transients to overcome the resolution barrier set by the typically applied Fourier transformation. We thus demonstrate that the isotopic fine structures of peptides may indeed improve the peptide and possibly protein identification, can be produced in a routine experiment by the state-of-the-art high resolution mass spectrometers, and can be potentially obtained on a chromatographic time-scale of a typical bottom-up proteomics experiment. The latter one requires at least an order of magnitude increase in sensitivity of ion detection, which presumably can be realized using high-field Orbitrap FTMS and/or future generation of ultrahigh magnetic field FT-ICR MS equipped with harmonized ICR cells.     

De novo peptide sequencing based on a divide-and-conquer algorithm and peptide tandem spectrum simulation

Mass spectrometry-based de novo peptide sequencing is generally more reliable on high-resolution instruments owing to their high resolution and mass accuracy. On a lower resolution instrument such as the more widely used quadrupole ion traps, de novo peptide sequencing is not so reliable or requires more MS(3) experiments. However, the peptide CID spectrum has been demonstrated to be quite reproducible on an ion trap instrument and can be predicted with good accuracy. A new de novo peptide sequencing technique, DACSIM, combining a divide-and-conquer algorithm for deriving sequence candidates and spectrum simulation for sequence refinement, is developed for spectra acquired on an ion trap instrument. When DACSIM was used to sequence peptides 500-1900 u in mass generated from proteolytic digests of hemoglobin and myoglobin, the success rate was 70% with a false positive rate of only 6%, when isoleucine and leucine residues were not distinguished.     

Investigation of the unusual behavior of metolachlor under chemical ionization in a hybrid 3D ion trap mass spectrometer

This article describes the strange behavior of the widely used herbicide metolachlor under chemical ionization conditions in a hybrid source ion trap mass spectrometer in gas chromatography/mass spectrometry (GC/MS) coupling. With the use of ammonia as the reagent gas, metolachlor provides a chlorinated ion at m/z 295/297, almost as abundant as the protonated molecule at m/z 284/286, which cannot be isolated to perform tandem mass spectrometry (MS(n)) experiments. Curiously, this ion at m/z = M + 12 is not observed for the herbicides acetochlor and alachlor, which present very similar chemical structures. The chemical structure of the m/z 295/297 ions and the explanation of the observed phenomenon based on the metastable behavior of these ions were elucidated on the basis of experiments including isotopic labeling and modifications of the operating conditions of the ion trap mass spectrometer. This work allows one to give new recommendations for an optimized use of hybrid source ion trap mass spectrometers.     

Filter diagonalization method-based mass spectrometry for molecular and macromolecular structure analysis

Molecular and macromolecular structure analysis by high resolution and accurate mass spectrometry (MS) is indispensable for a number of fundamental and applied research areas, including health and energy domains. Comprehensive structure analysis of molecules and macromolecules present in the extremely complex samples and performed under time-constrained experimental conditions demands a substantial increase in the acquisition speed of high resolution MS data. We demonstrate here that signal processing based on the filter diagonalization method (FDM) provides the required resolution for shorter experimental transient signals in ion cyclotron resonance (ICR) MS compared to the Fourier transform (FT) processing. We thus present the development of a FDM-based MS (FDM MS) and demonstrate its implementation in ICR MS. The considered FDM MS applications are in bottom-up and top-down proteomics, metabolomics, and petroleomics.