Optical detection and charge-state analysis of MALDI-generated particles with molecular masses larger than 5 MDa

Charged polystyrene nanoparticles are generated by matrix-assisted laser desorption/ionization (MALDI) and detected by laser-induced fluorescence (LIF) in a quadrupole ion trap. Employing the LIF technique, observations of individual fluorescent nanospheres (27 nm in diameter and containing 180 fluorescein dye equivalents) have been achieved with an average signal-to-noise ratio of approximately 10. With the trap operating at a frequency around 5 kHz, charge state analysis of the particles reveals that the number of charges carried by the spheres is between 1 and 10. It suggests a mass-to-charge ratio (m/z) in the range of 10(5)-10(6) for the MALDI-generated particles. To effectively trap such large particles (m > 5 MDa), damping of the particles' motions by using approximately 50 mTorr He buffer gas is absolutely required. Similar findings are obtained for particles with a nominal size of 1 microm in diameter, demonstrating that production of charged particles with a molecular mass as high as 10(12) Da is possible using the MALDI technique.     

Frequency-scanning MALDI linear ion trap mass spectrometer for large biomolecular ion detection

This study presents the first report on the development of a matrix-assisted laser desorption ionization (MALDI) linear ion trap mass spectrometer for large biomolecular ion detection by frequency scan. We designed, installed, and tested this radio frequency (RF) scan linear ion trap mass spectrometer and its associated electronics to dramatically extend the mass region to be detected. The RF circuit can be adjusted from 300 to 10 kHz with a set of operation amplifiers. To trap the ions produced by MALDI, a high pressure of helium buffer gas was employed to quench extra kinetic energy of the heavy ions produced by MALDI. The successful detection of the singly charged secretory immunoglobulin A ions indicates that the detectable mass-to-charge ratio (m/z) of this system can reach ~385 000 or beyond.     

Single-particle mass spectrometry of polystyrene microspheres and diamond nanocrystals.

High-resolution mass spectra of single submicrometer-sized particles are obtained using an electrospray ionization source in combination with an audio frequency quadrupole ion-trap mass spectrometer. Distinct from conventional methods, light scattering from a continuous Ar-ion laser is detected for particles ejected out of the ion trap. Typically, 10 particles are being trapped and interrogated in each measurement. With the audio frequency ion trap operated in a mass-selective instability mode, analysis of the particles reveals that they all differ in mass-to-charge ratio (m/z), and the individual peak in the observed mass spectrum is essentially derived from one single particle. A histogram of the spectra acquired in 10(2) repetitions of the experiment is equivalent to the single spectrum that would be observed when an ion ensemble of 10(3) particles is analyzed simultaneously using the single-particle mass spectrometer (SPMS). To calibrate such single-particle mass spectra, secular frequencies of the oscillatory motions of the individual particle within the trap are measured, and the trap parameter qz at the point of ejection is determined. A mass resolution exceeding 10(4) can readily be achieved in the absence of ion ensemble effect. We demonstrate in this work that the SPMS not only allows investigations of monodisperse polystyrene microspheres, but also is capable of detecting diamond nanoparticles with a nominal diameter of 100 nm, as well.     

MALDI of individual biomolecule-containing airborne particles in an ion trap mass spectrometer

Individual airborne biomolecule-containing particles were detected and characterized in near real-time by matrix-assisted laser desorption/ionization (MALDI) with an ion trap mass spectrometer. Biomolecule-containing particles were laboratory-generated and passed through a heated region containing a solution of matrix in equilibrium with the gas phase. Passage into a cooler region created a supersaturation, resulting in rapid deposition of the matrix vapor onto the biomolecule-containing particles, whereupon they were sampled into the inlet of our spectrometer. The coated particles were collimated and individually sized by light-scattering-based time-of-flight. When the sized particle reached the center of the ion trap, it was irradiated with a focused 266-nm laser, and the resulting ions were mass-analyzed. Mass spectra of leucine enkephalin, bradykinin, substance P, and polylysine-containing particles were determined with attomole sensitivity. Structural information of the peptides contained in an individual particle was obtained by tandem mass spectrometry. Analysis of the results yields insights into the aerosol laser ablation ionization process that suggests an optically limited mechanism for ion production that has interesting ramifications on the utility of aerosol-based MALDI as an analytical technique.     

MALDI ion trap mass spectrometer with charge detector for large biomolecule detection

Up to now, all commercial matrix-assisted laser desorption/ionization (MALDI) mass spectrometers still can not efficiently analyze very large biomolecules. In this work, we report the development of a novel MALDI ion trap mass spectrometer which can enrich biomolecular ions to enhance the detection sensitivity. A charge detector was installed to measure the large ions directly. With this design, we report the first measurement of IgM with the mass-to-charge ratio (m/z) at 980?000. In addition, quantitative measurements of the number of ions can be obtained. A step function frequency scan was first developed to get a clear signal in the m/z range from 200,000 to 1,000,000.     

Automatic identification of proteins with a MALDI-quadrupole ion trap mass spectrometer.

A matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer of new design is described. The instrument is based on a commercial Finnegan LCQ ion trap mass spectrometer to which we have added a MALDI ion source that incorporates a sample stage constructed from a compact disk and a new ion transmission interface. The ion interface contains a quadrupole ion guide installed between the skimmer and the octapoles of the original instrument configuration, allowing for operation in both MALDI and electrospray ionization modes. The instrument has femtomole sensitivity for peptides and is capable of collecting a large number of MALDI MS and MALDI MS/MS spectra within a short period of time. The MALDI source produces reproducible signals for 10(4)-10(5) laser pulses, enabling us to collect MS/MS spectra from all the discernible singly charged ions detected in a MS peptide map. We describe the different modes of the instrument operation and algorithms for data processing as applied to challenging protein identification problems.     

Atmospheric pressure MALDI/ion trap mass spectrometry

A new sample ionization technique, atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI), was coupled with a commercial ion trap mass spectrometer. This configuration enables the application-specific selection of external atmospheric ionization sources: the electrospray/APCI (commercially available) and AP MALDI (built in-house), which can be readily interchanged within minutes. The detection limit of the novel AP MALDI/ion trap is 10-50 fmol of analyte deposited on the target surface for a four-component mixture of peptides with 800-1700 molecular weight. The possibility of peptide structural analysis by MS/MS and MS3 experiments for AP MALDI-generated ions was demonstrated for the first time.     

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.     

Fourier transform time-of-flight mass spectrometry in an electrostatic ion beam trap

We report on the application of an electrostatic ion beam trap as a mass spectrometer. The instrument is analogous to an optical resonator; ions are trapped between focusing mirrors. The storage time is limited by the residual gas pressure and reaches up to several seconds, resulting in long ion flight paths. The oscillation of ion bunches between the mirrors is monitored by nondestructive image charge detection in a field-free region and mass spectra are obtained via Fourier transform. The principle of operation is demonstrated by measuring the mass spectrum of trapped Ar+ and Xe+ particles, produced by a standard electron impact ion source. Also, mass spectra of heavier PEGnNa+ and bradykinin ions from a pulsed MALDI ion source were obtained. The long ion flight path, combined with mass-independent charge detection, makes this system particularly interesting for the investigation of large molecules.     

Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer

We describe an approach to the quantitative analysis of complex protein mixtures using a MALDI quadrupole time-of-flight (MALDI QqTOF) mass spectrometer and isotope coded affinity tag reagents (Gygi, S. P.; et al. Nat. Biotechnol. 1999, 17, 994-9.). Proteins in mixtures are first labeled on cysteinyl residues using an isotope coded affinity tag reagent, the proteins are enzymatically digested, and the labeled peptides are purified using a multidimensional separation procedure, with the last step being the elution of the labeled peptides from a microcapillary reversed-phase liquid chromatography column directly onto a MALDI sample target. After addition of matrix, the sample spots are analyzed using a MALDI QqTOF mass spectrometer, by first obtaining a mass spectrum of the peptides in each sample spot in order to quantify the ratio of abundance of pairs of isotopically tagged peptides, followed by tandem mass spectrometric analysis to ascertain the sequence of selected peptides for protein identification. The effectiveness of this approach is demonstrated in the quantification and identification of peptides from a control mixture of proteins of known relative concentrations and also in the comparative analysis of protein expression in Saccharomyces cerevisiae grown on two different carbon sources.     

Chemical characterization of individual, airborne sub-10-nm particles and molecules

A nanoaerosol mass spectrometer (NAMS) is described for real-time characterization of individual airborne nanoparticles. The NAMS includes an aerodynamic inlet, quadrupole ion guide, quadrupole ion trap, and time-of-flight mass analyzer. Charged particles in the aerosol are drawn through the aerodynamic inlet, focused through the ion guide, and captured in the ion trap. Trapped particles are irradiated with a high-energy laser pulse to reach the "complete ionization limit" where each particle is thought to be completely disintegrated into atomic ions. In this limit, the relative signal intensities of the atomic ions give the atomic composition. The method is first demonstrated with sucrose particles produced with an electrospray generator. Under the conditions used, the particle detection efficiency (fraction of charged particles entering the inlet that are subsequently analyzed) reaches a maximum of 10(-4) at 9.5 nm in diameter and the size distribution of trapped particles has a geometric standard deviation of 1.1 based on a log-normal distribution. A method to deconvolute overlapping multiply charged ions (e.g. C3+ and O4+) is presented. When applied to sucrose spectra, the measured C/O atomic ratio is 1.1, which matches the expected ratio from the molecular formula. The spectra of singly charged bovine serum albumin (BSA) molecules are also presented, and the measured and expected C/N/O atomic ratios are within 15% of the each other. Also observed in the BSA spectra are signals from 13C and 32S which arise from 40 and approximately 34 atoms per molecule (particle), respectively. Potential applications of NAMS to atmospheric chemistry and biotechnology are briefly discussed.     

Unbiased high-throughput screening of reactive metabolites on the linear ion trap mass spectrometer using polarity switch and mass tag triggered data-dependent acquisition

Constant neutral loss (CNL) and precursor ion (PI) scan have been widely used for the in vitro screening of glutathione conjugates derived from reactive metabolites, but these two methods are only applicable to triple quadrupole or hybrid triple quadrupole mass spectrometers. Additionally, the success of CNL and PI scanning largely depends on structure and CID fragmentation pathways of GSH conjugates. In the present study, a highly efficient methodology has been developed as an alternative approach for high-throughput screening and structural characterization of reactive metabolites using the linear ion trap mass spectrometer. In microsomal incubations, a mixture of glutathione [GSH, gamma-glutamyl-cystein-glycin] and the stable-isotope labeled compound [GSX, gamma-glutamyl-cystein-glycin-(13)C2-(15)N] was used to trap reactive metabolites, resulting in formation of both labeled and unlabeled conjugates at a given isotopic ratio. A mass difference of 3.0 Da between the natural and labeled GSH conjugate (mass tag) at a fixed isotopic ratio constitutes a unique mass pattern that can selectively trigger the data-dependent MS(2) scan of both isotopic partner ions, respectively. In order to eliminate the response bias of GSH adducts in the positive and negative mode, a polarity switch is executed between the mass tag-triggered data dependent MS(2) scan, and thus ESI- and ESI+ MS(2) spectra of both labeled and nonlabeled GSH conjugates are obtained in a single LC-MS run. Unambiguous identification of glutathione adducts was readily achieved with great confidence by MS(2) spectra of both labeled and unlabeled conjugates. Reliability of this method was vigorously validated using several model compounds that are known to form reactive metabolites. This approach is not based on the appearance of a particular product ion such as MH(+) - 129 and anion at m/z 272, whose formation can be structure-dependent and sensitive to the collision energy level; therefore, the present method can be suitable for unbiased screening of any reactive metabolites, regardless of their CID fragmentation pathways. Additionally, this methodology can potentially be applied to triple quadrupole or hybrid triple quadrupole mass spectrometers.     

Quantitative comparison of proteomic data quality between a 2D and 3D quadrupole ion trap

A 2D ion trap has a greater ion trapping efficiency, greater ion capacity before observing space-charging effects, and a faster ion ejection rate than a traditional 3D ion trap mass spectrometer. These hardware improvements should result in a significant increase in protein identifications from complex mixtures analyzed using shotgun proteomics. In this study, we compare the quality and quantity of peptide identifications using data-dependent acquisition of tandem mass spectra of peptides between two commercially available ion trap mass spectrometers (an LTQ and an LCQ XP Max). We demonstrate that the increased trapping efficiency, increased ion capacity, and faster ion ejection rate of the LTQ results in greater than 5-fold more protein identifications, better identification of low-abundance proteins, and higher confidence protein identifications when compared with a LCQ XP Max.     

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.     

Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer

Design and performance of a novel hybrid mass spectrometer is described. It couples a linear ion trap mass spectrometer to an orbitrap mass analyzer via an rf-only trapping quadrupole with a curved axis. The latter injects pulsed ion beams into a rapidly changing electric field in the orbitrap wherein they are trapped at high kinetic energies around an inner electrode. Image current detection is subsequently performed after a stable electrostatic field is achieved. Fourier transformation of the acquired transient allows wide mass range detection with high resolving power, mass accuracy, and dynamic range. The entire instrument operates in LC/MS mode (1 spectrum/s) with nominal mass resolving power of 60,000 and uses automatic gain control to provide high-accuracy mass measurements, within 2 ppm using internal standards and within 5 ppm with external calibration. The maximum resolving power exceeds 100,000 (fwhm). Rapid, automated data-dependent capabilities enable real-time acquisition of up to three high-mass accuracy MS/MS spectra per second.     

C60 secondary ion mass spectrometry with a hybrid-quadrupole orthogonal time-of-flight mass spectrometer

A hybrid quadrupole orthogonal time-of-flight mass spectrometer optimized for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization has been equipped with a C 60 cluster ion source. This configuration is shown to exhibit a number of characteristics that improve the performance of traditional time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments for the analysis of complex organic materials and, potentially, for chemical imaging. Specifically, the primary ion beam is operated as a continuous rather than a pulsed beam, resulting in up to 4 orders of magnitude greater ion fluence on the target. The secondary ions are extracted at very low voltage into 8 mTorr of N 2 gas introduced for collisional focusing and cooling purposes. This extraction configuration is shown to yield secondary ions that rapidly lose memory of the mechanism of their birth, yielding tandem mass spectra that are identical for SIMS and MALDI. With implementation of ion trapping, the extraction efficiency is shown to be equivalent to that found in traditional TOF-SIMS machines. Examples are given, for a variety of substrates that illustrate mass resolution of 12,000-15,600 with a mass range for inorganic compounds to m/ z 40,000. Preliminary chemical mapping experiments show that with added sensitivity, imaging in the MS/MS mode of operation is straightforward. In general, the combination of MALDI and SIMS is shown to add capabilities to each technique, providing a robust platform for TOF-SIMS experiments that already exists in a large number of laboratories.     

Sensitive fluorometric nanoparticle assays for cell counting and viability

We have developed easy-to-use homogeneous methods utilizing time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence quenching for quantification of eukaryotic cells. The methods rely on a competitive adsorption of cells and fluorescently labeled protein onto citrate-stabilized colloidal gold nanoparticles or carboxylate-modified polystyrene nanoparticles doped with an Eu(III) chelate. In the gold nanoparticle sensor, the adsorption of the labeled protein to the gold nanoparticles leads to quenching of the fluorochrome. Eukaryotic cells reduce the adsorption of labeled protein to the gold particles increasing the fluorescence signal. In the Eu(III) nanoparticle sensor, the time-resolved fluorescence resonance energy transfer between the nanoparticles and an acceptor-labeled protein is detected; a decrease in the magnitude of the time-resolved energy transfer signal (sensitized time-resolved fluorescence) is proportional to the cell-nanoparticle interaction and subsequent reduced adsorption of the labeled protein. Less than five cells were detected and quantified with the nanoparticle sensors in the homogeneous microtiter assay format with a coefficient of variation of 6% for the gold and 12% for the Eu(III) nanoparticle sensor. The Eu(III) nanoparticle sensor was also combined with a cell impermeable nucleic acid dye assay to measure cell viability in a single tube test with cell counts below 1000 cells/tube. This sensitive and easy-to-use nanoparticle sensor combined with a viability test for a low concentration of cells could potentially replace existing microscopic methods in biochemical laboratories.     

Gold nanoparticles as selective and concentrating probes for samples in MALDI MS analysis

MALDI mass spectrometry is used widely in various fields because it has the characteristics of speed, ease of use, high sensitivity, and wide detectable mass range, but suppression effects between analyte molecules and interference from the sample matrix frequently arise during MALDI analysis. The suppression effects can be avoided if target species are isolated from complicated matrix solutions in advance. Herein, we proposed a novel method for achieving such a goal. We describe a strategy that uses gold nanoparticles to capture charged species from a sample solution. Generally, ionic agents, such as anionic or cationic stabilizers, encapsulate gold nanoparticles to prevent their aggregation in solution. These charged stabilizers at the surface of the gold particles are capable of attracting oppositely charged species from a sample solution through electrostatic interactions. We have employed this concept to develop nanoparticle-based probes that selectively trap and concentrate target species in sample solutions. Additionally, to readily isolate them from solution after attracting their target species, we used gold nanoparticles that are adhered to the surface of magnetic particles through S-Au bonding. A magnet can then be employed to isolate the Au@magnetic particles from the solution. The species trapped by the isolated particles were then characterized by MALDI MS after a simple washing. We demonstrate that Au@magnetic particles having negatively charged surfaces are suitable probes for selectively trapping positively charged proteins from aqueous solutions. In addition, we have employed Au@magnetic particle-based probes successfully to concentrate low amounts of peptide residues from the tryptic digest products of cytochrome c (10(-7) M).     

Microchip flow cytometry using electrokinetic focusing

Flow cytometry of fluorescently labeled and unlabeled latex particles is demonstrated on a microfabricated device. The latex particles were detected and counted using laser light scattering and fluorescence coincidence measurements. Sample confinement was accomplished using electrokinetic focusing at a cross intersection, and detection occurred 50 μm downstream from the intersection. Particles with diameters of 1 and 2 μm were analyzed and distinguished from each other based on their light scattering intensity and fluorescence. A maximum sample throughput of 34 particles/s was achieved. Sample mixtures with varying proportions of fluorescently labeled and unlabeled particles were also analyzed and found to be within experimental error of the expected ratios.