High mass measurement accuracy determination for proteomics using multivariate regression fitting: application to electrospray ionization time-of-flight mass spectrometry

Important factors that limit the mass measurement accuracy from a mass spectrometer are related to (1) the type of mass analyzer used and (2) the data processing/calibration methods used to obtain mass values from the raw data. Here, two data processing methods are presented that correct for systematic deviations when the mass of ions is measured using a time-of-flight (TOF) mass spectrometer. The first fitting method is one where m/z values are obtained from fitting peak distributions using double Gaussian functions. A second calibration method takes into account the slight nonlinear response of the TOF analyzer in addition to the drift in the calibration over time. Using multivariate regression, both of these two effects can be corrected for using a single calibration formula. Achievable performance was evaluated with a trypsin digestion of serum albumin and proteins from the organism D. radiodurans that was analyzed using gradient reversed-phase liquid chromatography combined with an electrospray ionization orthogonal TOF mass spectrometer. The root-mean-square deviation between the theoretical and experimental m/z values for serum albumin tryptic peptides was found to be 8 ppm using the double Gaussian-multivariate method compared to 29 ppm determined using linear calibration and normal peak centroiding. An advantage of the methods presented here is that no calibrant compounds need to be added to the mobile phase, thereby avoiding interference effects and signal suppression of analytes.     

Bidirectional ion transfer between quadrupole arrays: MSn ion/ion reaction experiments on a quadrupole/time-of-flight tandem mass spectrometer

Methods for bidirectional ion transmission between distinct quadrupole arrays were developed on a quadrupole/time-of-flight tandem mass spectrometer (QqTOF) containing three quadrupoles (ion guide Q0, mass filter Q1, and collision cell Q2) and a reflectron TOF analyzer, for the purpose of implementing multistage ion/ion reaction experiments. The transfer efficiency, defined as the percentage of ions detected after two transfer steps relative to the initial ion abundance, was found to be about 60% between Q2 and Q0 (with passage through the intermediate array (Q1)) and almost 100% between Q2 and Q1. Efficient ion transfer enabled new means for executing MSn experiments on an instrument of this type by operating Q1 in rf/dc mode for performing multiple steps of precursor/product ion isolation while passing ions through Q1 or trapping ions in Q1. In the latter case, the Q1 functioned as a linear ion trap. Either collision induced dissociation (CID) or ion/ion reactions can be conducted in between each stage of mass analysis. MS3 or MS4 experiments were developed to illustrate the charge increase of peptide ions via two steps of charge inversion ion/ion reactions, CID of electron-transfer dissociation (ETD) products and CID of a metal-peptide complex formed from ion/ion reactions.     

Multiplexed rectilinear ion trap mass spectrometer for high-throughput analysis

A multichannel mass spectrometer based on the rectilinear ion trap (RIT) analyzer was designed and constructed for simultaneous high-throughput analysis of multiple samples. The instrument features four parallel ion source/mass analyzer/detector channels assembled in a single vacuum chamber and operated using a common set of control electronics, including a single rf amplifier and transformer coil. This multiplexed RIT mass spectrometer employs an array of four millimeter-sized ion traps (x(o) = 5.0 mm and y(o) = 4.0 mm, where x(o) and y(o) are the half-distances in the x and y dimensions, respectively). Mass spectra are acquired from four different samples simultaneously. The available mass/charge range is m/z 15-510 with excellent linearity of the mass calibration (R2 = 0.999 999). The peak width is less than 0.3 mass/charge units at m/z 146, corresponding to a resolution of approximately 500. Simultaneous MS/MS of ions due to four compounds (3-fluoroanisole, 4-fluoroanisole, 2-fluorobenzyl alcohol, 2,6-dimethylcyclohexanone) with the same nominal molecular radical cation but distinctive fragmentation patterns was demonstrated. Isolation and fragmentation efficiencies were approximately 25 and approximately 75%, respectively, measured in the typical case of the molecular radical cation of acetophenone. Preacquisition differential data were obtained by real-time subtraction of the ion signals from two channels of the multiplexed mass spectrometer. The differential experiment presented offers proof of principle of comparative mass spectra in high-throughput screening applications while reducing data storage requirements.     

Ion trap/ion mobility/quadrupole/time-of-flight mass spectrometry for peptide mixture analysis

An ion trap/ion mobility/quadrupole/time-of-flight mass spectrometer has been developed for the analysis of peptide mixtures. In this approach, a mixture of peptides is electrosprayed into the gas phase. The mixture of ions that is created is accumulated in an ion trap and periodically injected into a drift tube where ions separate according to differences in gas-phase ion mobilities. Upon exiting the drift tube, ions enter a quadrupole mass filter where a specific mass-to-charge (m/z) ratio can be selected prior to collisional activation in an octopole collision cell. Parent and fragment ions that exit the collision cell are analyzed using a reflectron geometry time-of-flight mass spectrometer. The overall configuration allows different species to be selected according to their mobilities and m/z ratios prior to collision-induced dissociation and final MS analysis. A key parameter in these studies is the pressure of the target gas in the collision cell. Above a critical pressure, the well-defined mobility separation degrades. The approach is demonstrated by examining a mixture of tryptic digest peptides of ubiquitin.     

High-throughput miniature cylindrical ion trap array mass spectrometer

A fully multiplexed cylindrical ion trap (CIT) array mass spectrometer with four parallel ion source/mass analyzer/detector channels has been built to allow simultaneous high-throughput analysis of multiple samples. A multielement external chemical ionization/electron ionization source was coupled to a parallel array of CITs each of equal size (internal radius 2.5 mm), and the signal was recorded using an array of four miniature (2-mm inner diameter) electron multipliers. Using external electron ionization, the spectra of four separate samples were recorded simultaneously in real time using a four-channel preamplifier system and a data acquisition program written using LabVIEW software. These experiments mark the first demonstration of externally generated ions being successfully trapped in a miniature CIT mass analyzer. The instrument currently provides mass/charge range of approximately m/z 50-500. Average peak width is m/z 0.3, corresponding to a resolution of 1000 at m/z 300. The four-channel mass spectrometer is housed in a single vacuum manifold and operated with a single set of control electronics. The modular design of this instrument allows scale-up to many more channels of analysis for future applications in the areas of industrial process monitoring and combinatorial analysis and in the fields of proteomics and metabolomics.     

Limitation of time-of-flight resolution in the ultra high mass range

In this work, we have examined the reason for the deterioration of resolution and mass accuracy of time-of-flight mass analyzers with increasing mass after the expansion-induced kinetic energy has been eliminated by collisional cooling in an ion guide. Theoretically, removing the expansion-induced kinetic energy by collisional cooling permits the ions to travel along the ion guide axes without significant deviation so that they can be injected into the analyzer in a well-collimated ion beam with well-defined kinetic energy. If the ions can be injected into an orthogonal acceleration time-of-flight mass analyzer (oa-TOF) in this manner, high-resolution mass analysis can be obtained regardless of mass or m/z. Unfortunately, high resolution did not result. It is our contention that the effusive expansion out of the first ion guide yields dispersive axial ejection that reduces TOF resolving power with increasing mass not m/z.     

Rectilinear ion trap mass spectrometer with atmospheric pressure interface and electrospray ionization source

A rectilinear ion trap (RIT) mass analyzer was incorporated into a mass spectrometer fitted with an electrospray ionization source and an atmospheric pressure interface. The RIT mass spectrometer, which was assembled in two different configurations, was used for the study of biological compounds, for which performance data are given. A variety of techniques, including the use of a balanced rf, elevated background gas pressure, automatic gain control, and resonance ejection waveforms with dynamically adjusted amplitude, were applied to enhance performance. The capabilities of the instrument were characterized using proteins, peptides, and pharmaceutical drugs. Unit resolution and an accuracy of better than m/z 0.2 was achieved for mass-to-charge (m/z) ratios up to 2000 Th at a scan rate of approximately 3000 amu/(charge.s) while reduced scan rates gave greater resolution and peak widths of less than m/z 0.5 over the same range. The mass discrimination in trapping externally generated ions was characterized over the range m/z 190-2000 and an optimized low mass cutoff value of m/z 120-140 was found to give equal trapping efficiencies over the entire range. The radial detection efficiency was measured as a function of m/z ratio and found to rise from 35% at low m/z values to more than 90% for ions of m/z 1800. The way in which the ion trapping capacity depends on the dc trapping potential was investigated by measuring the mass shift due to space charge effects, and it was shown that low trapping potentials minimize space charge effects by increasing the useful volume of the device. The collision-induced dissociation (CID) capabilities of the RIT instrument were evaluated by measuring isolation efficiency as a function of mass resolution as well as measuring peptide CID efficiencies. Overall CID efficiencies of more than 60% were easily reached, while isolation of an ion with unit resolution at m/z 524 was achieved with high rejection (>95%) of the adjacent ions. The overall analytical capabilities of the ESI-RIT instrument were demonstrated with the analysis of a mixture of pharmaceutical compounds using multiple-stage mass spectrometry.     

Implementation of ion/ion reactions in a quadrupole/time-of-flight tandem mass spectrometer

A commercial quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been adapted for ion/ion reaction studies. To enable mutual storage of oppositely charged ions in a linear ion trap, the oscillating quadrupole field of the second quadrupole of the system (Q2) serves to store ions in the radial dimension while auxiliary radio frequency is superposed on the end lenses of Q2 during the reaction period to create barriers in the axial dimension. A pulsed dual electrospray (ESI) source is directly coupled to the instrument interface for the purpose of proton transfer reactions. Singly and doubly charged protein ions as high in mass as 66 kDa are readily formed and observed after proton-transfer reactions. For the modified instrument, the mass resolving power is approximately 8000 for a wide m/z range, and the mass accuracy is approximately 20 ppm for external calibration and approximately 5 ppm for internal calibration after ion/ion reactions. Parallel ion parking is demonstrated with a six-component protein mixture, which shows the potential application of reducing spectral complexity and concentrating certain charge states. The current system has high flexibility with respect to defining MS(n) experiments involving collision-induced dissociation (CID) and ion/ion reactions. Protein precursor and CID product masses can be determined with good accuracy, providing an attractive platform for top-down proteomics. Electron transfer dissociation ion/ion reactions are implemented by using a pulsed nano-ESI/atmospheric pressure chemical ionization dual source for ionization. The reaction between protonated peptide ions and radical anions of 1,3-dinitrobenzene formed exclusively c- and z-type fragment ions.     

Narrow mass extraction of time-of-flight data for quantitative analysis of proteins: determination of insulin-like growth factor-1

Methods for quantitative analysis of proteins by mass spectrometry have progressed dramatically. While isotope-dilution approaches using selected reaction monitoring of tryptic peptides (also known as bottom up) have become common, the potential to use narrow mass extraction of high-resolution mass spectra provides a compelling alternative. We investigated the relationships between instrument performance and data processing with the aim of determining whether this approach can lead to robust bioanalytical assays for proteins. Our approach utilized off-line sample preparation combined with online sample extraction coupled to HPLC with the effluent from the analytical column directed to a high-resolution, high-mass accuracy quadrupole time-of-flight (qTOF) mass spectrometer operated in full scan mode. Narrow mass extraction of a single isotope from IGF-1 in the 7+ charge state (m/z 1093.5209) was used to generate extracted ion chromatograms. We found that with appropriate attention to instrument performance and data processing, quantitative protein assays with good sensitivity, high selectivity, and excellent analytical performance can be developed.     

Chip-based capillary electrophoresis/mass spectrometry determination of carnitines in human urine

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the CE separation and on-line electrospray detection of carnitine and selected acylcarnitines from mixtures of analytical standards as well as extracts of fortified human urine. Chip-based CE/MS experiments in two different laboratories were carried out using a triple-quadrupole mass spectrometer and a quadrupole time-of-flight (QTOF) mass spectrometer, respectively. The glass chips used with both systems were comparably equipped with a microfabricated capillary electrophoresis (CE) channel but with different electrosprayers. The quadrupole chip-based CE/MS experiments employed a miniature coupled microsprayer, which allowed coupling of the microelectrospray process via a micro liquid junction at the exit of the CE capillary channel. Selected ion monitoring (SIM) CE/MS experiments were employed for all of the quadrupole CE/MS work. The QTOF CE/MS full-scan single MS and MS/MS experiments were carried out in another laboratory using accurate mass measurement TOF mass spectrometry techniques. The electrospray process that was employed with the QTOF system differed in that an inserted nanoelectrospray capillary needle was carefully affixed into a flat-bottomed hole that was aligned with the CE channel exit orifice. SIM CE/MS using the described quadrupole system provided acceptable ion current electropherograms from fmole levels from analytical standard solutions of carnitine and acylcarnitines that were manually injected (loaded) onto the chip. In addition, the corresponding electropherograms for human urine fortified with the target carnitine and acylcarnitines at a 10-20 microg/mL (35-124 microM) level were obtained via SIM CE/MS techniques. The measured CE separation efficiency for the SIM CE/MS electropherograms was determined to be 2860 plates (peak width at half-height method or N = 5.54(T/WO.5(2)), and carnitine and three acylcarnitines were separated in less than 48 s. In contrast, using quadrupole-TOF technologies, the same samples could be diluted by a factor of 2-4 to obtain a comparable detector response for the target compounds. In the full-scan, single mass analyzer mode (m/z 150-500), the CE separation efficiency was measured to be 2600 plates, but mass measurement accuracy was less than 5.0 ppm for the quaternary cations. In the CE/MS/MS mode, full-scan collision-induced dissociation (CID) mass spectra were obtained with a mass accuracy of < or =10 ppm for the higher mass ions and < or =27 ppm for the lower mass product ions. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric detection for these important compounds in synthetic mixtures, as well as in human urine extracts.     

Tandem parallel fragmentation of peptides for mass spectrometry

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.     

Direct analysis and identification of Helicobacter and Campylobacter species by MALDI-TOF mass spectrometry.

Campylobacter jejuni, Campylobacter fetus, and Campylobacter coli were compared with Helicobacter pylori and Helicobacter mustelae by direct analysis of individual cultured colonies in 50% methanol-water with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). H. pylori and Campylobacter species from blood agar culture produced unique, complex spectra with over 25 different ions in mass/charge (m/z) range from 2,000 to 62,000. A biomarker for H. pylori was centered around m/z 58,268, and H. mustelae was distinguished from H. pylori by its ions at m/z 49,608 and 57,231. Campylobacters could be distinguished from Helicobacters by their lack of ions around m/z 58,000 and 61,000 as well as distinguishing biomarkers of lower m/z: 10,074 and 25,478 for C. coli; m/z 10,285 and 12,901 for C. jejuni; m/z 10,726 and 11,289 for C. fetus. MALDI-TOF MS is a rapid and direct method for detection of these potentially pathogenic bacteria from culture.     

Surface-induced dissociation on a MALDI-ion mobility-orthogonal time-of-flight mass spectrometer: sequencing peptides from an "in-solution" protein digest

Peptide sequencing by surface-induced dissociation (SID) on a MALDI-ion mobility-orthogonal TOF mass spectrometer is demonstrated. SID of approximately 100-fmol amounts of model peptides HLGLAR (m/z 666.8), gramicidin S (m/z 1142.5), and bovine insulin b chain (m/z 3495.5) was accomplished using hydrocarbon-coated gold grids and approximately 20-eV collision energies. The current version of the instrument achieves a mobility resolution of approximately 20 and TOF mass resolution better than 200. Peptide sequences of four peptides from a tryptic digest of cytochrome c (approximately 1 pmol deposited) were obtained. The advantage of IM-SID-o-TOF-MS is that a single experiment can be used to simultaneously measure the molecular weights of the tryptic peptide fragments (e.g., peptide mass mapping) and partial sequence analysis, (e.g., real-time tandem mass spectrometry.)     

An automated matrix-assisted laser desorption/ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometer for "bottom-up" proteomics

Here we describe a new quadrupole Fourier transform ion cyclotron resonance hybrid mass spectrometer equipped with an intermediate-pressure MALDI ion source and demonstrate its suitability for "bottom-up" proteomics. The integration of a high-speed MALDI sample stage, a quadrupole analyzer, and a FT-ICR mass spectrometer together with a novel software user interface allows this instrument to perform high-throughput proteomics experiments. A set of linearly encoded stages allows sub-second positioning of any location on a microtiter-sized target with up to 1536 samples with micrometer precision in the source focus of the ion optics. Such precise control enables internal calibration for high mass accuracy MS and MS/MS spectra using separate calibrant and analyte regions on the target plate, avoiding ion suppression effects that would result from the spiking of calibrants into the sample. An elongated open cylindrical analyzer cell with trap plates allows trapping of ions from 1000 to 5000 m/z without notable mass discrimination. The instrument is highly sensitive, detecting less than 50 amol of angiotensin II and neurotensin in a microLC MALDI MS run under standard experimental conditions. The automated tandem MS of a reversed-phase separated bovine serum albumin digest demonstrated a successful identification for 27 peptides covering 45% of the sequence. An automated tandem MS experiment of a reversed-phase separated yeast cytosolic protein digest resulted in 226 identified peptides corresponding to 111 different proteins from 799 MS/MS attempts. The benefits of accurate mass measurements for data validation for such experiments are discussed.     

Image charge detection mass spectrometry: pushing the envelope with sensitivity and accuracy

A novel image charge detection mass spectrometer (CDMS) with improved sensitivity and mass accuracy is described. The improved detector design and method of data analysis allow us to measure a reliable mass for a single macroion that is an order of magnitude smaller than previously achieved with CDMS. The apparatus employs an image charge detector array consisting of 22 detectors. The detectors are divided into two groups that can be floated at different potentials. The signals from the detector array are analyzed using a correlation approach to yield the velocities in the two groups of detectors and the charge. These quantities, together with the voltage difference between the two groups of detectors, provide a value for the mass. The mass, m/z, and charge distributions recorded for 300 kDa poly(ethylene oxide) (PEG) are presented. The mass distribution shows a peak at around 300 kDa with a width close to that expected from the polymer size distribution. In addition, there are broad peaks in the mass distribution at around 100 and 500 MDa. The 300 kDa ions have m/z ratios of ～2 kDa/e, and the 100 and 500 MDa ions have m/z ratios of ～40 kDa/e. The 100 and 500 MDa ions probably result from PEG aggregates that are either present in solution or the residue of large electrospray droplets.     

Tandem ion trap design with enhanced mass analysis capabilities for large populations of ions

A new arrangement consisting of two separate radio frequency (rf) quadrupole ion traps is used to analyze large populations of ions over a wide mass-to-charge (m/z) range. The setup consists of an "accumulation" trap that is maintained at a higher pressure than the second high-performance "analyzer" trap. The two traps are scanned simultaneously, with a mass difference between that determines the residence time and mass range of ions in the analytical trap. Initially, all ions are trapped in the accumulation trap and then mass-selectively ejected into the analyzer trap. As ions arrive in the analyzer trap, they cool through collisions with the buffer gas and then are mass selectively ejected toward the detector. This concurrent linked mass scanning reduces the total number of ions present in the analyzer trap during mass analysis, thereby reducing space charge effects and leading to improved resolution and mass accuracy of analytical spectra.     

The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer

A new matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometer is described for sequencing peptides. This instrument combines the advantages of high sensitivity for peptide analysis associated with MALDI and comprehensive fragmentation information provided by high-energy collision-induced dissociation (CID). Unlike the postsource decay technique that is widely used with MALDI-TOF instruments and typically combines as many as 10 separate spectra of different mass regions, this instrument allows complete fragment ion spectra to be obtained in a single acquisition at a fixed reflectron voltage. To achieve optimum resolution and focusing over the whole mass range, it may be desirable to acquire and combine three separate sections. Different combinations of MALDI matrix and collision gas determine the amount of internal energy deposited by the MALDI process and the CID process, which provide control over the extent and nature of the fragment ions observed. Examples of peptide sequencing are presented that identify sequence-dependent features and demonstrate the value of modifying the ionization and collision conditions to optimize the spectral information.     

Positive ion transmission mode ion/ion reactions in a hybrid linear ion trap

A triple quadrupole mass spectrometer capable of ion trapping experiments has been adapted for ion/ion reaction studies. The instrument is based on a commercially available linear ion trap (LIT) tandem mass spectrometer (i.e., an MDS SCIEX 2000 Q TRAP) that has been modified by mounting an atmospheric sampling glow discharge ionization (ASGDI) source to the side of the vacuum manifold for production of singly charged anions. The ASGDI source is located line of sight to the side of the third quadrupole of the triple quadrupole assembly (Q3). Anions are focused into the side of the rod array (i.e., anion injection occurs orthogonal to the normal ion flight path). A transmission mode method to perform ion/ion reactions has been developed whereby positive ions are transmitted through the pressurized collision quadrupole (Q2) while anions are stored in Q2. The Q2 LIT is used to trap negative ions whereas the Q3 LIT is used to accumulate positive ions transmitted from Q2. Anions are injected to Q3 and transferred to Q2, where they are stored and collisionally cooled. Multiply charged protein/peptide ions, formed by electrospray, are then mass selected by the first quadrupole assembly (Q1) operated in the rf/dc mode and injected into Q2. The positive ions, including the residual precursor ions and the product ions arising from ion/ion proton-transfer reactions, are accumulated in Q3 until they are analyzed via mass-selective axial ejection for mass analysis. The parameters that affect ion/ion reactions are discussed, including pressure, nature of the gas in Q2, and operation of Q2 as a linear accelerator. Ion/ion reactions in this mode can be readily utilized to separate ions with the same m/z but largely different mass and charge, e.g., +1 bradykinin and +16 myoglobin, in the gas phase.     

Infrared multiphoton dissociation in quadrupole time-of-flight mass spectrometry: top-down characterization of proteins

The first implementation of infrared multiphoton dissociation (IRMPD) for a hybrid quadrupole time-of-flight (QqTOF) mass spectrometer is reported. Ions were trapped in the radio frequency-only quadrupole (q2), which normally serves as a collision cell, and irradiated by a continuous CO2 IR laser. The laser beam was introduced coaxially with the quadrupoles in order to maximize overlap with the ion path. The resolution of the TOF mass analyzer allowed direct charge state determination for fragments smaller than 7 kDa. For larger fragments, the charge state could be assigned using the multiple losses of water, characteristic for IRMPD of proteins. The analytical performance is demonstrated by top-down sequencing of several representative proteins (equine myoglobin, bovine casein, and human insulin and chaperonin 10). Various post-translational modifications such as phosphorylation, acetylation, formation of disulfide bridges, and removal of N-terminal methionine followed by acetylation are detected and characterized. The utility of IRMPD for the analysis of biological samples is demonstrated in a study of a recently identified potential marker for endometrial cancer, chaperonin 10.