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.     

Initial implementation of external accumulation liquid chromatography/electrospray ionization Fourier transform ion cyclotron resonance with automated gain control

Capillary liquid chromatography (LC) separation coupled with external accumulation Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has recently been demonstrated to have significant potential for proteomics research. Accumulation of an excessive space charge external to the FTICR cell ion trap has been shown to result in increased mass measurement error, undesirable ion discrimination and/or fragmentation, potentially causing misrepresentation or incorrect assignments of lower abundance peptides in the acquired mass spectra. In this work we report on the capability of data-dependent adjustment of ion accumulation times in the course of LC separations, further referred to as automated gain control (AGC). Three different AGC approaches were evaluated based on the number of putative peptides from a tryptic digest of four casein proteins detected in the course of LC/FTICR separations. When compared with the conventional technique, AGC was found to increase, up to a factor of 3, the total number of peptides identified.     

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.     

Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing-mass spectrometry

The human liver alcohol dehydrogenase (ADH) isoenzymes are currently believed to play a major role in ethanol metabolism, accounting for most of the ethanol oxidized in the liver. They have similar molecular masses and similar isoelectric point (pI) values (the 13 possible isoenzymes having pIs in the range of 8.26-8.87), making their characterization a significant analytical challenge. Capillary isoelectric focusing (CIEF) coupled on-line with electrospray ionization - Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was applied to separate and characterize mixtures of alphaalpha, beta1beta1 and beta3beta3 ADH isoenzymes. Seven different species were resolved by the separation in the pI 8.26-8.67 range. ESI-FTICR analysis of native ADHs revealed that each noncovalent ADH complex contains two monomeric protein units and four zinc atoms. The combination of CIEF separations with mass spectrometry appears well-suited for detailed characterization of ADH isozymes, and the attomole level sensitivity of FTICR should allow very small samples to be addressed.     

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.     

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.     

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.     

Interfacing matrix-assisted laser desorption/ionization mass spectrometry with column and planar separations

This paper presents a critical review of off-line and on-line coupling of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to liquid column separations (e.g. HPLC, GPC and CE) and planar separations (e.g. PAGE and TLC). Off-line MALDI analysis of fractions collected from HPLC, GPC and CE or spots scraped and extracted from TLC and PAGE has already become a routine practice for many laboratories. MALDI has also been used to obtain mass spectra directly from TLC plates and PAGE. The direct analysis methods range from dot-blotted samples to two-dimensional scanning of the entire gels/plates. Various combinations of on-line coupling of MALDI with column separations are also reviewed. The review discusses the strengths and limitations associated with different off-line and on-line coupling approaches.     

Interfacing matrix-assisted laser desorption/ionization mass spectrometry with column and planar separations

This paper presents a critical review of off-line and on-line coupling of matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry to liquid column separations (e.g. HPLC, GPC and CE) and planar separations (e.g. PAGE and TLC). Off-line MALDI analysis of fractions collected from HPLC, GPC and CE or spots scraped and extracted from TLC and PAGE has already become a routine practice for many laboratories. MALDI has also been used to obtain mass spectra directly from TLC plates and PAGE. The direct analysis methods range from dot-blotted samples to two-dimensional scanning of the entire gels/plates. Various combinations of on-line coupling of MALDI with column separations are also reviewed. The review discusses the strengths and limitations associated with different off-line and on-line coupling approaches.     

Obtaining more accurate Fourier transform ion cyclotron resonance mass measurements without internal standards using multiply charged ions

Space-charge effects produce frequency shifts in Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and correction for these shifts is necessary for obtaining accurate mass measurements. We report a novel method for obtaining accurate mass calibration to correct for space-charge induced mass shifts without the requirement for internal calibrants. The new approach is particularly well suited for electrospray ionization-FTICR mass spectra that contain multiple charge states of the same molecular species. This method, deconvolution of Coulombic affected linearity (DeCAL), is described and presented with several examples demonstrating the increased mass measurement accuracy obtained. DeCAL provides the basis for more routinely obtaining higher mass accuracy measurements in conjunction with chromatographic separations for complex mixture analysis, and obviates the need for internal calibration in many applications.     

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.     

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 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.     

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.     

Phytochemical analysis of traditional Chinese medicine using liquid chromatography coupled with mass spectrometry

Traditional Chinese medicine (TCM) is commonly considered to operate due to the synergistic effects of all the major and minor components in the medicines. Hence sensitive and comprehensive analytical techniques are needed to acquire a better understanding of the pharmacological basis of the herb and to enhance the product quality control. The present review mainly focuses on the phytochemical analysis of TCMs using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are the two commonly used ion sources. Triple quadrupole, ion trap (IT), Fourier transform ion cyclotron resonance (FTICR) and time-of-flight (TOF) mass spectrometers are used as on-line analyzer. The relationship between structural features and fragmentation patterns should be investigated as thoroughly as possible and hence be applied in the on-line analysis to deduce the structures of detected peaks. Characteristic fragmentation behaviors of the reference standards, as well as information regarding polarity obtained from retention time data, on-line UV spectra, data from the literature and bio-sources of the compounds allowed the identification of the phytochemical constituents in the crude extracts. Although a mass spectrometer is not a universal detector, high-performance liquid chromatography coupled with multistage mass spectrometry (HPLC-MS(n)) technique was still proved to be a rapid and sensitive method to analyze the majority of the many constituents in herbal medicines, particularly for the detection of those present in minor or trace amounts. The methods established using HPLC-MS techniques facilitate the convenient and rapid quality control of traditional medicines and their pharmaceutical preparations. However, the quantitative analysis is not the topic of this review.     

Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry.

The patterns of gene expression, post-translational modifications, protein/biomolecular interactions, and how these may be affected by changes in the environment, cannot be accurately predicted from DNA sequences. Approaches for proteome characterization are generally based upon mass spectrometric analysis of in-gel digested two dimensional polyacrylamide gel electrophoresis (2-D PAGE) separated proteins, allowing relatively rapid protein identification compared to conventional approaches. This technique, however, is constrained by the speed of the 2-D PAGE separations, the sensitivity limits intrinsic to staining necessary for protein visualization, the speed and sensitivity of subsequent mass spectrometric analyses for identification, and the limited ability for accurate quantitative measurements based on differences in spot intensity. We are presently developing alternative approaches for proteomics based upon the combination of fast capillary electrophoresis, or other suitable chromatographic separations, and the high mass accuracy and sensitivity obtainable with unique Fourier transform ion cyclotron resonance (FTICR) mass spectrometers available at our laboratory. Several approaches are presently being pursued; one based upon the analysis of intact proteins and the second upon approaches for global protein digestion and accurate peptide mass analysis. Quantitation of protein/peptide levels are based on using two or more stable-isotope labeled versions of proteomes which are combined to obtain precise quantitation of relative protein abundances. We describe the status of our efforts towards the development of a high-throughput proteomics capability and present initial results for application to several microorganisms and discuss our efforts for extending the developed capability to mammalian proteomes.     

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.     

A top-down LC-FTICR MS-based strategy for characterizing oxidized calmodulin in activated macrophages

A liquid chromatography-mass spectrometry (LC-MS)-based approach for characterizing the degree of nitration and oxidation of intact calmodulin (CaM) has been used to resolve ～250 CaM oxiforms using only 500 ng of protein. The analysis was based on high-resolution data of the intact CaM isoforms obtained by Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS) coupled with an on-line reversed-phase LC separation. Tentative identifications of post-translational modifications (PTMs), such as oxidation or nitration, have been assigned by matching observed protein mass to a database containing all theoretically predicted oxidation products of CaM and verified through a combination of tryptic peptide information (generated from bottom-up analyses) and on-line collisionally induced dissociation (CID) tandem mass spectrometry (MS/MS) at the intact protein level. The reduction in abundance and diversity of oxidatively modified CaM (i.e., nitrated tyrosines and oxidized methionines) induced by macrophage activation has been explored and semiquantified for different oxidation degrees (i.e., no oxidation, moderate, and high oxidation). This work demonstrates the power of the top-down approach to identify and quantify hundreds of combinations of PTMs for single protein target such as CaM and implicate competing repair and peptidase activities to modulate cellular metabolism in response to oxidative stress.     

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.