Patent Application
20100285446
The apparatuses and methods described herein generally relate to ionization sources for mass spectrometers and methods of mass spectrometry, and in particular, laser ablation electrospray ionization mass spectrometry as well as methods of making and using the same.
Infectious diseases and metabolic disorders cause death, disability, and social and economic distress for millions of people throughout the world. Examples of the causative agents of infectious diseases include, but are not limited to, human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), human T-lymphotropic virus type 1 (HTLV1),), human T-lymphotropic virus type 2 (HTLV2), human T-lymphotropic virus type 3 (HTLV3), human papillomavirus (HPV), hepatitis A, hepatitis B, hepatitis C, viral encephalitis, herpes virus, paramyxoviruses, influenza, and severe acute respiratory syndrome (SARS). Examples of metabolic disorders include, but are not limited to, diabetes, muscular dystrophy, phenylketonuria (PKU), Tay Sachs disease, leukodystrophies, lysosomal disorders, Wilson's disease, Lesch-Nyhan syndrome, urea cycle disorder, amyloidosis and lipid storage diseases. Genomic, proteomic, and metabolomic technologies have been used to identify biomarkers and other indicators of infectious diseases and metabolic disorders. Rapid monitoring of the metabolome may be more useful than monitoring the transcriptome or proteome because the metabolic composition of a cell or organism provides its actual biochemical condition.
Infectious diseases and metabolic disorders may alter the metabolism of a living cell or organism. In other words, the metabolism of a healthy cell may be different than the metabolism of an unhealthy cell of the same type. Metabolism generally refers to the chemical processes of a living cell or organism that support and maintain life. The products of these chemical processes may be generally referred to as metabolites. The metabolism and/or metabolites of a living cell or organism may change depending on its biological state, developmental stage, history, and/or environment. Viruses, for example, may alter the metabolism of an infected cell. The metabolisms and/or metabolites of a virally infected cell and an uninfected cell of the same type may be different. The identification and analysis of metabolites may facilitate the detection, prevention, and/or treatment of infectious diseases and metabolic disorders.
Mass spectrometry is an analytical technique that has been successfully used in chemistry, biology, and other fields for qualitative and quantitative analysis. The identification and analysis of metabolites by conventional methods of mass spectrometry may be problematic. For example, matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI), may suffer from time consuming and complex sample preparation, and in situ analysis of a sample may be difficult. Atmospheric pressure mass spectrometry methods, such as direct analysis in real time (DART), desorption electrospray ionization (DESI), atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), desorption atmospheric pressure chemical ionization (DAPCI), matrix-assisted laser desorption electrospray ionization (MALDESI), and electrospray laser desorption ionization (ELDI), may suffer from other limitations, including, but not limited to, complex and time consuming separation techniques, a narrower range of samples that may be analyzed, higher detection limits, sensitivity to surface properties, sampling errors, and/or lack of imaging and quantization capabilities. Mass spectrometry may be combined with separation techniques, such as gas chromatography, high performance liquid chromatography and capillary electrophoresis, however, these techniques may increase the analysis time and/or cost.
Therefore, more efficient and/or cost-effective mass spectrometers and methods of making and using the same are desirable.
In certain embodiments, more efficient and/or cost-effective mass spectrometers and methods of making and using the same are described.
According to certain embodiments, a method of mass spectrometry may generally comprise subjecting a sample comprising at least one indicator to laser ablation electrospray ionization mass spectrometry; determining a relative intensity of the indicator; and comparing the relative intensity of the indicator to a standard indicator intensity.
The various embodiments described herein may be better understood by considering the following description in conjunction with the accompanying drawing figures.
As generally used herein, the terms “consisting essentially of and “consisting of” are embodied in the term “comprising”.
As generally used herein, the articles “one”, “a”, “an” and “the” refer to “at least one” or “one or more”, unless otherwise indicated.
As generally used herein, the terms “including” and “having” mean “comprising”.
As used herein, the terms “LAESI” and “LAESI-MS” refer to laser ablation electrospray ionization mass spectrometry.
As used herein, the term “infection” refers to the invasion by, multiplication and/or presence of a virus or bacteria in a cell, tissue, organ and/or organism.
As used herein, the term “metabolic disorder” refers to any pathological condition in a cell, tissue, organ and/or organism resulting from an alteration in an organism's metabolism.
As used herein, the term “metabolome” refers to the total or partial set of metabolites in a cell, tissue, organ and/or organism at a specific time. An infection and/or metabolic disorder may cause certain metabolites to be upregulated or down-regulated.
As used herein, the term “pattern” refers to a set of metabolites and their intensities measured by a diagnostic method. The set of metabolites may be the total or partial set of metabolites in a cell, tissue, organ and/or organism at a specific time
As generally used herein, the terms “about” and “approximately” refer to an acceptable degree of error for the quantity measured, given the nature or precision of the measurements. Typical exemplary degrees of error may be within 20%, 10%, or 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” refer to values within an order of magnitude, potentially within 5-fold or 2-fold of a given value.
All numerical quantities stated herein are approximate unless stated otherwise; meaning that the term “about” may be inferred when not expressly stated. The numerical quantities disclosed herein are to be understood as not being strictly limited to the exact numerical values recited. Instead, unless stated otherwise, each numerical value is intended to mean both the recited value and a functionally equivalent range surrounding that value. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding the approximations of numerical quantities stated herein, the numerical quantities described in specific examples of actual measured values are reported as precisely as possible.
All numerical ranges stated herein include all sub-ranges subsumed therein. For example, a range of “1 to 10” is intended to include all sub-ranges between and including the recited minimum value of 1 and the recited maximum value of 10. Any maximum numerical limitation recited herein is intended to include all lower numerical limitations. Any minimum numerical limitation recited herein is intended to include all higher numerical limitations.
In the following description, certain details are set forth in order to provide a better understanding of various embodiments of mass spectrometers and methods for making and using the same. However, one skilled in the art will understand that the embodiments described herein may be practiced without these details. In other instances, well-known structures and methods associated with mass spectrometers and methods of mass spectrometry may not be shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments of this disclosure.
This disclosure describes various features, aspects, and advantages of various embodiments of mass spectrometers and methods for making and using the same. It is understood, however, that this disclosure embraces numerous alternative embodiments that may be accomplished by combining any of the various features, aspects, and advantages of the various embodiments described herein in any combination or sub-combination that one of ordinary skill in the art may find useful.
Infection of a cell or organism causes extensive changes at the gene, protein, and metabolite levels. These changes are usually followed by gene-expression profiling and proteomic analysis. Viruses, for example, rely on the metabolic network of their cellular hosts for survival and replication. Exploring the metabolic consequences of a viral infection may provide insight into the causes and/or treatment of viral infection. The insight gained by such studies may depend on the target sample, treatment procedures, and detection techniques used. Conventionally, biofluids, such as blood and urine, have been used to follow metabolic changes after infection, but in many cases they may complicate the analysis due to the pooling of changes in different cell types and the variations between individuals. Ultimately, direct analysis of a sample is a more straightforward way to understand the actual disease-associated metabolic changes in and at the site of an infection. In such cases, a direct detection technique may offer key advantages.
Metabolites are small molecules of diverse physico-chemical properties with greatly different abundance levels that make their identification and analysis challenging. Typically, optical spectrometry, such as Fourier transform infrared spectrometry, nuclear magnetic resonance (NMR), and mass spectrometric techniques in combination with separation techniques, such as gas chromatography, high performance liquid chromatography (HPLC) and capillary electrophoresis, have been used for metabolomic studies. Mass spectrometry (MS) is a versatile technique that, combined with chromatographic separations, may provide qualitative and quantitative analyses of complex samples with high selectivity and sensitivity, as well as a broad dynamic range. Conventional mass spectrometric methods, however, are time consuming and involve extensive sample preparation. The application of direct sampling methods, such as flow injection electrospray ionization (ESI), may avoid chromatographic separation, but not the extensive sample preparation that may affect sample integrity and, in some cases, may lead to metabolite degradation. Therefore, these techniques generally restrict the choice of samples and discourage their in situ analysis.
Some of these problems may be mitigated by the use of atmospheric pressure ion sources. Recent advances in atmospheric pressure ion sources, such as direct analysis in real time (DART), desorption electrospray ionization (DESI), atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), and laser ablation electrospray ionization (LAESI) may enable direct analysis of cell and tissue samples without extensive sample preparation. Analysis of cells, cell cultures, and cell extracts using DART, DESI, and MALDI techniques may have their own limitations, such as coverage of analytes, sampling of the surface only, and quantitation restrictions. According to certain embodiments, LAESI-MS may provide for in situ cell and tissue analysis, and may sample the entire volume of the cells for metabolites and lipid components with tissue imaging and quantitation capabilities.
Previous studies of viral infection indicate that glycolysis, the citrate (TCA) cycle, pyrimidine nucleotide biosynthesis, and lipid metabolism are the main metabolic changes generally involved with viral infection. The metabolic intermediates of these pathways may increase in response to the infection, e.g., faster uptake of glucose and glutamine, greater accumulation of citrate, and increasing excretion of lactate and glutamate in infected cells. The efflux from infected cells may be enriched in metabolites related to nucleotide and fatty acid biosynthesis. The fatty acid biosynthesis in infected cells may be considered an antiviral response. Previous studies of HIV infected cells indicate a reduction of glutathione levels, changes in lipid metabolism, and polyamine pools (putrescine, spermidine and spermine). However, these studies were carried out using conventional MS-based techniques involving both sample extraction and chromatographic separation. According to certain embodiments, LAESI-MS provides rapid and direct identification of metabolic changes in HTLV1 infected T lymphocytes and reduces and/or eliminates the need for extensive sample preparation.
Human T cell leukemia virus type I (HTLV1), a member of the delta-retroviridae subfamily, was the first human pathogenic retrovirus discovered. HTLV1 may contribute to cancer development. Infection with HTLV1 may result in the development of adult T-cell leukemia (ATL), a CD4+T lymphoproliferative malignancy. Estimates of worldwide HTLV1 infections may be between 15 million to 25 million individuals. However, infected individuals develop ATL after a long latent period and at a 3-5% incidence rate. Infection with HTLV1 may result in HTLV1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and several inflammatory diseases, including polymyositis, uveitis, and lymphocyte alveolitis. The development of ATL from HTLV1 infection may be a multi-hit occurrence with initial transformation due to the viral protein Tax. Recent studies have indicated that the use of novel treatments, including monoclonal antibodies against the interleukin-2 receptor (IL-2R) and the combination therapy of interferon-alpha (IFN-α) and zidovudine (AZT), to be effective, but only in a small percentage of ATL patients. Therefore, new therapies for the treatment of ATL, and in particular, HTLV1 infection, are desirable.
According to certain embodiments, mass spectrometers and methods of mass spectrometry for identifying and analyzing changes in the metabolic profiles of a cell, tissue, and/or organism after infection and/or development of a metabolic disorder. The metabolic changes in virally infected cells and tissues may be monitored using high throughput metabolomic and complementary transcriptomic and proteomic technologies, such as LAESI-MS, as described herein. In certain embodiments, LAESI-MS may be used to identify and analyze changes in the metabolic profiles of a cell, tissue, and/or organism after HTLV1 infection, HTLV3 infection, Tax1 expression, and Tax3 expression as well as comparing the metabolic profiles of cells and tissues transfected with either HTLV3 molecular clone or Tax3 and HTLV1 transformed cells. Understanding the role of Tax in destabilizing key regulators, such as proteins, in metabolism and cell cycle control, may help identify molecular markers that contribute to ATL development and define new therapeutic strategies. Certain embodiments comprise in situ metabolite profiling of infected cells. Certain embodiments comprise facilitating the identification of virus-induced perturbations in the biochemical processes of a host cell.
Certain embodiments of the mass spectrometers and methods of making and using the same described herein may provide certain advantages over other approaches of mass spectrometric analysis. The advantages may include, but are not limited to, in situ analysis, simultaneous detection of multiple samples, independent optimization of ablation conditions and ionization conditions, a wider dynamic range of samples that may be used, operation under ambient conditions, simpler sample preparation, minimal sample manipulation, minimal sample degradation, improved sampling time, positional sensitivity, improved sensitivity to surface properties, and/or improved detection limits.
According to certain embodiments, a mass spectrometer for laser ablation electrospray ionization mass spectrometry may generally comprise a laser system, an electrospray apparatus, and a mass spectrometer. The laser system may comprise a laser and a focusing system comprising fiber optics, coupling lenses, and/or focusing lenses, and an x-y-z translation stage having a sample mount. The laser may be selected from the group consisting of an Er:YAG laser, an Nd:YAG laser driven optical parametric oscillator and a free electron laser. The electrospray apparatus may comprise an electrospray ionization emitter having a power supply and a syringe pump. The mass spectrometric ion source may comprise a solid state camera. The mass spectrometric ion source may comprise a shroud to enclose the sample, the sample holder, and/or the electrospray emitter. The translation stage and the sample environment may be temperature controlled and/or atmosphere controlled. This is to maintain sample integrity and to avoid condensation of moisture from the environment. The atmosphere may comprise ambient atmosphere. The temperature may ranges from −10° C. to 60° C. The relative humidity may range from 10% to 90%.
In certain embodiments, the atmosphere and/or the electrospray solution may comprise a reactive component to facilitate the ionization and/or fragmentation of certain constituents of the sample. For example, the electrospray solution may comprise Li2SO4 to facilitate the structural identification of lipids by inducing structure specific fragmentation in collision induced dissociation experiments. Examples of reactive gases include, but are not limited to, ammonia, SO2, and NO2.
Referring to
Referring to
In certain embodiments, an ablation plume (LA) may intersect an electrospray plume (ES). Referring to
In certain embodiments, the laser may comprise an infrared laser. The infrared laser may operate at a wavelength from 2600 nm to 3450 nm, such as 2800 nm to 3200 nm, and 2930 nm to 2950 nm. The laser may comprise a mid-infrared pulsed laser operating at a wavelength from 2600 nm to 3450 nm, a repetition rate from 1 Hz to 100 Hz, and a pulse width from 0.5 ns to 50 ns. In at least one embodiment, the laser may comprise a diode pumped Nd:YAG laser-driven optical parametric oscillator (OPO) (Opolette 100, Opotek, Carlsbad, Calif.) operating at 2940 nm, 100 Hz repetition rate, and 5 ns pulse width. The optical fiber may comprise a germanium oxide (GeO2)-based optical fiber (450 μm core diameter, HP Fiber, Infrared Fiber Systems, Inc., Silver Spring, Md.) with its tip etched to a radius of curvature from 1 μm to 50 μm, such as 5 μm to 25 μm, and 10 μm to 15 μm. In at least one embodiment, the radius of curvature may be 15 μm. The optical fiber may deliver the laser pulse to the sample. The energy of a laser pulse before coupling into the optical fiber may be from 0.1 mJ to 6 mJ, thus the pulse-to-pulse energy stability generally corresponds to 2% to 10%. In at least one embodiment, the energy of a laser pulse before coupling into the optical fiber may be 554±26 μJ, thus the pulse-to-pulse energy stability generally corresponds to 5%. The laser system may be operated at 100 Hz from 0.01 seconds to 20 seconds to ablate a sample. In at least one embodiment, laser system may be operated at 100 Hz for 1 second to ablate a sample. In certain embodiments, 1 to 100 laser pulses may be delivered to a sample for analysis.
In certain embodiments, the electrospray source may comprise a low noise syringe pump (Physio 22, Harvard Apparatus, Holliston, Mass.) to supply the electrospray solution to a tapered stainless steel emitter (inner diameter 50 μm, MT320-50-5-5, New Objective, Woburn, Mass.). The low noise syringe pump may supply the electrospray solution at a rate from 10 mL/min to 10 μL/min. In at least one embodiment, the low noise syringe pump may supply the electrospray solution at 200 mL/min. The tapered stainless steel emitter may have an outside diameter from 100 μm to 500 μm and an insider diameter from 10 μm to 200 μm. The power supply may comprise a regulated power supply (PS350, Stanford Research Systems, Sunnyvale, Calif.), to provide a stable high voltage from 2.5 to 5 kV to the electrospray emitter. The power supply may be mounted on a manual translation stage to optimize the LAESI signal by adjusting the relative position of the sample, electrospray emitter, and/or inlet orifice of the mass spectrometer. The electrospray solution may comprise at least one of 50% methanol with 0.1% (v/v) acetic acid, 50% methanol with 0.1% (v/v) formic acid, 50% methanol with 0.1% (v/v) trifluoroacetic acid, 50% methanol with 0.1% (v/v) ammonium acetate. The electrospray solution may be applied at an angle from 0° to 90°, such as 30°, 45°, and 60°, into the ablation plume. The angle may be adjusted from 0° to 90° to optimize ion production. In at least one embodiment, the electrospray solution may be applied at a right angle (90°) into the ablation plume.
According to certain embodiments, the mass spectrometer orifice may be on the same or a different axis as the electrospray emitter of the LAESI ion source. The angle between the mass spectrometer orifice and electrospray emitter of the LAESI ion source may be from 0° to 90°, such as 30°, 45°, and 60°. The distance from the mass spectrometer orifice to the electrospray emitter tip may be from 1 mm to 20 mm, such as 5 mm to 15 mm. In at least one embodiment, the distance from the mass spectrometer orifice to the electrospray emitter tip may be 12 mm. The sample may be placed onto a pre-cleaned microscope glass slide (catalog no. 125496, Fisher Scientific, Pittsburgh, Pa.). The sample may be placed onto a stepper motor-driven three axis precision flexure stage (NanoMax TS, Thorlabs, Newton, N.J.). The sample may be 1 mm to 30 mm below the spray axis, such as 5 mm to 25 mm, and 10 mm to 20 mm. In at least one embodiment, the sample may be 15 mm below the spray axis. In one experiment, no ions were detected by the mass spectrometer when the ESI was off, indicating that no ions directly induced by the laser were collected. This observation may result from the large (>15 mm) distance between the orifice of the mass spectrometer and the ablated sample.
The positive ions produced by the LAESI ion source may be analyzed by a mass spectrometer. The mass spectrometer may comprise an orthogonal acceleration time-of-flight mass spectrometer (QTOF Premier, Waters Co., MA). The orifice of the mass spectrometer may have an inner diameter from 100 μm to 500 μm, such as 225 μm to 375 μm. In at least one embodiment, the orifice of the mass spectrometer may have an inner diameter from 100 μm to 200 μm, such as 127 μm. The orifice of the mass spectrometer may be extended by a straight or curved extension tube having a similar inner diameter as the orifice of the mass spectrometer and a length from 20 mm to 500 mm. The interface block temperature may be from ambient temperature to 150° C., such as 23° C. to 90° C., and 60° C. to 80° C. In at least one embodiment, the interface block temperature may be 80° C. The potential may be from −100 V to 100 V, such as −70 V to 70 V. In at least one embodiment, the potential may be −70 V. Tandem mass spectra may be obtained by collision activated dissociation (CAD) with a collision gas, such as argon, helium or nitrogen, at a collision cell pressure from 10−6 mbar to 10−2 mbar, and with collision energies from 10 eV to 200 eV. In at least one embodiment, the collision gas may be argon, the collision cell pressure may be 4×10−3 mbar, and the collision energies may be from 10 eV to 25 eV.
In certain embodiments, the laser beam may be steered by gold-coated minors (PF10-03-M01, Thorlabs, Newton, N.J.) and coupled into the cleaved end of the optical fiber by a plano-convex calcium fluoride lens (Infrared Optical Products, Farmingdale, N.Y.) having a focal length from 2 mm to 100 mm, such as 25 mm to 75 mm, and 40 mm to 60 mm. In at least one embodiment, the focal length may be 50 mm. The optical fiber may be held by a bare fiber chuck (BFC300, Siskiyou Corporation, Grants Pass, Oreg.). The optical fiber may be positioned by a five-axis translator (BFT-5, Siskiyou Corporation, Grants Pass, Oreg.).
In certain embodiments, the optical fiber may comprise a GeO2-based glass fiber, a fluoride glass fiber, and a chalcogenide fiber. The optical fiber may have a high laser-damage threshold due to its high glass transition temperature. The Hytrel and polyimide coatings may be stripped off both ends of the fiber by the application of 1-methyl-2-pyrrolidinone (at 130° C. to 150° C. for 1 min). After stripping off the Hytrel and the polyimide coatings, the fiber ends may be cleaved with a Sapphire blade (KITCO Fiber Optics, Virginia Beach, Va.) by scoring and gently snapping them. Chemical etching of the GeO2-based glass fiber tip may be achieved by dipping one of the cleaved fiber ends 0.5 mm deep into 24° C. 1% HNO3 solution in a wide beaker to provide a low meniscus curvature. The meniscus formed at the fiber end may gradually etch the 450 μm diameter core into a sharp tip having a radius of curvature (R) of 15 μm. Prior to use, the etched tips may be washed with deionized water. In certain embodiments, no visible change of the fiber tip may be observed after performing the LAESI technique which may indicate the absence of damage or contamination.
In certain embodiments, the etched end of the fiber may be attached to a micromanipulator (MN-151, Narishige, Tokyo, Japan) to move the etched end of the fiber closer to the sample. The distance from the etched end of the fiber and the sample may be from contact (0 μm) to 50 μm. In at least one embodiment, the coordinate system may be aligned so that the x-y plane coincides with the sample and the x-axis is parallel with the emitter, the optical fiber is positioned at an azimuth angle from 20° to 160° and a zenith angle from 20° to 70°. In at least one embodiment, the azimuth angle may be 135° and the zenith angle may be 45°. The zenith angle of 45° may provide an acceptable trade-off between the shape of the ablation mark and signal intensity reduction by blocking the expanding plume. A thin sample material deposit may be observed on the fiber tip after ablation. In these cases, the fiber may be retracted from the surface and elevated laser pulse energy may be used to clean the tip. In at least one embodiment, the distance between the fiber tip and the sample surface (h) may be 2 R. This may result in an ablation mark with an average diameter of 2.5 R. In at least one embodiment, the distance between the fiber tip and the sample surface may be 30 μM, resulting in an ablation mark with an average diameter of 37.5 μm. Microscope images of the ablation marks may be obtained by an upright microscope (BX 51, Olympus America Inc., Center Valley, Pa.) in either reflected or transmitted mode and by an inverted microscope.
In certain embodiments, the mass spectrometer ion source may comprise a visualization system. The distance between the fiber tip and sample surface may be monitored by a long distance video microscope (InFocus Model KC, Infinity, Boulder Colo.) with a 5× infinity corrected objective lens (M Plan Apo 5×, Mitutoyo Co., Kanagawa, Japan), and the image may be captured by a CCD camera (Marlin F131, Allied Vision Technologies, Stadtroda, Germany). With the environmental vibration in the low micrometer range, an approximate distance from 30 μm to 40 μm may be maintained between the tip and the sample. A similar video microscope system may be used at a right angle to the sample surface to align the fiber tip over the location of interest in the sample for ablation. The visualization system may comprise a 7× precision zoom optic (Edmund Optics, Barrington, N.J.), fitted with a 10× infinity-corrected long working distance objective lens (M Plan Apo 10×, Mitutoyo Co., Kanagawa, Japan) and a CCD camera (Marlin F131, Allied Vision Technologies, Stadtroda, Germany).
In certain embodiments, the sample may comprise any sample that comprises water, including, but not limited to, cells, tissues, organs, biofilms, aqueous solutions, organic materials, inorganic materials, synthetic materials, biomedical samples, forensic samples, biological warfare agents, and wetted surfaces. For example, the sample may be selected from the group consisting of biomolecules (such as metabolites, lipids, nucleic acids, proteins, peptides, and carbohydrates), organic and inorganic molecules (such as pharmaceuticals, polymers, dendrimers and other macromolecules), and mixtures thereof. The sample may comprise a biofilm comprising an aggregate of microorganisms in which cells adhere to each other and/or to a surface. The sample may comprise a single cell, cells, small cell populations, cell lines, and tissues. The single cell may have a smallest dimension less than 100 micrometers, such as less than 50 μm, less than 25 μm, and/or less than 10 μm. The single cell may have a smallest dimension from 1 μm to 100 μm, such as from 5 μm to 50 μm, and from 10 μm to 25 μm. In at least one embodiment, the single cell may have a smallest dimension from 1 μm to 10 μm. The small cell population may comprise 10 to 1 million cells, such as 50 cells to 100,000 cells, and 100 cells to 1,000 cells. The sample may comprise a cell infected with at least one of a virus and a bacterium. The sample may comprise a virally infected living cell and/or tissue. The sample may comprise a cell having a metabolic disorder.
Referring to
According to certain embodiments, the indicator may comprise a biomolecule, an organic molecule, a molecular complex and/or a xenobiotic. The indicator may be selected from the group consisting of metabolites, lipids, lipid precursors, lipid components, nucleic acids, proteins, peptides, carbohydrates, and combinations thereof. The indicator may comprise intracellular metabolites from a single cell. The indicator may comprise a biomarker. The biomarker may be related to a metabolic change. The biomarker may be related to a disease state. The disease state may be associated with a viral infection, a bacterial infection, and/or a metabolic disorder.
Viruses may include, but are not limited to, human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), human T-lymphotropic virus type 1 (HTLV1), human T-lymphotropic virus type 2 (HTLV2), human T-lymphotropic virus type 3 (HTLV3), human papillomavirus (HPV), paramyxoviruses, hepatitis A, hepatitis B, hepatitis C, viral encephalitis, herpes virus, influenza, and/or severe acute respiratory syndrome (SARS). Bacteria may include, but are not limited to, Bacillus anthracis (the causative agent of anthrax), Yersinia pestis (the causative agent of bubonic plague), Mycobacterium tuberculosis (the causative agent of tuberculosis) and Vibrio cholera (the causative agent of cholera). The metabolic disorder may comprise diabetes, muscular dystrophy, phenylketonuria (PKU), Tay Sachs disease, leukodystrophies, lysosomal disorders, Wilson's disease, Lesch-Nyhan syndrome, urea cycle disorder, amyloidosis and/or lipid storage diseases. In certain embodiments, the disease state may comprise human immunodeficiency virus (HIV), human T-lymphotropic virus type 1 (HTLV1), and/or human T-lymphotropic virus type 3 (HTLV3) as well as Tax1 and/or Tax3 expressing cells, and the indicator may comprise glutathione, spermine, spermidine, putrescine, arginine, creatine, choline, phosphocholine, glycerophosphocholine, glycerophosphocholine lipids, ATP, ADP, AMP, cAMP, dopamine, dopamine metabolites, and/or any combination thereof. In certain embodiments, the indicator may be related to a metabolic change caused by exposure to a toxin. For example, the activity of an enzyme, butyrylcholinesterase, in blood may be used as a biomarker for nerve agent exposure.
According to certain embodiments, the method of mass spectrometry may comprise classifying the sample as belonging to or not belonging to the standard indicator intensity. A sample not belonging to the standard indicator intensity may indicate that the sample comprises a metabolic change. The metabolic change may be associated with a viral infection, a bacterial infection, and/or a metabolic disorder. A sample not belonging to the standard indicator intensity may indicate that the sample is predicted to comprise a disease state. The disease state may be associated with a viral infection, a bacterial infection, and/or a metabolic disorder.
In certain embodiments, the indicator may comprise a plurality of indicators. The method of mass spectrometry may comprise determining the relative intensity of each indicator to form a sample metabolite pattern and comparing the sample metabolite pattern to a standard metabolite pattern comprising the standard indicator intensity of each of the plurality of indicators. The method of mass spectrometry may comprise classifying the sample as belonging to or not belonging to the standard metabolite pattern. A sample not belonging to the standard metabolite pattern may indicate that the sample comprises a metabolic change. A sample not belonging to the standard metabolite pattern may indicate that the sample is predicted to comprise a disease state.
Referring to
In certain embodiments, determining the relative intensity of the indicator may comprise determining the relative intensity of an indicator comprising multiple charge states. The indicator may comprise a plurality of indicators. The method of mass spectrometry may comprise determining the relative intensity of each indicator to form a sample metabolite pattern and comparing the sample metabolite pattern to a standard metabolite pattern comprising the standard indicator intensity of each of the plurality of indicators. The method of mass spectrometry may comprise classifying the sample as belonging to or not belonging to the standard metabolite pattern. A sample not belonging to the standard metabolite pattern may indicate that the sample comprises a metabolic change. A sample not belonging to the standard metabolite pattern may indicate that the sample is predicted to comprise a disease state. The standard indicator intensity may comprise an internal reference and/or an external reference.
In certain embodiments, the metabolic state may be selected from the group consisting of developmental stages, for example, the stages of a cell cycle, environments, nutritional supplies, taxonomic units, genetic units, infected and uninfected states, diseased and healthy states, and different stages of pathogenicity.
According to certain embodiments, the method of determining a metabolic state may comprise ablating a second sample comprising the indicator with an infrared laser under ambient conditions to form a second ablation plume; intercepting the second ablation plume by a second electrospray plume; detecting the indicator of the second sample by mass spectrometry; determining the profile of the indicator of the second sample; comparing the first indicator and second indicator to each other and/or the standard indicator intensity; and classifying at least one of the first sample and the second sample as belonging to or not belonging to the standard indicator intensity. A sample not belonging to the standard indicator intensity may indicate that the sample comprises a metabolic change. A sample not belonging to the standard indicator intensity may indicate that the sample is predicted to comprise a disease state. The metabolic change and/or disease state may be associated with a viral infection, a bacterial infection, and/or a metabolic disorder. In certain embodiments, comparing the first indicator and second indicator to each other may indicate that the sample comprises a metabolic change. In certain embodiments, comparing the first indicator and second indicator to each other may indicate that the sample is predicted to comprise a disease state.
In certain embodiments, the method of mass spectrometry may comprise determining temporal and/or spatial information from a sample. The second sample may comprise the first sample at a different time. For example, the first sample may comprise a cell and/or tissue and the second sample may comprise the same cell and/or tissue at a later time, such as after a predetermined period of time or at different stages in the cell cycle. The second sample may comprise a different portion of the first sample. For example, the first sample may comprise a first portion of a cell and/or tissue and the second sample may comprise another portion of the same cell and/or tissue.
According to certain embodiments, the metabolic changes in virally infected cells/tissues may be monitored using LAESI-MS. In certain embodiments, changes in metabolites and lipids may be directly detected from uninfected T lymphocytes, human T-lymphotropic virus type 1 (HTLV1) transformed cells, and human T-lymphotropic virus type 3 (HTLV3) transformed cells, and Tax1 and Tax3 expressing cell lines T lymphocytes. The mass spectra of uninfected and infected cells may be compared to identify any metabolic changes. Glycerophosphocholine (PC) lipid components may be dominant in the non-HTLV1 transformed cells and PC(O-32:1) and PC(O-34:1) plasmalogens may be displaced by PC(30:0) and PC(32:0) species in the HTLV1 transformed cells. In HTLV1 transformed cells choline, phosphocholine, spermine and glutathione, among others, may be downregulated, whereas creatine, dopamine, arginine and AMP may be upregulated. In certain embodiments, individual measurements on the T-cells may take a few seconds enabling high throughput studies using LAESI-MS. Analysis of different cell lines transfected with either the HTLV3 molecular clone or Tax3 may reveal metabolite changes that correlate to HTLV1 and HTLV3 infected cells, whereas others may be unique to HTLV1.
According to certain embodiments, the method of mass spectrometry may comprise identifying virus-induced perturbations in the biochemical processes of the host cell by high throughput in situ metabolite profiling of HTLV1 and HTLV3 transformed cells. This method may be used to better understand the molecular mechanisms of HTLV1 and HTLV3 infections, which in turn may result in drug development and/or new treatment strategies.
According to certain embodiments, the method of mass spectrometry may comprise analyzing cell-to-cell metabolic variations and/or analyzing cells at different stages of the cell cycle. In certain embodiments, a method of mass spectrometry may comprise identifying metabolic changes in HTLV1 and Tax1 transformed T lymphocytes and HTLV3 and Tax3 transformed kidney epithelial cells. The indicators may comprise glutathione, spermine, choline, phosphocholine, glycerophosphocholine, thioacetamide, proline, taurine, carbamoyl phosphate, methoxytyramine and 8-hydroxy guanosine, creatine, arginine, dopamine, homovanillic acid and AMP. The levels of glutathione, spermine, choline, phosphocholine, glycerophosphocholine, thioacetamide, proline, taurine, carbamoyl phosphate, methoxytyramine and 8-hydroxy guanosine may be downregulated in C81 cells. The levels of creatine, arginine, dopamine, homovanillic acid and AMP may be upregulated in infected cells.
In certain embodiments, the method of mass spectrometry may comprise comparing metabolic changes detected in H9-Tax1 cells and H9 cells as well as HUT102 cells and H9 cells. Some of the key metabolic changes detected in C81 cells were also observed in H9-Tax1 cells vs. H9 cells as well as HUT102 cells vs. H9 cells. Without wishing to be bound to any particular theory, these indicators participate in biochemical pathways, such as polyamine biosynthesis, creatine biosynthesis, AMP biosynthesis, dopamine metabolism, lipid metabolism, redox reactions etc. Comparing HTLV1 and HTLV3 transfected cells revealed that part of the metabolic response was similar, but there were several changes specific to HTLV1. For example, the changes in the levels of putrescine, taurine, arginine and AMP were consistent among HTLV1 transformed cells.
The various embodiments described herein may be better understood when read in conjunction with the following representative examples. The following examples are included for purposes of illustration and not limitation.
The uninfected T lymphocyte cells (CEM and H9) and kidney epithelial cells (293T), HTLV1 infected cells (C81 and HUT102), and H9 cells stably transfected with Tax1 of HTLV1 (H9-Tax1) and 293T cells infected with HTLV3 (293T-HTLV3) and expressing Tax3 (293T-Tax3) were maintained in RPMI 1640 medium containing fetal bovine serum, L-glutamine (2 mM), penicillin (100 units/mL) and streptomycin (100 μg/mL). The medium solutions and buffers were procured from Quality Biological Inc. (Gaithersburg, Md.) and the solvents were HPLC grade available from Acros Organics (Geel, Belgium). The glacial acetic acid was procured from Fluka (Munich, Germany).
According to certain embodiments, the method of mass spectrometry was performed by a infrared laser system. An optical parametric oscillator (OPO) (Opolette 100, Opotek, Carlsbad, Calif.) converted the output of a 100 Hz repetition rate Nd:YAG laser to mid-IR pulses of 5 ns duration at 2940 nm wavelength. Beam steering and focusing was accomplished by gold coated mirrors (PF10-03-M01, Thorlabs, Newton, N.J.) and a 150 mm focal length CaF2 lens (Infrared Optical Products, Farmingdale, N.Y.), respectively. At 5-6 mm downstream from the tip of the spray capillary, the laser beam having average output energy of 0.3 mJ/pulse was used to ablate the tissue sample at a right angle (90°). The laser spot size was determined by optical microscopy of the burn pattern produced on a photographic paper. The laser spot size had a 300 μm diameter.
According to certain embodiments, the electrospray system comprised a low-noise syringe pump (Physio 22, Harvard Apparatus, Holliston, Mass.) to feed a 50% methanol solution containing 0.1% (v/v) acetic acid through a stainless steel emitter with tapered tip having an outside diameter of 320 μm and an inside diameter of 50 μm. (MT320-50-5-5, New Objective Inc., Woburn, Mass.). Stable high voltage (2800 V) was generated by a regulated power supply (PS350, Stanford Research Systems, Inc., Sunnyvale, Calif.). The regulated power supply was directly applied to the emitter. The orifice of the sampling cone was on-axis with the electrospray emitter at a distance of 12 mm from its tip.
According to certain embodiments, the cells of interest were grown to populations to produce 106 cells/pellet before subjecting the sample to LAESI-MS. The cells were washed twice with phosphate buffered saline (PBS) and pelleted by spindown (2000 rpm). The supernatant PBS was removed without disturbing the pellet, and 10 μL of the pellet was loaded onto a microscope slide and presented to the mass spectrometer for direct LAESI analysis. The microscope slide with the cell pellet was positioned 15 mm below the spray axis under ambient conditions (in air at ambient temperature and pressure). The microscope slide was mounted on a computer-controlled stepper motor-driven three-axis precision flexure stage (Nanomax TS, Thorlabs, Newton, N.Y.) for rastering and geometry optimization.
According to certain embodiments, the ion source was mounted on a Q-TOF Premier mass spectrometer (Waters, Milford, Mass.). Full scan mass spectra were recorded over the mass range of m/z 50-2,000 using a time-of-flight (TOF) analyzer at a resolution of 8,000 (FWHM). Individual measurements on the samples generally took a few seconds. For structure identification of individual metabolites, collision activated dissociation spectra was recorded by selecting the precursor ion using a quadrupole analyzer (transmission window 2 Da) and the product ions were resolved by the TOF analyzer. Argon was used as the collision gas at a collision cell pressure of 4×10−3 mbar and a collision energy set from 5 to 25 eV. Accurate masses were determined using an internal standard method. Glycine, methionine, N-acetyl phenylalanine, leucine enkephalin and glufibrinopeptide were dissolved in the electrospray solution to concentrations from 50 μM to 200 μM and used as internal standards. Averages of the LAESI spectra collected under similar experimental conditions for a fixed time were considered so that the approximate number of cells used for obtaining LAESI spectra were approximately the same for most of the samples.
The human metabolome database (HMDB; www.hmdb.ca), the MassBank high resolution mass spectral database (www.massbank.jp), the NIST/EPA/NIH mass spectral library, and the MetaCyc database (http://metacyc.org) were used with a mass tolerance ranging from 0.1 Da to 0.01 Da for the metabolite searches and identifications.
For verification purposes, according to certain embodiments, arginase activity was measured using the QuantiChrom Arginase Assay Kit (BioAssay Systems, Hayward, Calif.) according to the manufacturer's instructions. CEM and C81 cell lysates (10 μg and 100 μg) were measured in triplicates. The concentration of cAMP was measured using the CatchPoint Cyclic-AMP Fluorescent Assay Kit (Molecular Devices, Sunnyvale, Calif.) according to the manufacturer's instructions. CEM and C81 cell lysates (10 μg and 100 μg) were measured in triplicates. Glutathione reductase from CEM and C81 cell lysates (10 μg and 200 μg) were measured utilizing the Glutathione Reductase Assay Kit (Sigma, St. Louis, Mo.) according to the manufacturer's instructions.
According to certain embodiments, the sample may comprise populations of uninfected (CEM) and HTLV1 infected (C81) T lymphocytes. These non-adherent cells were grown in an RPMI medium comprising inorganic salts, sugar, amino acids, vitamins and antibiotics. To minimize the interfering peaks from the medium in the LAESI spectra, the cells were washed with PBS and the cell pellet was loaded onto a microscope slide. The cells were directly ablated by multiple laser shots and the average LAESI spectra of 10-15 scans were used. The resulting positive ion spectra exhibited various cell related metabolite ions in the range of m/z 20-1500, but also included interfering peaks from PBS and the medium left in the cell pellet. Referring to
The spectra of CEM T lymphocytes and HTLV1 infected C81 T lymphocytes with Tax1 expression cells showed a similar set of ions, except for the lipid peaks, but consistent differences were identified in their relative ion yields. Referring to
Representative mass spectra of degradation/fragmentation products of putrescine, spermidine and spermine are shown in
According to certain embodiments, the relative abundances of the detected ions in the mass spectra of non-HTLV1 transformed cells and HTLV1 transformed cells may be used to determine the extent of metabolic changes between them. The background peaks from PBS/medium solution were used as internal standards. The relative abundance ratios for each ion detected in CEM and C81 are listed in
According to certain embodiments, the mass spectra of T lymphocytes comprised glucose (relative abundance <3%), the major component of the medium (11 mM). The mass of protonated spermine (203.2236) was close to that of sodiated glucose species (203.059), but these two peaks may be well separated. This, however, may raise the issue of possible contribution of medium-related peaks to the spectra detected from T lymphocytes. The mass spectrum of the medium alone showed that arginine (m/z 175), choline (m/z 104), and glutathione (m/z 308) contributed to the signal from the related metabolites in T lymphocytes. The glutathione and choline peaks were less than <2% with respect to the glucose peak (m/z 203, base peak), whereas the arginine peak was 25-30%. These ratios were consistent with values from diluted medium (100 times). The glucose peak appeared in both CEM and C81 cells (<3%) with an abundance ratio for m/z 203 close to unit value. The arginine peak that was negligible in CEM cells may be much higher than the glucose peak in C81 cells. This may confirm that the arginine interferences from the medium are negligible and the arginine levels are indeed upregulated in C81 cells.
According to certain embodiments, the relative mass spectra in the low mass region (<m/z 500) may be corrected for the medium and electrospray related background. After correction, 43 ions were exclusively related to T lymphocytes, and 37 of these ions corresponded to the 21 metabolites shown in
According to certain embodiments, representative mass spectra of the T lymphocytes may comprise glycerophosphocholine (PC) lipids. Referring to
As shown in
According to certain embodiments, the changes in metabolite levels between the non-HTLV1 transformed T lymphocytes and HTLV1 transformed T lymphocytes may be verified at the protein level by quantifying the enzymes or proteins involved in the related metabolic pathway. The levels of cAMP, arginase, and glutathione reductase in CEM and C81 cells were measured using biochemical assays. Referring to
According to certain embodiments, the sample may comprise other cell lines, e.g., non-HTLV1 transformed T lymphocytes (H9), their Tax1-transfected counterparts (H9-Tax1), and HTLV1 transformed cells (HUT102 cells). The metabolic changes upon transfection are listed in
According to certain embodiments, the sample may comprise other cell lines to determine the specificity of the observed metabolite changes to HTLV1 transformation, e.g., uninfected 293T kidney epithelial cells, and on HTLV3 and Tax3 transfected 293T cells. The metabolic changes upon HTLV1 transformation and the presence of Tax3 are listed in
According to certain embodiments, multiple abundant ionic species of glutathione may be detected in non-HTLV1 transformed T lymphocytes reflecting high concentrations, whereas in HTLV1 transformed cells there may be a 2 to 5-fold decrease in their abundance. The reduced form of glutathione (GSH) may be the most predominant thiol present in mammalian cells with concentrations up to 12 mM. GSH may serve several important functions, such as antioxidant (protection against oxidative stress), cofactor in isomerization reactions, transport and storage form of cysteine, and regulator of intracellular redox status, cell proliferation and apoptosis. Biologically, the oxidized glutathione (GSSG) may be converted to GSH by the enzyme glutathione reductase. The ratio of GSH and GSSG may serve as a representative indicator of the antioxidative capacity of the cell. Cellular GSH concentrations may be reduced in response to protein malnutrition, oxidative stress, and other pathological conditions. Intracellular GSH levels may regulate T lymphocyte function, and deficiency of GSH may be associated with HIV infection.
According to certain embodiments, the GSH level in HTLV1 transformed T lymphocytes was decreased. The levels of glutathione reductase in both non-HTLV1 transformed cells and HTLV1 transformed cells were measured using an enzyme assay, and GSH was reduced in HTLV1 transformed cells and GSH was reduced in HTLV3 transformed 293T cells.
According to certain embodiments, the sample may comprise spermine, spermidine and putrescine, which belong to the polycationic compounds named polyamines. These polyamines may be involved in genetic processes, such as DNA synthesis and gene expression, and play a major role in cell proliferation, cell differentiation, and programmed cell death. Referring to
According to certain embodiments, the arginine levels in HTLV1 transformed cells were increased. The transcriptional upregulation of arginase in infected cells, which may be confirmed by the enzyme assay, was consistent with elevated levels of arginine upon viral infection. The role of arginine may play a role in the survival of endothelial cells during oxidative stress. Deprivation of arginine may cause serious disturbances in cellular function and enhances apoptosis. Arginine availability may contribute to the regulation of T lymphocyte function in cancer. Referring to
According to certain embodiments, the mass spectra of T lymphocyte cells and kidney epithelial cells show that choline containing metabolites, e.g., choline, phosphocholine, glycerophosphocholine, and several glycerophosphocholine lipids, may be downregulated upon transformation by HTLV1, HTLV3, Tax1 or Tax3 when compared to non-transformed cells, except in the HUT102 case. Referring to
In vivo NMR spectroscopy may be used to monitor choline containing metabolites, however, it may be difficult to determine which specific metabolites are altered. According to certain embodiments, choline containing metabolites may be directly subjected to LAESI-MS to determine which specific metabolites are altered. The mass spectra of non-transformed cells and transformed cells may provide information about the precursors and lipid components simultaneously. In one experiment, a decrease in the glycerophosphocholine lipid content in HTLV1 transformed cells confirmed increased lipid catabolism to produce fatty acids. Apart from a few lipids (PC(30:0), PC(32:5), PC(32:3), PC(32:0), and PC(34:6) that remained at higher levels in HTLV1 transformed cells, most of the glycerophosphocholine lipids present in non-HTLV1 transformed cells were downregualated in the HTLV1 transformed cells.
Levels of phosphorylated adenosine nucleotides, including ATP, ADP, and AMP, may define the energy state in living cells. Quantitation of individual adenine nucleotides may be used for the assessment of the energy state of cells. The level of exogenous ATP in the body may be increased in various inflammatory and shock conditions. Extracellular ATP may be important for cell-to-cell communication and in the immune system. In one experiment, AMP was detected directly from cells subjected to LAESI-MS. Significantly elevated AMP abundance was observed in HTLV1 and Tax1 transformed cells. Referring to
According to certain embodiments, the sample may comprise cAMP. cAMP is a second messenger and activates several protein kinases that may be involved in significant biochemical processes. The amount of cAMP known to be present in T lymphocytes is 6 pmol/107 cells. In one experiment, glutathione interfered with the detection of cAMP. Referring to
According to certain embodiments, the sample may comprise dopamine, a neuromodulator, and its metabolites, methoxytyramine and homovanillic acid, in T lymphocytes. Dopamine belongs to the group of catecholamines, and may be involved in the neuroimmunological network. T lymphocytes may be activated by neurotransmitters via neurotransmitter receptors that may elicit crucial functions. Catecholamines may be synthesized in mouse lymphocytes. Increased levels of catecholamines may indicate an activated state. As shown in
All documents cited herein are, in relevant part, incorporated herein by reference, but only to the extent that the incorporated material does not conflict with existing definitions, statements, or other documents set forth herein. To the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern. The citation of any document is not to be construed as an admission that it is prior art with respect to this document.
While particular embodiments of mass spectrometers and methods of making and using the same have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific apparatuses and methods described herein, including alternatives, variants, additions, deletions, modifications and substitutions. This disclosure including the appended claims is therefore intended to cover all such changes and modifications that are within the scope of this invention.
This is a continuation-in-part of U.S. application Ser. No. 12/176,324, filed on Jul. 18, 2008, which claims priority to U.S. provisional application Ser. No. 60/951,186, filed on Jul. 20, 2007, and U.S. application Ser. No. 12/323,276, filed on Nov. 25, 2008, each of the foregoing applications are hereby incorporated herein by reference in their entireties.
Portions of this invention were made with United States government support under Grant Nos. 0415521 and 0719232 awarded by the National Science Foundation. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60951186 | Jul 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12323276 | Nov 2008 | US |
| Child | 12774533 | US | |
| Parent | 12176324 | Jul 2008 | US |
| Child | 12323276 | US |
