Improved Mass Measurement Accuracy Research Articles

Sidebands in Fourier transform ion cyclotron resonance mass spectra

Sidebands in mass spectra are an intrinsic feature of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Appearance of the sidebands there is detrimental for the analytical performance, especially in case of complex mixtures analyzed at high resolution. Yet, the sidebands have a practical potential as well. Specifically, they can be applied for fine tuning of ICR cells and were previously employed to improve mass measurement accuracy for small molecules and atoms in fundamental physics experiments. Moreover, experimental characteristics of sidebands allow evaluating the theory of the ICR signal, which provides the metrological basis in FT-ICR MS. Here, we revisit the sidebands phenomenon in the conventional FT-ICR MS, specifically applied to macromolecules. We extend the previous reports on sidebands by examining the appearance of sidebands as functions of ICR cell trapping potentials, resolution, and number of charges for the first three harmonics of the reduced cyclotron frequency. Next, we develop an analytical model of sidebands that contributes to the existing theory of the ICR signal by showing the origin of sidebands to be the result of the broadband amplitude-phase modulation occurring in the ICR signal. Finally, we evaluate the theory of the ICR signal on the basis of the obtained experimental sidebands. Further progress in the theory of the ICR signal shall outline the way for further improvements in FT-ICR MS performance.

Enhanced Analyte Detection Using In-Source Fragmentation of Field Asymmetric Waveform Ion Mobility Spectrometry-Selected Ions in Combination with Time-of-Flight Mass Spectrometry

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (FAIMS) is used for the selective transmission of differential mobility-selected ions prior to in-source collision-induced dissociation (CID) and time-of-flight mass spectrometry (TOFMS) analysis. The FAIMS-in-source collision induced dissociation-TOFMS (FISCID-MS) method requires only minor modification of the ion source region of the mass spectrometer and is shown to significantly enhance analyte detection in complex mixtures. Improved mass measurement accuracy and simplified product ion mass spectra were observed following FAIMS preselection and subsequent in-source CID of ions derived from pharmaceutical excipients, sufficiently close in m/z (17.7 ppm mass difference) that they could not be resolved by TOFMS alone. The FISCID-MS approach is also demonstrated for the qualitative and quantitative analysis of mixtures of peptides with FAIMS used to filter out unrelated precursor ions thereby simplifying the resulting product ion mass spectra. Liquid chromatography combined with FISCID-MS was applied to the analysis of coeluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching. The potential of FISCID-MS for the quantitative determination of a model peptide spiked into human plasma in the range of 0.45-9.0 μg/mL is demonstrated, showing good reproducibility (%RSD < 14.6%) and linearity (R(2) > 0.99).

Improving mass measurement accuracy in mass spectrometry based proteomics by combining open source tools for chromatographic alignment and internal calibration

Accurate mass determination enhances peptide identification in mass spectrometry based proteomics. We here describe the combination of two previously published open source software tools to improve mass measurement accuracy in Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). The first program, msalign, aligns one MS/MS dataset with one FTICRMS dataset. The second software, recal2, uses peptides identified from the MS/MS data for automated internal calibration of the FTICR spectra, resulting in sub-ppm mass measurement errors.

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.

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.

In situ tissue analysis of neuropeptides by MALDI FTMS in-cell accumulation.

Herein we report the first application of Fourier transform mass spectrometry for the analysis of neuropeptides directly from neuronal tissues. Sample preparation protocols and instrumentation conditions are developed to allow in situ neuropeptide analysis of the neuroendocrine organs freshly isolated from a marine organism Cancer borealis. The utility of a previously developed in-cell accumulation (ICA) technique is extended for peptide analysis in complex tissue samples. With the ICA procedure, ion signals from multiple laser shots are accumulated in the analyzer cell prior to detection. This procedure allows the accumulation of ion signals without accumulating noise, thus improving the signal-to-noise ratio and enhancing the sensitivity for the detection of trace-level endogenous neuropeptides. De novo sequencing of peptides directly from tissue samples becomes more feasible through this improvement. Additionally, an integrated pulse sequence is constructed to cover a wide mass range from m/z 215 to 9000 by centering quadrupole collection of ions at different masses for successive laser shots. Finally, improved mass measurement accuracy (2 ppm) for tissue peptide analysis is achieved using ICA by incorporating calibrants on a separate spot from the sample of interest without premixing calibration standards with the analytes.

Accurate mass measurements by Fourier transform mass spectrometry

AbstractThe capability for accurate mass measurements is an important attribute of Fourier transform mass spectrometry (FTMS). Unlike other instrumental methods in mass spectrometry, FTMS still offers significant opportunities to improve mass measurement accuracy (MMA), making it an area of research. This review covers the published literature in FTMS from the late 1970s to the present. We discuss the development and evolution of mass calibration that give accuracies in the low ppm range. We sketch the derivation and show the common foundation of these mass calibration procedures. We also describe the relation of mass calibration and the fundamentals of ion motion and space‐charge effects, and we review efforts to improve the basic calibration procedure particularly those that correct for effects of space charge. The advent of matrix‐assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) have opened the door for FTMS to be used for analyzing at high performance biopolymers, including proteins, oligodeoxynucleotides (ODNs), oligosaccharides, and synthetic polymers. We also discuss the utility of FTMS for accurate mass measurement in these areas and some practical ways to improve MMA. © 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:286–309, 2005

Determination of a correction to improve mass measurement accuracy of isotopically unresolved polymerase chain reaction amplicons by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

The experimental determination of average mass by mass spectrometry is limited for large molecules due to the negative bias introduced by the natural distribution of isotopic abundances. This results in the measurement of the top-of-centroid (ToC) as opposed to the true centroid. We have developed a practical correction factor that is applied to the ToC measurement to largely remove the systematic bias introduced by nature. The correction factor is calculated easily using the average molecular mass (<100 kDa) of the analyte molecule and the full-width half maximum resolving power (<3,500) of the measurement. In addition, an approach to calculating resolving power is described that accurately predicts resolving power achievable for Fourier transform ion cyclotron resonance (FT-ICR) mass analysis of large molecules. A combination of internal calibration with a dual-electrospray source and application of the correction factor to average mass measurements improved the mass error from 192.5 to -35.0 ppm for a 44 kDa PCR amplicon.

Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures.

Capillary isoelectric focusing (CIEF) can provide high-resolution separations of complex protein mixtures, but until recently it has primarily been used with conventional UV detection. This technique would be greatly enhanced by much more information-rich detection methods that can aid in protein characterization. We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and its application to proteome characterization. Studies have revealed 400-1000 putative proteins in the mass range of 2-100 kDa from total injections of approximately 300 ng protein in single CIEF-FTICR analyses of cell lysates for both Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans). We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for quantitative proteome-wide measurements of protein expression. The ability to make such comprehensive and precise measurements of differences in protein expression in response to cellular perturbations should provide new insights into complex cellular processes.

Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Unlike the genome, the proteome is exquisitely sensitive to cellular conditions and will consist of proteins having abundances dependent upon stage in the cell cycle, cell differentiation, response to environmental conditions (nutrients, temperature, stress etc.), or disease state(s). Therefore, the study of proteomes under well-defined conditions can provide a better understanding of complex biological processes and inference of protein function. Thus, much faster, more sensitive, and precise capabilities for the characterization of cellular constituents are desired. We describe progress in the development and initial application of the powerful combination of capillary isoelectric focusing (CIEF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for measurements of the proteome of the model system Escherichia coli. Isotope depletion of the growth media has been used to improve mass measurement accuracy, and the comparison of CIEF-FTICR results for the analysis of cell lysates harvested from E. coli cultured in normal and isotopically depleted media are presented. The initial studies have revealed 400-1000 putative proteins in the mass range 2-100 kDa from total injections of approximately 300 ng of E. coli proteins in a single CIEF-FTICR analysis.

Comparative Mapping of Recombinant Proteins and Glycoproteins by Plasma Desorption and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

The mass spectrometric (MS) techniques of 252Cf-plasma desorption (PD) and matrix-assisted laser desorption/ionization (MALDI) are compared in the molecular weight determination and the mapping analysis of several recombinant proteins and glycoproteins. MALDI MS analysis exhibited better sensitivity and mass measurement accuracy and a remarkably short analysis time compared with PD MS analysis. The latter was not successful in the analysis of rhIFN-gamma and the higher mass mammalian cell-derived IL-5 glycoproteins. Mapping of the Escherichia coli-derived rhIFN alpha-2b and rhIL-4 proteins, by direct PD or MALDI MS analysis of the trypsin-generated peptide mixtures provided signals for ca. 95% and 88% of the expected tryptic peptides, respectively. Peptide signals below m/z 1500 were generally more intense in the PD mass spectra, while higher mass signals were more intense in the MALDI mass spectra. Both PD and MALDI MS analyses provided a rapid confirmation of the existing two and three disulfide bonds in the rhIFN alpha-2b and rhIL-4 proteins, respectively. In the mapping of the CHO IL-4 glycoprotein, detection of the trypsin-generated glycopeptides was only possible by MALDI, where their detection was greatly improved by using the super-DHB (sDHB) matrix, a 9:1 mixture of 2,5-dihydroxybenzoic acid (DHB) with 2-hydroxy-5-methoxybenzoic acid. This sDHB matrix also generated significantly enhanced and better resolved MALDI peptide signals, which in turn resulted in a much improved mass measurement accuracy.

Improved accuracy of mass measurement with a quadrupole ion‐trap mass spectrometer

AbstractMass Measurement accuracy of better than 0.015% on protonated gramicidin S (nominal chemical average molecular weight 1141.5 Da) and its first three isotopes has been achieved using reasonant ion ejection in the quadrupole ion‐trap mass spectrometer operated at unit resolution. Analyte and Calibrant ions are subjected to identical injection and trapping conditions when desorbed by Cs+ ions from a split prone‐tip and co‐injected into the trap. When the mass analysis scan rate is slowed, enhanced resolution is achieved for all ions but the mass range accessed in a single scan of the digital‐to‐analog converter, which controls the mass analysis scan, is limited making calibration difficult. A peak‐matching procedure is described for use at these higher resolution conditions in which the offset voltage which determines the origin of the RF amplitude ramp is adjusted in such a fashion that the analyte and calibrant ions occur at the same point in the mass analysis scan. This procedure gave an improved mass measurement accuracy of better than 0.007% for the protonated gramicidin S. ions.

Quadrupole ion trap mass spectrometry: Current applications and future directions for peptide analysis

The capabilities of an ion trap mass spectrometer (ITMS) in determining the molecular weights and characterizing the primary sequences of peptides are explored. Ionization is accomplished by Cs+ desorption of the sample in a liquid matrix or by laser desorption from a solid matrix and is performed externally to the ion trap, which is operated with mass/charge range extension using resonance ejection. The mass spectra recorded in this way are dominated by the protonated molecules and are relatively free of the chemical noise normally associated with liquid secondary ion mass spectrometry (SIMS) or fast atom bombardment ionization. The matrix-assisted laser desorption spectra are of comparable quality to those obtained with Cs+ SIMS. The molecular weights of a variety of model peptides are measured with a resolution of 103 (FWHM) and a mass measurement accuracy of 0.1%, using mass analysis scan speeds in the 103–104 Da s−1 range. By slowing the scan speed by factors as large as 1000, better than unit resolution is achieved for all the compounds examined and, in the case of gramicidin S, a resolution of more than 105 has been recorded. Tandem mass spectrometry (MS/MS) is performed by isolating the protonated molecule in the trap using a reverse-then-forward scan and then irradiating it by resonance excitation to cause collision-induced dissociation. The product ion tandem mass spectrum is then recorded by the usual resonant ejection scan. Dissociation efficiencies are very high (>50%) and the product spectra are dominated by the fragments of amide bond scission together with associated CO losses (y, b and a-type fragments). In addition, internal cleavage products are also observed. Peptides such as prepro-VIP/PHM, renin substrate and glu-ribinopeptide give tandem spectra which are dominated by y ions, whereas other peptides, such as α-endorphin, give principally b-type fragment ions. The cyclic peptides, gramicidin S and the antibiotic actinomycin D, give spectra dominated by amide bond cleavages. Additional structural information, especially in the low-mass region, is sometimes desirable and can be provided by recording MS3 spectra. This can be done using little additional sample or analysis time. The high-resolution capabilities available by slowing the mass analysis scan time are applied to the MS/MS experiment, allowing unit resolution to be achieved, both in selecting the molecular ion and in recording the product ion spectrum. It is also shown that the higher-resolution resonant excitation experiment does not require isolation of the individual isotopic forms of the protonated molcule in order to provide an isotopically selective product spectrum. Sample sizes loaded onto the probe for MS/MS experiments are typically < 1 pmol and the data acquisition time is usually much shorter than the sample life-time. Shortcomings of the method include limitations common to desorption ionization (analyte suppression effects and reactions in the matrix) as well as the need to calibrate the mass scale with an external standard which results in mass measurement accuracies which, at present, are ±0.1%. A summary is given of steps which are being taken to improve mass measurement accuracy, to deposit increased internal energies to cause more extensive fragmentation, and to improve our understanding of ion traps through simulations of ion motion.

An elongated trapped-ion cell for ion cyclotron resonance mass spectrometry with a superconducting magnet

A one-region trapped-ion analyzer cell that is elongated in the dimension parallel to the magnetic field is described and shown to be well suited for ion cyclotron resonance (ICR) experiments using a solenoidal-geometry superconducting magnet. An analysis of the static electric fields inside the elongated cell shows that the resonance frequency of the ions is far less affected by the trapping voltages than with a cubic cell or the original trapped-ion cell. The main advantages of the elongated cell are improved mass measurement accuracy and increased dynamic range.