Overview of Target Enrichment Strategies.

Target enrichment is commonly used in next generation sequencing (NGS) workflows to eliminate genomic DNA regions that are not of interest for a particular experiment. By only targeting specific regions such as exons, one can obtain greater depth of DNA sequencing coverage for regions of interest or increase the sampling numbers of individuals, thereby saving both time and cost. This overview of target enrichment strategies provides a high-level review of distinct approaches to capture specific sequences: (a) hybridization-based strategies, (b) transposon-mediated fragmentation (tagmentation), (c) molecular inversion probes (MIPs), and (d) singleplex and multiplex polymerase chain reaction (PCR) target enrichment. Strategies for assay design and performance criteria are also discussed. Other platforms currently in development are also briefly described.

Note: Optimized circuit for excitation and detection with one pair of electrodes for improved Fourier transform ion cyclotron resonance mass spectrometry.

A conventional Fourier transform-Ion Cyclotron Resonance (ICR) detection cell is azimuthally divided into four equal sections. One pair of opposed electrodes is used for ion cyclotron excitation, and the other pair for ion image charge detection. In this work, we demonstrate that an appropriate electrical circuit facilitates excitation and detection on one pair of opposed electrodes. The new scheme can be used to minimize the number of electrically independent ICR cell electrodes and/or improve the electrode geometry for simultaneously increased ICR signal magnitude and optimal post-excitation radius, which results in higher signal-to-noise ratio and decreased space-charge effects.

Fourier transform ion cyclotron resonance: state of the art.

This short review summarizes recent and projected advances in Fourier transform ion cyclotron resonance mass spectrometry instrumentation and applications, ranging from petroleomics to proteomics. More details are available from the cited primary literature and topical reviews.

Kendrick mass defect spectrum: a compact visual analysis for ultrahigh-resolution broadband mass spectra.

At currently achievable Fourier transform ion cyclotron resonance broadband mass spectrometry resolving power (m/deltam50% > 350,000 for 200 < m/z < 1,000), it would be necessary to spread out a conventional mass spectrum over approximately 200 m in order to provide visual resolution of the most closely resolved peaks. Fortunately, there are natural gaps in a typical mass spectrum, spaced 1 Da apart, because virtually no commonly encountered elemental compositions yield masses at those values. Thus, it is possible to break a broadband mass spectrum into 1-Da segments, rotate each segment by 90 degrees, scale each segment according to its mass defect (i.e., difference between exact and nominal mass), and then compress the spacing between the segments to yield a compact display. For hydrocarbon systems, conversion from IUPAC mass to "Kendrick" mass (i.e., multiplying each mass by 14.00000/14.01565) further simplifies the display by rectilinearizing the peak patterns. The resulting display preserves not only the "coarse" spacings (e.g., approximately 1 Da between odd and even masses, corresponding to either even vs odd number of nitrogens or 12C(c) vs 12C(c-1)13C1 elemental compositions of the same molecule; approximately 2-Da separations, corresponding to a double bond or ring; approximately 14 Da separations, corresponding to one CH2 group) but also the "fine structure" (i.e., different mass defects for different elemental compositions) across each 1-Da segment. The method is illustrated for experimental electrospray ionization FTICR ultrahigh-resolution mass spectra of a petroleum crude oil. Several thousand elemental compositions may be resolved visually in a single one-page two-dimensional display, and various compound families-class (NnOoSs), type (Z in C(c)H2(c+z)NnOoSs), and alkylation series-may be identified visually as well.

High-sensitivity electron capture dissociation tandem FTICR mass spectrometry of microelectrosprayed peptides.

Electron capture dissociation (ECD) has previously been shown by other research groups to result in greater peptide sequence coverage than other ion dissociation techniques and to localize labile posttranslational modifications. Here, ECD has been achieved for 10-13-mer peptides microelectrosprayed from 10 nM (10 fmol/microL) solutions and for tryptic peptides from a 50 nM unfractionated digest of a 28-kDa protein. Tandem Fourier transform ion cyclotron resonance (FTICR) mass spectra contain fragment ions corresponding to cleavages at all possible peptide backbone amine bonds, except on the N-terminal side of proline, for substance P and neurotensin. For luteinizing hormone-releasing hormone, all but two expected backbone amine bond cleavages are observed. The tandem FTICR mass spectra of the tryptic peptides contain fragment ions corresponding to cleavages at 6 of 12 (1545.7-Da peptide) and 8 of 21 (2944.5-Da peptide) expected backbone amine bonds. The present sensitivity is 200-2000 times higher than previously reported. These results show promise for ECD as a tool to produce sequence tags for identification of peptides in complex mixtures available only in limited amounts, as in proteomics.

High sensitivity Fourier transform ion cyclotron resonance mass spectrometry for biological analysis with nano-LC and microelectrospray ionization.

Modifications to a 7 T nano-LC micro-ESI FT-ICR mass spectrometer, including a shorter octopole, approximately 100% duty cycle, improved nano-LC micro-ESI emitter tips, and reverse-phase HPLC resins that require no ion-pairing agent, combine to achieve attomole detection limit. Three peptides in a mixture totaling 500 attomoles (amol) each in water (10 microL, 50 amol/microL) are separated and detected, demonstrating detection from a mixture at low endogenous biological concentration. Two peptides in a mixture totaling 500 amol each in artificial cerebrospinal fluid (1 microL, 500 amol/microL) are separated and detected, demonstrating detection from a mixture at a biological concentration in a biological solvent. The highest sensitivity is attained with arg8-vasotocin, in which a total of 300 amol is detected in artificial cerebrospinal fluid (1 microL, 300 amol/microL) and a total of 100 amol in water (1 microL, 100 amol/microL). Arg8-vasotocin isolated from the pineal gland of rainbow trout is detected, demonstrating the ability of FT-ICR to detect and identify a true endogenous biological analyte.

Baseline mass resolution of peptide isobars: a record for molecular mass resolution.

Baseline resolution of two peptides, RVMRGMR and RSHRGHR, of neutral monoisotopic mass, approximately 904 Da, has been achieved by microelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry at a mass resolving power of approximately 3 300 000. The elemental compositions of these molecules differ by N40 vs. S2H8 (0.000 45 Da), which is less than one electron's mass (0.000 55 Da)! This result establishes a new record for the smallest resolved mass difference between any two molecules. This achievement is made possible by a combination of high magnetic field (9.4 T), large-diameter (4-in.) Penning trap, and low ion density. The implications for proteomics based on accurate mass measurements are discussed briefly.

Competitive binding to the oligopeptide binding protein, OppA: in-trap cleanup in an Fourier transform ion cyclotron resonance mass spectrometer.

This communication demonstrates that gentle infrared laser heating can remove unwanted buffer adducts from a gas-phase protein complex without dissociating the complex itself. Specifically, noncovalent complexes of the oligopeptide-binding protein, OppA, bound to either (Ala)3 or LysTrpLys were electrosprayed from aqueous buffer solution into a 9.4 tesla Fourier transform ion cyclotron resonance mass spectrometer. In addition to the intact complexes, several additional buffer adduct species were produced under the conditions of the experiment. Irradiation of the trapped ion population with a continuous-wave infrared CO2 laser at relatively low power (2.5 W) for 1 s dissociated the buffer adducts but retained the intact protein:peptide complexes. Adduct-free complex(es) were then readily identified, and signal-to-noise ratio also increased by an order of magnitude because the same number of protein ions are distributed over fewer species. Higher IR power (5 W for 1 s) dissociated the adduct-free complex(es) without internal fragmentation. The present in-trap clean-up technique may prove especially useful for identifying and screening the combinatorial library ligands most strongly bound to a receptor in the gas phase.

Stable isotope incorporation triples the upper mass limit for determination of elemental composition by accurate mass measurement.

By comparing electrospray ionization Fourier-transform ion cyclotron resonance (FT-ICR) mass spectra and collision-induced dissociation (CID) FT-ICR mass spectra of a phospholipid (851 Da) extracted from natural abundance and 99% 13C bacterial growth media, we are able to reduce its number of possible elemental compositions (based on +/-10 ppm externally calibrated mass accuracy and biologically relevant compositional constraints) from 394 to 1. The basic idea is simply that the mass of a molecule containing N carbon atoms increases by N Da when 12C is replaced by 13C. Once the number of carbons is known, the number of possible combinations of other atoms in the molecule is greatly reduced. We demonstrate the method for a stored-waveform inverse Fourier transform-isolated phospholipid from an extract of membrane lipids from Rhodococcus rhodochrous hydrocarbon-degrading bacteria grown on either natural abundance or 99% 13C-enriched mixtures of n-hexadecane and n-octadecane. We project that this method raises the upper mass limit for unique determination of elemental composition from accurate mass measurement by a factor of at least 3, thereby extending "chemical formula" determination to identification and sequencing of larger synthetic and bio-polymers: phospholipids, oligopeptides of more than three to four amino acids, DNA or RNA of more than two nucleotides, oligosaccharides of more than three sugars, etc. The method can also be extended to determination of the number of other atoms for which heavy isotopes are available (e.g., 15N, 34S, 18O, etc.).

Unequivocal determination of metal atom oxidation state in naked heme proteins: Fe(III)myoglobin, Fe(III)cytochrome c, Fe(III)cytochrome b5, and Fe(III)cytochrome b5 L47R.

Unambiguous determination of metal atom oxidation state in an intact metalloprotein is achieved by matching experimental (electrospray ionization 9.4 tesla Fourier transform ion cyclotron resonance) and theoretical isotopic abundance mass distributions for one or more holoprotein charge states. The ion atom oxidation state is determined unequivocally as Fe(III) for each of four gas-phase unhydrated heme proteins electrosprayed from H2O: myoglobin, cytochrome c, cytochrome b5, and cytochrome b5 L47R (i.e., the solution-phase oxidation state is conserved following electrospray to produce gas-phase ions). However, the same Fe(III) oxidation state in all four heme proteins is observed after prior reduction by sodium dithionite to produce Fe(II) heme proteins in solution: thus proving that oxygen was present during the electrospray process. Those results bear directly on the issue of similarity (or lack thereof) of solution-phase and gas-phase protein conformations. Finally, infrared multiphoton irradiation of the gas-phase Fe(III)holoproteins releases Fe(III)heme from each of the noncovalently bound Fe(III)heme proteins (myoglobin, cytochrome b5 and cytochrome b5 L47R), but yields Fe(II)heme from the covalently bound heme in cytochrome c.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

The basic principles and recent advances in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry are reviewed. A brief history of electrospray ionization is provided, along with a complete technical description of the technique, electrospray ionization variations, and advantages. Next, the fundamental principles of Fourier transform ion cyclotron resonance mass spectrometry are covered, including ion cyclotron motion, ion cyclotron resonance excitation, and image current detection. Instrumentation and methods used to couple these techniques are then described. Topics include ion source configuration, ion transport through a strong magnetic field gradient, and ion trapping methods. The article concludes with selected applications that highlight the strengths of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Posttranslational heterocyclization of cysteine and serine residues in the antibiotic microcin B17: distributivity and directionality.

To produce the antibiotic Microcin B17, four Cys and four Ser residues are converted into four thiazoles and four oxazoles by the three subunit Microcin B17 synthetase. High-resolution mass spectrometry (MS) was used to monitor the kinetics of posttranslational heterocyclic ring formation (-20 Da per ring) and demonstrated the accumulation of all intermediates, from one to seven rings, indicating distributive processing. All of the intermediates could be converted by the enzyme to the eight ring product. Enzymatic chemoselectivity (Cys vs Ser cyclization rates) was assessed using iodoacetamido-salicylate to alkylate unreacted cysteines (+193 Da) in the 8 kDa biosynthetic intermediates; three of the first four rings formed were thiazoles, and by the five ring stage, all four of the cysteines had been heterocyclized while three of the original four serines remained uncyclized. Finally, tandem MS using a 9.4 T Fourier transform instrument with electrospray ionization was used to elaborate the major processing pathway: the first two rings formed are at the most amino proximal sites (Cys(41) then Ser(40)) followed by the remaining three cysteines at positions 48, 51, and 55. The cyclization of serines at positions 56, 62, and 65 then follows, with Ser(62) and Ser(65) the last to heterocyclize and the first of these at a slower rate. Thus, despite free dissociation of intermediates after each of seven ring-forming catalytic cycles, there is an overall directionality of ring formation from N-terminal to C-terminal sites. This remarkable regioselectivity is determined more by the substrate than the enzyme, due to a combination of (1) initial high-affinity binding of the posttranslational catalyst to the N-terminal propeptide of substrate 88mer, and (2) a chemoselectivity for thiazole over oxazole formation. This mechanism is consistent with antibiotic biosynthesis in vivo, yielding microcin with six, seven, and eight rings, all with bioactivity.

Identification of intact proteins in mixtures by alternated capillary liquid chromatography electrospray ionization and LC ESI infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.

Here we propose a novel method for rapidly identifying proteins in complex mixtures. A list of candidate proteins (including provision for posttranslational modifications) is obtained by database searching, within a specified mass range about the accurately measured mass (e.g., +/- 0.1 Da at 10 kDa) of the intact protein, by capillary liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS). On alternate scans, LC ESI infrared multiphoton dissociation (IRMPD) FT-ICR MS yields mostly b and y fragment ions for each protein, from which the correct candidate is identified as the one with the highest "hit" score (i.e., most b and y fragments matching the candidate database protein amino acid sequence masses) and sequence "tag" score (based on a series of fragment sequences differing in mass by 1 or 2 amino acids). The method succeeds in uniquely identifying each of a mixture of five proteins treated as unknowns (melittin, ubiquitin, GroES, myoglobin, carbonic anhydrase II), from more than 1000 possible database candidates within a +/- 500 Da mass window. We are also able to identify posttranslational modifications of two of the proteins (mellitin and GroES). The method is simple, rapid, and definitive and is extendable to a mixture of affinity-selected proteins, to identify proteins with a common biological function.

Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Protein tyrosine phosphatases (PTPase) play important roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. Here, solvent accessibility is determined for backbone amide protons from various segments of wild-type Yersinia PTPase in the presence or absence of 220 microM vanadate, a competitive inhibitor, as well as an active site mutant in which the essential cysteine 403 has been replaced by serine (C403S). The method consists of solution-phase H/D exchange, followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform ion cyclotron resonance mass spectrometry. Proteolytic segments spanning approximately 93.5% of the primary sequence are analyzed. Binding of vanadate reduces the H/D exchange rate throughout the protein, both for the WpD loop and for numerous other residues that are shielded when that loop is pulled down over the active site on binding of the inhibitor. The single active site C403S mutation reduces solvent access to the WpD loop itself, but opens up the structure in several other segments. Although the 3D structure of the ligand-bound C403S mutant is similar to that of the wild-type PTPase, and the C403S mutant and the wild-type enzyme display similar affinities for vanadate, the thermodynamics for binding of vanadate is different for the two proteins. Collectively, these results establish the flexibility of the WpD loop (previously inferred by comparing PTPase X-ray single-cyrstal diffraction structures in the presence and absence of a tungstate inhibitor), as well as several other signficant changes in segment exposure and/or flexibility that are not evident from X-ray structures.

High-resolution electrospray ionization Fourier transform mass spectrometry with infrared multiphoton dissociation of glucokinase from Bacillus Stearothermophilus.

Glucokinase (GK, EC 2.7.1.2), a member of the enzyme family of hexokinases, has been shown to be linked to maturity-onset diabetes of the young type II (MODY-2). Although nucleotide and amino acid sequence information are available for the human varieties, they are not known for the variety from Bacillus stearothermophilus, which is often used in protein binding studies. Here, a combination of electrospray Fourier transform mass spectrometry (FTMS) and infrared multiphoton dissociation (IRMPD) is used to obtain accurate molecular weight and preliminary amino acid sequence information for the protein. Electrospray FTMS provides evidence of a solution phase dimer. In addition, dithiothreitol reduction shows no shift in high-resolution isotopic distributions, indicating a probable absence of disulfide bonds in the protein. The partial sequence information obtained from IRMPD could be the basis for creating a DNA probe for the protein.

Fourier transform ion cyclotron resonance mass spectrometry: a primer.

This review offers an introduction to the principles and generic applications of FT-ICR mass spectrometry, directed to readers with no prior experience with the technique. We are able to explain the fundamental FT-ICR phenomena from a simplified theoretical treatment of ion behavior in idealized magnetic and electric fields. The effects of trapping voltage, trap size and shape, and other nonidealities are manifested mainly as perturbations that preserve the idealized ion behavior modified by appropriate numerical correction factors. Topics include: effect of ion mass, charge, magnetic field, and trapping voltage on ion cyclotron frequency; excitation and detection of ICR signals; mass calibration; mass resolving power and mass accuracy; upper mass limit(s); dynamic range; detection limit, strategies for mass and energy selection for MSn; ion axialization, cooling, and remeasurement; and means for guiding externally formed ions into the ion trap. The relation of FT-ICR MS to other types of Fourier transform spectroscopy and to the Paul (quadrupole) ion trap is described. The article concludes with selected applications, an appendix listing accurate fundamental constants needed for ultrahigh-precision analysis, and an annotated list of selected reviews and primary source publications that describe in further detail various FT-ICR MS techniques and applications.

Counting individual sulfur atoms in a protein by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry: experimental resolution of isotopic fine structure in proteins.

A typical molecular ion mass spectrum consists of a sum of signals from species of various possible isotopic compositions. Only the monoisotopic peak (e.g., all carbons are 12C; all nitrogens are 14N, etc.) has a unique elemental composition. Every other isotope peak at approximately integer multiples of approximately 1 Da higher in nominal mass represents a sum of contributions from isotope combinations differing by a few mDa (e.g., two 13C vs. two 15N vs. one 13C and one 15N vs. 34S, vs. 18O, etc., at approximately 2 Da higher in mass than the monoisotopic mass). At sufficiently high mass resolving power, each of these nominal-mass peaks resolves into its isotopic fine structure. Here, we report resolution of the isotopic fine structure of proteins up to 15.8 kDa (isotopic 13C,15N doubly depleted tumor suppressor protein, p16), made possible by electrospray ionization followed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass analysis at 9.4 tesla. Further, a resolving power of m/Deltam50% approximately 8,000,000 has been achieved on bovine ubiquitin (8.6 kDa). These results represent a 10-fold increase in the highest mass at which isotopic fine structure previously had been observed. Finally, because isotopic fine structure reveals elemental composition directly, it can be used to confirm or determine molecular formula. For p16, for example, we were able to determine (5.1 +/- 0.3) the correct number (five) of sulfur atoms solely from the abundance ratio of the resolved 34S peak to the monoisotopic peak.

Application of micro-electrospray liquid chromatography techniques to FT-ICR MS to enable high-sensitivity biological analysis.

A microbore electrospray (ESI) injection system has been adapted to our 9.4-tesla ESI FT-ICR mass spectrometer, greatly enhancing the stability and sensitivity of the system. Spray was generated from micro-ESI needles made from sharply tapered, polished fused silica capillaries of 25 to 50 microns inner diameter. Micro-ESI permits low-level sample analysis by constant infusion at sub-microL/min flow rate over a wide range of solvent conditions in both positive- and negative-ion mode. The system is flexible and allows rapid conversion to allow routine LC/MS analysis on low-level mixtures presented in biological media. LC/MS analyses were accomplished by replacing micro-ESI needles with capillaries packed with reverse phase retention media to permit analyte concentration and purification prior to analysis (micro-ESI/LC). A unique nano-flow LC pumping system was developed, capable of producing a true unsplit solvent gradient at flow rates below 1 microL/min. The micro-ESI/LC FT-ICR system produces mass spectra from a mixture of three neuroactive peptides at a concentration of 500 amol/microL (5 fmol each total loaded) in biological salts with baseline separation, signal-to-noise ratio of > 10:1 and mass resolving power > 5000. These results represent a reduction in detection limit by a factor of approximately 2 x 10(6) over the best previously published LC/FT-ICR MS data.

Identification, Composition, and Asymmetric Formation Mechanism of Glycidyl Methacrylate/Butyl Methacrylate Copolymers up to 7000 Da from Electrospray Ionization Ultrahigh-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Glycidyl methacrylate (GMA) and butyl methacrylate (BMA) have the same nominal mass (142 Da) but differ in exact mass by 0.036 Da (CH(4) vs O). Therefore, copolymers formed from the two isobaric monomers exhibit a characteristic isobaric distribution due to different monomer compositions. Here, we show that electrospray ionization FT-ICR mass spectrometry at 9.4 T resolves the isobaric components of copolymers as large as 7000 Da with a resolving power (m/Δm(50%)) of ∼500 000 in a gel permeation chromatography fractionated polymer sample. That resolution provides for complete and unequivocal component analysis of such copolymers of the size used for high solid content automobile coatings. All five possible copolymer products predicted by the polymerization mechanism are resolved and identified in the mass spectrum. Two of those polymer series (each with saturated end group) were previously unresolved by mass spectrometry because they differ in mass from the two other unsaturated products by only 0.0089 Da. Finally, analysis of the asymmetrical isobaric distribution for the copolymer n-mers, (GMA)(m)(BMA)(n)(-)(m), 0≤ m ≤ n, in which species with adjacent values of m differ from each other in mass by 36 mDa (i.e., the mass difference, CH(4) vs O, between GMA and BMA) proves that GMA is less reactive than BMA in the polymerization process.

Electrospray ionization Fourier transform ion cyclotron resonance at 9.4 T.

We present the first results from a new electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer operated at a magnetic field of 9.4 T (i.e. > or = 2.4 T higher than for any prior FTICR instrument). The 9.4 T instrument provides substantially improved performance for large molecules (> or = 50% increase in mass resolving power) and complex mixtures (> or = 100% increase in dynamic range) compared to lower-field (< or = 6 T) instruments. The higher magnetic field makes possible larger trapped-ion population without introduction of significant space--charge effects such as spectral peak shift and/or distortion, and coalescence of closely-spaced resonances. For bovine ubiquitin (8.6 kDa) we observe accurate relative isotopic abundances at a signal-to-noise ratio greater than 1000:1, whereas a complete nozzle-skimmer dissociation electrospray ionization (ESI) FTICR mass spectrum of bovine carbonic anhydrase (29 kDa) is achieved from a single scan with a signal-to-noise ratio of more than 250:1. Finally, we are able to obtain mass resolving power, m/delta m > 200,000, routinely for porcine serum albumin (67 kDa). The present performance guides further modifications of the instrument, which should lead to significant further improvements.