Targeted tandem mass spectrometry for high-throughput comparative proteomics employing NanoLC-FTICR MS with external ion dissociation

Targeted tandem mass spectrometry (MS/MS) is an attractive proteomic approach that allows selective identification of peptides exhibiting abundance differences, e.g., between culture conditions and/or diseased states. Herein, we report on a targeted LC-MS/MS capability realized with a hybrid quadrupole-7 tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer that provides data-dependent ion selection, accumulation, and dissociation external to the ICR trap, and a control software that directs intelligent MS/MS target selection based on LC elution time and m/z ratio. We show that the continuous on-the-fly alignment of the LC elution time during the targeted LC-MS/MS experiment, combined with the high mass resolution of FTICR MS, is crucial for accurate selection of targets, whereas high mass measurement accuracy MS/MS data facilitate unambiguous peptide identifications. Identification of a subset of differentially abundant proteins from Shewanella oneidensis grown under suboxic versus aerobic conditions demonstrates the feasibility of such approach.     

External accumulation of ions for enhanced electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry provides for mass analysis of biological molecules with unrivaled mass accuracy, resolving power and sensitivity. However, ESI FTICR MS performance with on-line separation techniques such as liquid chromatography (LC) and capillary electrophoresis has to date been limited primarily by pulsed gas assisted accumulation and the incompatibility of the associated pump-down time with the frequent ion beam sampling requirement of on-line chromatographic separation. Here we describe numerous analytical advantages that accrue by trapping ions at high pressure in the first rf-only octupole of a dual octupole ion injection system before ion transfer to the ion trap in the center of the magnet for high performance mass analysis at low pressure. The new configuration improves the duty cycle for analysis of continuously generated ions, and is thus ideally suited for on-line chromatographic applications. LC/ESI FTICR MS is demonstrated on a mixture of 500 fmol of each of three peptides. Additional improvements include a fivefold increase in signal-to-noise ratio and resolving power compared to prior methods on our instrument.     

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics

We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dual-channel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1-5% deviation in uncorrected elution times), repeatability (<20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ion-energy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10(3), and >10(5) for peptide abundances in the overall separation.     

Electrospray ionization-Fourier transform ion cyclotron mass spectrometry using ion preselection and external accumulation for ultrahigh sensitivity

The dynamic range of Fourier transform ion cyclotron mass spectrometry (FTICR) is typically limited by the useful charge capacity of an FTICR cell (to approximately 10(6) to 10(7) elementary charges) and the minimum number of ions required to produce a useful signal (approximately 10(2) elementary charges). We show that the expansion of the dynamic range by 2 orders of magnitude can be achieved by preselecting lower abundance species in a quadrupole interface to an electrospray ionization (ESI) source. Ion preselection is then followed by ion accumulation in external to the FTICR cell a linear (2-D) quadrupole trap and subsequent transfer to the region of high magnetic field for gated trapping in the FTICR cell. Two modes of ion preselection, using either the quadrupole filtering mode or rf-only dipolar excitation, were studied and mass resolutions of 30 to 100 were achieved for selective external ion accumulation of peptides and proteins with molecular weights ranging from 500 to 17,000 Da. The ability to selectively eject the most abundant species before trapping in the FTICR has enormous practical benefits for increasing the sensitivity and dynamic range of measurements.     

A new technique for unbiased external ion accumulation in a quadrupole two-dimensional ion trap for electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

External ion accumulation in a two-dimensional (2D) multipole trap has been shown to increase the sensitivity, dynamic range and duty cycle of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. However, it is important that trapped ions be detected without significant bias at longer accumulation times in the external 2D multipole trap. With increasing ion accumulation time pronounced m/z discrimination was observed when trapping ions in an accumulation quadrupole. In this work we show that superimposing lower rf-amplitude dipolar excitation over the main rf-field in the accumulation quadrupole results in disruption of the m/z discrimination and can potentially be used to achieve unbiased external ion accumulation with FTICR.     

Automatic internal calibration in liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry of protein digests

Accurately measured peptide masses can be used for large-scale protein identification from bacterial whole-cell digests as an alternative to tandem mass spectrometry (MS/MS) provided mass measurement errors of a few parts-per-million (ppm) are obtained. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) routinely achieves such mass accuracy either with internal calibration or by regulating the charge in the analyzer cell. We have developed a novel and automated method for internal calibration of liquid chromatography (LC)/FTICR data from whole-cell digests using peptides in the sample identified by concurrent MS/MS together with ambient polydimethylcyclosiloxanes as internal calibrants in the mass spectra. The method reduced mass measurement error from 4.3 +/- 3.7 ppm to 0.3 +/- 2.3 ppm in an E. coli LC/FTICR dataset of 1000 MS and MS/MS spectra and is applicable to all analyses of complex protein digests by FTICRMS.     

A dynamic ion cooling technique for FTICR mass spectrometry

A fast dynamic ion cooling technique based upon the adiabatic invariant phenomenon for Fourier transform ion cyclotron resonance mass spectrometry (FTICR) is presented. The method cools ions in the FTICR trap more efficiently, within a few hundred milliseconds without the use of a buffer gas, and results in a substantial signal enhancement. All performance aspects of the FTICR spectrum, e.g., peak intensities, mass resolution, and mass accuracy, improve significantly compared with cooling based on ion-ion interactions. The method may be useful in biological applications of FTICR, such as in proteomic studies involving extended on-line liquid chromatography (LC) separations, in which both the duty cycle and mass accuracy are crucially important.     

A novel mass spectrometry cluster for high-throughput quantitative proteomics

We have developed and implemented a novel mass spectrometry (MS) platform combining the advantages of high mass accuracy and resolving power of Fourier transform ion cyclotron resonance (FTICR) with the economy and speed of multiple ion traps for tandem mass spectrometry. The instruments are integrated using novel algorithms and software and work in concert as one system. Using chromatographic time compression, a single expensive FTICR mass spectrometer can match the throughput of multiple relatively inexpensive ion trap instruments. Liquid chromatography (LC)-mass spectrometry data from the two types of spectrometers are aligned and combined to hybrid datasets, from which peptides are identified using accurate mass from the FTICR data and tandem mass spectra from the ion trap data. In addition, the high resolving power and dynamic range of a 12 tesla FTICR also allows precise label-free quantitation. Using two ion traps in parallel with one LC allows simultaneous MS/MS experiments and optimal application of collision induced dissociation and electrontransfer dissociation throughout the chromatographic separation for increased proteome coverage, characterization of post-translational modifications and/or simultaneous measurement in positive and negative ionization mode. An FTICR-ion trap cluster can achieve similar performance and sample throughput as multiple hybrid ion trap-FTICR instruments, but at a lower cost. We here describe the first such FTICR-ion trap cluster, its performance and the idea of chromatographic compression.     

Increased proteome coverage for quantitative peptide abundance measurements based upon high performance separations and DREAMS FTICR mass spectrometry

A primary challenge in proteome measurements is to be able to detect, identify, and quantify the extremely complex mixtures of proteins. The relative abundances of interest span at least six orders of magnitude for mammalian proteomes, and this constitutes an intractable challenge for high throughput proteome studies. We have recently described a new approach, Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS), which is based upon the selective ejection of the most abundant species to expand the dynamic range of Fourier transform ion cyclotron resonanace (FTICR) measurements. The basis of our approach is on-the-fly data-dependent selective ejection of highly abundant species, followed by prolonged accumulation of remaining low-abundance species in a quadrupole external to the FTICR ion trap. Here we report the initial implementation of this approach with high efficiency capillary reverse phase LC separations and high magnetic field electrospray ionization FTICR mass spectrometry for obtaining enhanced coverage in quantitative measurements for mammalian proteomes. We describe the analysis of a sample derived from a tryptic digest of proteins from mouse B16 cells cultured in both natural isotopic abundance and 15N-labeled media. The FTICR mass spectrometric analysis allows the assignment of peptide pairs (corresponding to the two distinctive versions of each peptide), and thus provides the basis for quantiative measurements when one of the two proteomes in the mixture is perturbed or altered in some fashion. We show that implementation of the DREAMS approach allows assignment of approximately 80% more peptide pairs, thus providing quantitative information for approximately 18,000 peptide pairs in a single analysis.     

A new and sensitive on-line liquid chromatography/mass spectrometric approach for top-down protein analysis: the comprehensive analysis of human growth hormone in an E. coli lysate using a hybrid linear ion trap/Fourier transform ion cyclotron resonance mass spectrometer

A sensitive, integrated top-down liquid chromatography/mass spectrometry (LC/MS) approach, suitable for the near complete characterization of specific proteins in complex protein mixtures, such as inclusion bodies of an E. coli lysate, has been successfully developed using a hybrid linear ion trap/Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. In particular, human growth hormone (hGH) (200 fmol) was analyzed with high sequence coverage (>95%), including the sites of disulfide linkages. The high mass accuracy and resolution of the FTICR mass spectrometer was used to reveal high charge state ions of hGH (22 kDa). The highly charged intact protein ions (such as the 17+ species) were captured and fragmented in the linear ion trap cell. The fragment ions from MS/MS spectra were then successfully analyzed in the FTICR cell in an on-line LC/MS run. Peptide fragments from the N-terminal and C-terminal regions, as well as large interior fragments, were captured and identified. The results allowed the unambiguous assignment of disulfide bonds Cys53-Cys165 and Cys182-Cys189, indicative of proper folding of hGH. The disulfide bond assignments were also confirmed by analysis of the tryptic digest of a sample of hGH purified from inclusion bodies. On-line LC/MS with the linear ion trap/FTICR yields high mass accuracy in both the MS and MS/MS modes (within 2 ppm with external calibration). The approach should prove useful in biotechnology applications to characterize correctly folded proteins, both in the early protein expression and the later processed stages, using only a single automated on-line LC/MS top-down method.     

Optimal pressure conditions for unbiased external ion accumulation in a two-dimensional radio-frequency quadrupole for Fourier transform ion cyclotron resonance mass spectrometry.

When combined with on-line separations (e.g., capillary liquid chromatography (LC)), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides a powerful tool for biological applications, and particularly proteomic studies. The sensitivity, dynamic range, and duty cycle provided by FTICR-MS have been shown to be increased by ion trapping and accumulation in a two-dimensional (2D) radio-frequency (rf)-only multipole positioned externally to an FTICR cell. However, it is important that ions be detected across the desired m/z range without a significant bias. In this work we found that pressure inside the accumulation rf-quadrupole plays an important role in obtaining "unbiased" ion accumulation. Pressure optimization was performed in both pulsed and continuous modes. It was found that unbiased accumulation in a 2D rf-only quadrupole could be achieved in the pressure range of 5 x 10(-4) to 5 x 10(-3) Torr. External ion accumulation performed at the optimal pressure resulted in an increase in both the spectrum acquisition rates and dynamic range.     

Magnetic field focused ion accumulation for an internal bore liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer using a central trapping electrode

Presented is the application and evaluation of a magnetic field focusing central trapping electrode ion accumulation cell for a capillary liquid chromatography electrospray Fourier transform ion cyclotron (LC-ESI/FTICR) mass spectrometer. The ESI source and accumulation cell are located within the magnetic field to confine the radial motion of the ions, eliminating the need for elaborate focusing optics to transport the ions to the low-pressure analyzer cell for analysis. The central trapping electrode accumulation cell increases sensitivity by providing the necessary potential well in a confined volume to capture ions currently lost during the detection event of LC/FTICR experiments. With this electrode geometry the time needed to gate the ions into the analyzer cell is reduced and pump down delays are minimized. The decreased scan time improves LC resolution and increases the number of mass spectral scans per eluted component while maintaining appropriate base pressures for high performance ESI/FTICR. Results achieved with the central trapping electrode accumulation cell include an effective duty cycle increase from 10% to 40%, a S/N increase by a factor of 30, and a mass resolution increase of 80%.     

Chromatographic alignment of LC-MS and LC-MS/MS datasets by genetic algorithm feature extraction

Liquid chromatography-mass spectrometry (LC-MS) datasets can be compared or combined following chromatographic alignment. Here we describe a simple solution to the specific problem of aligning one LC-MS dataset and one LC-MS/MS dataset, acquired on separate instruments from an enzymatic digest of a protein mixture, using feature extraction and a genetic algorithm. First, the LC-MS dataset is searched within a few ppm of the calculated theoretical masses of peptides confidently identified by LC-MS/MS. A piecewise linear function is then fitted to these matched peptides using a genetic algorithm with a fitness function that is insensitive to incorrect matches but sufficiently flexible to adapt to the discrete shifts common when comparing LC datasets. We demonstrate the utility of this method by aligning ion trap LC-MS/MS data with accurate LC-MS data from an FTICR mass spectrometer and show how hybrid datasets can improve peptide and protein identification by combining the speed of the ion trap with the mass accuracy of the FTICR, similar to using a hybrid ion trap-FTICR instrument. We also show that the high resolving power of FTICR can improve precision and linear dynamic range in quantitative proteomics. The alignment software, msalign, is freely available as open source.     

Combined infrared multiphoton dissociation and electron capture dissociation with a hollow electron beam in Fourier transform ion cyclotron resonance mass spectrometry

An electron injection system based on an indirectly heated ring-shaped dispenser cathode has been developed and installed in a 7 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This new hardware design allows high-rate electron capture dissociation (ECD) to be carried out by a hollow electron beam coaxial with the ion cyclotron resonance (ICR) trap. Infrared multiphoton dissociation (IRMPD) can also be performed with an on-axis IR-laser beam passing through a hole at the centre of the dispenser cathode. Electron and photon irradiation times of the order of 100 ms are required for efficient ECD and IRMPD, respectively. As ECD and IRMPD generate fragments of different types (mostly c, z and b, y, respectively), complementary structural information that improves the characterization of peptides and proteins by FTICR mass spectrometry can be obtained. The developed technique enables the consecutive or simultaneous use of the ECD and IRMPD methods within a single FTICR experimental sequence and on the same ensemble of trapped ions in multistage tandem (MS/MS/MS or MS(n)) mass spectrometry. Flexible changing between ECD and IRMPD should present advantages for the analysis of protein digests separated by liquid chromatography prior to FTICRMS. Furthermore, ion activation by either electron or laser irradiation prior to, as well as after, dissociation by IRMPD or ECD increases the efficiency of ion fragmentation, including the w-type fragment ion formation, and improves sequencing of peptides with multiple disulfide bridges. The developed instrumental configuration is essential for combined ECD and IRMPD on FTICR mass spectrometers with limited access into the ICR trap.     

Controlled ion fragmentation in a 2-D quadrupole ion trap for external ion accumulation in ESI FTICR mass spectrometry

Undesired fragmentation of electrospray generated ions in an rf multipole traps can be problematic in many applications. Of special interest here is ion dissociation in a 2-D quadrupole ion trap external to a Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) used in proteomic studies. In this work, we identified the experimental parameters that determine the efficiency of ion fragmentation. We have found that under the pressure conditions used in this study there is a specific combination of the radial and axial potential well depths that determines the fragmentation threshold. This combination of rf and dc fields appears to be universal for ions of different mass-to-charge ratios, molecular weights, and charge states. Such universality allows the fragmentation efficiency of the trapped ions in the course of capillary liquid chromatography (LC) separation studied to be controlled and can increase the useful duty cycle and dynamic range of a FTICR mass spectrometer equipped with an external rf only 2-D quadrupole ion trap.     

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

High performance fourier transform ion cyclotron resonance mass spectrometric detection for capillary electrophoresis

We describe the current state of the on-line combination of capillary electrophoresis (CE) electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS), and discuss aspects of the FTICR technique relevant to its use as a detection scheme for on-line separations. Aspects including sensitivity, mass resolution, duty cycle, and tandem mass spectrometric capabilities are discussed in the context of online separations with examples from the authors' laboratory.     

ESI-FTICR mass spectrometry employing Data-dependent external ion selection and accumulation

Data-dependent external m/z selection and accumulation of ions is demonstrated in use with ESI-FTICR instrumentation, with two different methods for ion selection being explored. One method uses RF/DC quadrupole filtering and is described in use with an 11.5 tesla (T) FTICR instrument, while the second method employs RF-only resonance dipolar excitation selection and is described in use with a 3.5 T FTICR instrument. In both methods ions are data-dependently selected on the fly in a linear quadrupole ion guide, then accumulated in a second linear RF-only quadrupole trap that immediately follows. A major benefit of ion preselection prior to external accumulation is the enhancement of ion populations for low-level species. This development is expected to expand the dynamic range and sensitivity of FTICR for applications including analysis of complex polypeptide mixtures (e.g., proteomics).     

Restrained ion population transfer: a novel ion transfer method for mass spectrometry

Fourier transform ion cyclotron resonance (FTICR) mass spectrometers function such that the ion accumulation event takes place in a region of higher pressure outside the magnetic field which allows ions to be thermally cooled before being accelerated toward the ICR cell where they are decelerated and re-trapped. This transfer process suffers from mass discrimination due to time-of-flight effects. Also, trapping ions with substantial axial kinetic energy can decrease the performance of the FTICR instrument compared with the analysis of thermally cooled ions located at the trap center. Therefore, it is desirable to limit the energy imparted to the ions which results in lower applied trap plate potentials and reduces the spread in axial kinetic energy. The approach presented here for ion transfer, called restrained ion population transfer or RIPT, is designed to provide complete axial and radial containment of an ion population throughout the entire transfer process from the accumulation region to the ICR cell, eliminating mass discrimination associated with time-of-flight separation. This was accomplished by use of a number of quadrupole segments arranged in series with independent control of the direct current (DC) bias voltage applied to each segment of the quadrupole ion guide. The DC bias voltage is applied in such a way as to minimize the energy imparted to the ions allowing transfer of ions with low kinetic energy from the ion accumulation region to the ICR cell. Initial FTICR mass spectral data are presented that illustrate the feasibility of RIPT. A larger m/z range for a mixture of peptides is demonstrated compared with gated trapping. The increase in ion transfer time (3 ms to 130 ms) resulted in an approximately 11% decrease in the duty cycle; however this can be improved by simultaneously transferring multiple ion populations with RIPT. The technique was also modeled with SIMION 7.0 and simulation results that support our feasibility studies of the ion transfer process are presented.     

Precision profiling and identification of human serum peptides using Fourier transform ion cyclotron resonance mass spectrometry

Many biomarker discovery studies are based on matrix-assisted laser desorption/ionisation (MALDI) peptide profiles. In this study, 96 human serum samples were analysed on a Bruker solariX(TM) MALDI Fourier transform ion cyclotron resonance (FTICR) system equipped with a 15 tesla magnet. Isotopically resolved peptides were observed in ultrahigh resolution FTICR profiles up to m/z 6500 with mass measurement errors (MMEs) of previously identified peptides at a sub-ppm level. For comparison with our previous platform for peptide profile mass analysis (i.e. Ultraflex II) the corresponding time-of-flight (TOF) spectra were obtained with isotopically resolved peptides up to m/z 3500. The FTICR and TOF systems performed rather similar with respect to the repeatability of the signal intensities. However, the mass measurement precision improved at least 10-fold in ultrahigh resolution data and thus simplified spectral alignment necessary for robust and quantitatively precise comparisons of profiles in large-scale clinical studies. From each single MALDI-FTICR spectrum an m/z-list was obtained with sub-ppm precision for all different species, which is beneficial for identification purposes and interlaboratory comparisons. Furthermore, the FTICR system allowed new peptide identifications from collision-induced dissociation (CID) spectra using direct infusion of reversed-phase (RP) C(18)-fractionated serum samples on an electrospray ionisation (ESI) source.