Metabolites are downstream end products of gene and protein activity that closely correlate with ... more Metabolites are downstream end products of gene and protein activity that closely correlate with the phenotype of a biological organism. 1-7 Therefore, by observing specific metabolic changes, one can gain insight into perturbations underlying disease. 8 Consequently, increasing attention has been dedicated to analyzing metabolites using MS in the context of clinical diagnostics, understanding disease mechanisms, and identifying new therapeutic targets.5 , 9 , 10 The ability to analyze metabolites directly from biofluids and tissues continues to challenge current MS technology, largely because of the limits imposed by the complexity of these samples, which contain thousands to tens of thousands of metabolites. 11 A new technology being developed to address this challenge is Nanostructure-Initiator MS (NIMS), a desorption/ionization approach that does not require the application of matrix and thereby facilitates small-molecule (i.e., metabolite) identification. 12 Surface-based mass analysis has seen a resurgence in the past decade, with new MS technologies focused on increasing sensitivity, minimizing background, and reducing sample preparation. 4,6,7,13, 14 MALDI is one of the primary MS platforms currently used for the analysis of biological samples.2 , 15 , 16 However, the application of a MALDI matrix can add significant background at <1000 Da that complicates analysis of the low-mass range (i.e., metabolites).17 In addition, the size of the resulting matrix crystals limits the spatial resolution that can be achieved in tissue imaging.14 Because of these limitations, several matrix-free desorption/ionization approaches have been applied to the analysis of biofluids and tissues.17 Secondary ion MS (SIMS) was one of the first matrix-free desorption/ ionization approaches used to analyze metabolites from biological samples.18 SIMS uses a high-energy primary ion beam to desorb and generate secondary ions from a surface. The primary advantage of SIMS is its high spatial resolution (as small as 50 nm), a powerful characteristic for tissue imaging with MS.19 However, SIMS has yet to be readily applied to the analysis of biofluids and tissues because of its limited sensitivity at >500 Da and analyte fragmentation generated by the high-energy primary ion beam.14 Desorption electrospray ionization (DESI) is a matrix-free technique for analyzing biological samples that uses a charged solvent spray to desorb ions from a surface.20 Advantages of DESI are that no special surface is required and the analysis is performed at ambient pressure with full access to the sample during acquisition.20 , 21 The main limitation of DESI is spatial resolution because "focusing" the charged solvent spray is difficult.22 However, a recent development termed laser ablation ESI (LAESI) is a promising approach to circumvent this limitation. 23 Desorption/ionization on silicon (DIOS), the precursor to NIMS, is another matrix-free laser-induced desorption/ionization approach.24 , 25 DIOS utilizes a porous silicon substrate to absorb the laser energy and vaporize and ionize analytes on the surface without extensive fragmentation.26 Because it does not use a matrix, it has the advantages of low chemical noise and high sensitivity in the low-mass range when used to analzye metabolites in Contact Siuzdak at
Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell ... more Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of α-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.
Nanostructure-initiator mass spectrometry (NIMS) is a new surface-based MS technique that uses a ... more Nanostructure-initiator mass spectrometry (NIMS) is a new surface-based MS technique that uses a nanostructured surface to trap liquid (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;initiator&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;) compounds. Analyte materials adsorbed onto this &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;clathrate&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; surface are subsequently released by laser irradiation for mass analysis. In this protocol, we describe the preparation of NIMS surfaces capable of producing low background and high-sensitivity mass spectrometric measurement using the initiator compound BisF17. Examples of analytes that adsorb to this surface are small molecules, drugs, lipids, carbohydrates and peptides. Typically, NIMS is used to analyze samples ranging from simple analytical standards and proteolytic digests to more complex samples such as tissues, cells and biofluids. Critical experimental considerations of NIMS are described. Specifically, NIMS sensitivity is examined as a function of pre-etch cleaning treatment, etching current density, etching time, initiator composition, sample concentration, sample deposition method and laser fluence. Typically, NIMS surface preparation can be completed in less than 2 h. Subsequent sample preparation requires 1-5 min, depending on sample deposition method. Mass spectrometric data acquisition typically takes 1-30 s per sample.
Analytical and biological variability are issues of central importance to human metabolomics stud... more Analytical and biological variability are issues of central importance to human metabolomics studies. Here both types of variation are examined in human plasma and cerebrospinal fluid (CSF) using a global liquid chromatography/mass spectrometry (LC/MS) metabolomics strategy. The platform shows small analytical variation with a median coefficient of variation (CV) of 15-16% for both plasma and CSF sample matrixes when the integrated area of each peak in the mass spectra is considered. Analysis of biological variation shows that human CSF has a median CV of 35% and plasma has a median CV of 46%. To understand the difference in CV between the biofluids, we compared plasma and CSF independently obtained from different healthy humans. Additionally, we analyzed another group of patients from whom we compared matched CSF and plasma (plasma and CSF obtained from the same human subject). A similar number of features was observed in both biofluids, although the majority of features appeared with greater intensity in plasma. More than a dozen metabolites shared between the human CSF and plasma metabolomes were identified based on accurate mass measurements, retention times, and MS/MS spectra. The fold change in these metabolites was consistent with the median biological CV determined for all peaks. The measured median biological CV together with analysis of intragroup variation of healthy individuals suggests that fold changes above 2 in metabolomics studies investigating plasma or CSF are statistically relevant with respect to the inherent variability of a healthy control group. These data demonstrate the reproducibility of the global metabolomics platform using LC/MS and reveal the robustness of the approach for biomarker discovery.
Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolom... more Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI-MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,-trimethoxyphenyl)phosphonium acetic acid (TMPP-AA) to improve liquid chromatography (LC)/ESI-MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl-sn-glycerol, dihydrotachysterol, octadecanol, and alpha-tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI-MS instrumentation and demonstrates the potential for targeted metabolomics analysis. Published in 2009 by John Wiley & Sons, Ltd.
Nanostructure-initiator mass spectrometry (NIMS) is a highly sensitive, matrix-free technique tha... more Nanostructure-initiator mass spectrometry (NIMS) is a highly sensitive, matrix-free technique that is well suited for biofluid analysis and imaging of biological tissues. Here we provide a new technical variation of NIMS to analyze carbohydrates and steroids, molecules that are challenging to detect with traditional mass spectrometric approaches. Analysis of carbohydrates and steroids was accomplished by spray depositing NaCl or AgNO 3 on the NIMS porous silicon surface to provide a uniform environment rich with cationization agents prior to desporption of the fluorinated polymer initiator. Laser desorption/ionization of the ion-coated NIMS surface allowed for Na + cationization of carbohydrates and Ag + cationization of steroids. The reliability of the approach is quantitatively demonstrated with a calibration curve over the physiological range of glucose and cholesterol concentrations in human serum (1 -200 μM). Additionally we illustrate the sensitivity of the method by showing its ability to detect carbohydrates and steroids down to the 800-amol and 100-fmol levels, respectively. The technique developed is well suited for tissue imaging of biologically significant metabolites such as sucrose and cholesterol. To highlight its applicability, we used cation-enhanced NIMS to image the distribution of sucrose in a Gerbera jamesonii flower stem and the distribution of cholesterol in a mouse brain. The flower stem and brain sections were placed directly on the ioncoated NIMS surface without further preparation and analyzed directly. The overall results reported underscore the potential of NIMS to analyze and image chemically diverse compounds that have been traditionally challenging to observe with mass spectrometry-based techniques.
Nanostructure initiator mass spectrometry (NIMS) is a recently introduced matrix-free desorption/... more Nanostructure initiator mass spectrometry (NIMS) is a recently introduced matrix-free desorption/ ionization platform that requires minimal sample preparation. Its application to xenobiotics and endogenous metabolites in tissues is demonstrated, where clozapine and N-desmethylclozapine were observed from mouse and rat brain sections. It has also been applied to direct biofluid analysis where ketamine and norketamine were observed from plasma and urine. Detection of xenobiotics from biofluids was made even more effective using a novel NIMS on-surface extraction method taking advantage of the hydrophobic nature of the initiator. Linear response and limit of detection were also evaluated for xenobiotics such as methamphetamine, codeine, alprazolam, and morphine, revealing that NIMS can be used for quantitative analysis. Overall, our results demonstrate the capacity of NIMS to perform sensitive, simple, and rapid analyses from highly complex biological tissues and fluids.
Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolom... more Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI-MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,-trimethoxyphenyl)phosphonium acetic acid (TMPP-AA) to improve liquid chromatography (LC)/ESI-MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl-sn-glycerol, dihydrotachysterol, octadecanol, and alpha-tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI-MS instrumentation and demonstrates the potential for targeted metabolomics analysis. Published in
Journal of The American Society for Mass Spectrometry, 2007
The surface structure of porous silicon used in desorption/ionization on porous silicon (DIOS) ma... more The surface structure of porous silicon used in desorption/ionization on porous silicon (DIOS) mass analysis is known to play a primary role in the desorption/ionization (D/I) process. In this study, mass spectrometry and scanning electron microscopy (SEM) are used to examine the correlation between intact ion generation with surface ablation and surface morphology. The DIOS process is found to be highly laser energy dependent and correlates directly with the appearance of surface ions (Si n ϩ and OSiH ϩ ). A threshold laser energy for DIOS is observed (10 mJ/cm 2 ), which supports that DIOS is driven by surface restructuring and is not a strictly thermal process. In addition, three DIOS regimes are observed that correspond to surface restructuring and melting. These results suggest that higher surface area silicon substrates may enhance DIOS performance. A recent example that fits into this mechanism is the surface of silicon nanowires, which has a high surface energy and concomitantly requires lower laser energy for analyte desorption. (J Am Soc Mass Spectrom 2007, 18, 1945-1949
We demonstrate vibrational cooling of anions via collisions with a background gas in an ion trap ... more We demonstrate vibrational cooling of anions via collisions with a background gas in an ion trap attached to a cryogenically controlled cold head (10-400 K). Photoelectron spectra of vibrationally cold C60- anions, produced by electrospray ionization and cooled in the cold ion trap, have been obtained. Relative to spectra taken at room temperature, vibrational hot bands are completely eliminated, yielding well-resolved vibrational structures and a more accurate electron affinity for neutral C60. The electron affinity of C60 is measured to be 2.683+/-0.008 eV. The cold spectra reveal complicated vibrational structures for the transition to the C60 ground state due to the Jahn-Teller effect in the ground state of C60-. Vibrational excitations in the two Ag modes and eight Hg modes are observed, providing ideal data to assess the vibronic couplings in C60-
The strength of the low-barrier hydrogen bond in hydrogen maleate in the gas phase was investigat... more The strength of the low-barrier hydrogen bond in hydrogen maleate in the gas phase was investigated by low-temperature photoelectron spectroscopy and ab initio calculations. Photoelectron spectra of maleic and fumaric acid monoanions (cis-/trans-HO 2 CCHdCHCO 2 -) were obtained at low temperatures and at 193 nm photon energy. Vibrational structure was observed for trans-HO 2 CCHdCHCO 2due to the OCO bending modes; however, cis-HO 2 CCHdCHCO 2yielded a broad and featureless spectrum. The electron binding energy of cis-HO 2 CCHdCHCO 2is about 1 eV blue-shifted relative to trans-HO 2 CCHdCHCO 2due to the formation of intramolecular hydrogen bond in the cis-isomer. Theoretical calculations (CCSD(T)/ aug-cc-pVTZ and B3LYP/aug-cc-pVTZ) were carried out to estimate the strength of the intramolecular hydrogen bond in cis-HO 2 CCHdCHCO 2 -. Combining experimental and theoretical calculations yields an estimate of 21.5 ( 2.0 kcal/mol for the intramolecular hydrogen bond strength in hydrogen maleate.
Photoelectron spectroscopy of vibrationally cold singly and doubly charged higher fullerenes, C n... more Photoelectron spectroscopy of vibrationally cold singly and doubly charged higher fullerenes, C nand C n 2-(n ) 76, 78, and 84), has been investigated at several photon energies. Vibrationally resolved spectra are obtained for both the singly and doubly charged species, and for n ) 78 and 84, transitions from different isomers are also observed. The electron affinities (EAs) of C 76 , C 78 , and C 84 are accurately determined to be 2.975 ( 0.010 eV for C 76 , 3.20 ( 0.01 eV for C 78 (C 2V ), 3.165 ( 0.010 eV for C 78 (D 3 ), 3.23 ( 0.02 for C 78 (C 2V ′), 3.185 ( 0.010 eV for C 84 (D 2 ), and 3.26 ( 0.02 eV for C 84 (D 2d ). The second EA of the higher fullerenes, which represent the electronic stability of the doubly charged anions, are measured to be 0.325 ( 0.010 eV for C 76 , 0.44 ( 0.02 eV for C 78 (C 2v ), 0.53 ( 0.02 eV for C 78 (D 3 ), 0.60 ( 0.04 eV for C 78 (C 2v ′), 0.615 ( 0.010 eV for C 84 (D 2d ), and 0.82 ( 0.01 eV for C 84 (D 2 ). The spectra of the dianions are observed to be similar to those of the singly charged anions, suggesting that the charging induces relatively small structural changes to the fullerene cages. The onsite Coulomb repulsions in the doubly charged fullerenes are directly measured from the differences of the first and second EAs and reveal strong correlation effects between the two extra electrons. The repulsive Coulomb barriers in the doubly charged fullerenes are estimated from the cutoff in the photoelectron spectra and are found to be consistent with estimates from an electrostatic model.
A detailed understanding of the electronic structures of transition metal bis(dithiolene) centers... more A detailed understanding of the electronic structures of transition metal bis(dithiolene) centers is important in the context of their interesting redox, magnetic, and optical properties. The electronic structures of the series [M(mnt) 2 ] n-(M ) Fe − Zn; mnt ) 1,2-S 2 C 2 (CN) 2 ; n ) 1, 2) were examined by a combination of photodetachment photoelectron spectroscopy and density functional theory calculations, providing insights into changes in electronic structure with variation of the metal center and with oxidation. Significant changes were observed for the dianions [M(mnt) 2 ] 2due to stabilization of the metal 3d levels from Fe to Zn and the transition from square-planar to tetrahedral coordination about the metal center (Fe−Ni, D 2h f Cu D 2 f Zn, D 2d ). Changes with oxidation from [M(mnt) 2 ] 2to [M(mnt) 2 ] 1were largely dependent on the nature of the redox-active orbital in the couple [M(mnt) 2 ] 2-/1-. In particular, the first detachment feature for [Fe(mnt) 2 ] 2originated from a metal-based orbital (Fe II f Fe III ) while that for [Fe(mnt) 2 ] 1originated from a ligand-based orbital, a consequence of stabilization of Fe 3d levels in the latter. In contrast, the first detachment feature for both of [Ni(mnt) 2 ] 2and [Ni(mnt) 2 ] 1originated from the same ligand-based orbital in both cases, a result of occupied Ni 3d levels being stabilized relative those of Fe 3d and occurring below the highest energy occupied ligand-based orbital for both of [Ni(mnt) 2 ] 2and [Ni(mnt) 2 ] 1-. The combined data illustrate the subtle interplay between metal-and ligand-based redox chemistry in these species and demonstrate changes in their electronic structures with variation of metal center, oxidation, and coordination geometry.
The cysteine anion was produced in the gas phase by electrospray ionization and investigated by p... more The cysteine anion was produced in the gas phase by electrospray ionization and investigated by photoelectron spectroscopy at low temperature (70 K). The cysteine anion was found to exhibit the thiolate form [ -SCH 2 CH(NH 2 )CO 2 H], rather than the expected carboxylate form [HSCH 2 CH(NH 2 )CO 2 -]. This observation was confirmed by two control experiments, that is, methyl cysteine [CH 3 SCH 2 CH(NH 2 )CO 2 -] and cysteine methyl ester [ -SCH 2 CH(NH 2 )CO 2 CH 3 ]. The electron binding energy of [ -SCH 2 CH(NH 2 )CO 2 H] was measured to be about 0.7 eV blue-shifted relative to [ -SCH 2 CH(NH 2 )CO 2 CH 3 ] due to the formation of an intramolecular -S -‚‚‚HO 2 C-hydrogen bond in the cysteine thiolate. Theoretical calculations at the CCSD(T)/6-311++G-(2df,p) and B3LYP/6-311++G(2df,p) levels were carried out to estimate the strength of this intramolecular -S -‚‚‚HO 2 C-hydrogen bond. Combining experimental measurements and theoretical calculations yielded an estimated value of 16.4 ( 2.0 kcal/mol for the -S -‚‚‚HO 2 C-intramolecular hydrogen-bond strength.
Metabolites are downstream end products of gene and protein activity that closely correlate with ... more Metabolites are downstream end products of gene and protein activity that closely correlate with the phenotype of a biological organism. 1-7 Therefore, by observing specific metabolic changes, one can gain insight into perturbations underlying disease. 8 Consequently, increasing attention has been dedicated to analyzing metabolites using MS in the context of clinical diagnostics, understanding disease mechanisms, and identifying new therapeutic targets.5 , 9 , 10 The ability to analyze metabolites directly from biofluids and tissues continues to challenge current MS technology, largely because of the limits imposed by the complexity of these samples, which contain thousands to tens of thousands of metabolites. 11 A new technology being developed to address this challenge is Nanostructure-Initiator MS (NIMS), a desorption/ionization approach that does not require the application of matrix and thereby facilitates small-molecule (i.e., metabolite) identification. 12 Surface-based mass analysis has seen a resurgence in the past decade, with new MS technologies focused on increasing sensitivity, minimizing background, and reducing sample preparation. 4,6,7,13, 14 MALDI is one of the primary MS platforms currently used for the analysis of biological samples.2 , 15 , 16 However, the application of a MALDI matrix can add significant background at <1000 Da that complicates analysis of the low-mass range (i.e., metabolites).17 In addition, the size of the resulting matrix crystals limits the spatial resolution that can be achieved in tissue imaging.14 Because of these limitations, several matrix-free desorption/ionization approaches have been applied to the analysis of biofluids and tissues.17 Secondary ion MS (SIMS) was one of the first matrix-free desorption/ ionization approaches used to analyze metabolites from biological samples.18 SIMS uses a high-energy primary ion beam to desorb and generate secondary ions from a surface. The primary advantage of SIMS is its high spatial resolution (as small as 50 nm), a powerful characteristic for tissue imaging with MS.19 However, SIMS has yet to be readily applied to the analysis of biofluids and tissues because of its limited sensitivity at >500 Da and analyte fragmentation generated by the high-energy primary ion beam.14 Desorption electrospray ionization (DESI) is a matrix-free technique for analyzing biological samples that uses a charged solvent spray to desorb ions from a surface.20 Advantages of DESI are that no special surface is required and the analysis is performed at ambient pressure with full access to the sample during acquisition.20 , 21 The main limitation of DESI is spatial resolution because "focusing" the charged solvent spray is difficult.22 However, a recent development termed laser ablation ESI (LAESI) is a promising approach to circumvent this limitation. 23 Desorption/ionization on silicon (DIOS), the precursor to NIMS, is another matrix-free laser-induced desorption/ionization approach.24 , 25 DIOS utilizes a porous silicon substrate to absorb the laser energy and vaporize and ionize analytes on the surface without extensive fragmentation.26 Because it does not use a matrix, it has the advantages of low chemical noise and high sensitivity in the low-mass range when used to analzye metabolites in Contact Siuzdak at
Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell ... more Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of α-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.
Nanostructure-initiator mass spectrometry (NIMS) is a new surface-based MS technique that uses a ... more Nanostructure-initiator mass spectrometry (NIMS) is a new surface-based MS technique that uses a nanostructured surface to trap liquid (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;initiator&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;) compounds. Analyte materials adsorbed onto this &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;clathrate&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; surface are subsequently released by laser irradiation for mass analysis. In this protocol, we describe the preparation of NIMS surfaces capable of producing low background and high-sensitivity mass spectrometric measurement using the initiator compound BisF17. Examples of analytes that adsorb to this surface are small molecules, drugs, lipids, carbohydrates and peptides. Typically, NIMS is used to analyze samples ranging from simple analytical standards and proteolytic digests to more complex samples such as tissues, cells and biofluids. Critical experimental considerations of NIMS are described. Specifically, NIMS sensitivity is examined as a function of pre-etch cleaning treatment, etching current density, etching time, initiator composition, sample concentration, sample deposition method and laser fluence. Typically, NIMS surface preparation can be completed in less than 2 h. Subsequent sample preparation requires 1-5 min, depending on sample deposition method. Mass spectrometric data acquisition typically takes 1-30 s per sample.
Analytical and biological variability are issues of central importance to human metabolomics stud... more Analytical and biological variability are issues of central importance to human metabolomics studies. Here both types of variation are examined in human plasma and cerebrospinal fluid (CSF) using a global liquid chromatography/mass spectrometry (LC/MS) metabolomics strategy. The platform shows small analytical variation with a median coefficient of variation (CV) of 15-16% for both plasma and CSF sample matrixes when the integrated area of each peak in the mass spectra is considered. Analysis of biological variation shows that human CSF has a median CV of 35% and plasma has a median CV of 46%. To understand the difference in CV between the biofluids, we compared plasma and CSF independently obtained from different healthy humans. Additionally, we analyzed another group of patients from whom we compared matched CSF and plasma (plasma and CSF obtained from the same human subject). A similar number of features was observed in both biofluids, although the majority of features appeared with greater intensity in plasma. More than a dozen metabolites shared between the human CSF and plasma metabolomes were identified based on accurate mass measurements, retention times, and MS/MS spectra. The fold change in these metabolites was consistent with the median biological CV determined for all peaks. The measured median biological CV together with analysis of intragroup variation of healthy individuals suggests that fold changes above 2 in metabolomics studies investigating plasma or CSF are statistically relevant with respect to the inherent variability of a healthy control group. These data demonstrate the reproducibility of the global metabolomics platform using LC/MS and reveal the robustness of the approach for biomarker discovery.
Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolom... more Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI-MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,-trimethoxyphenyl)phosphonium acetic acid (TMPP-AA) to improve liquid chromatography (LC)/ESI-MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl-sn-glycerol, dihydrotachysterol, octadecanol, and alpha-tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI-MS instrumentation and demonstrates the potential for targeted metabolomics analysis. Published in 2009 by John Wiley & Sons, Ltd.
Nanostructure-initiator mass spectrometry (NIMS) is a highly sensitive, matrix-free technique tha... more Nanostructure-initiator mass spectrometry (NIMS) is a highly sensitive, matrix-free technique that is well suited for biofluid analysis and imaging of biological tissues. Here we provide a new technical variation of NIMS to analyze carbohydrates and steroids, molecules that are challenging to detect with traditional mass spectrometric approaches. Analysis of carbohydrates and steroids was accomplished by spray depositing NaCl or AgNO 3 on the NIMS porous silicon surface to provide a uniform environment rich with cationization agents prior to desporption of the fluorinated polymer initiator. Laser desorption/ionization of the ion-coated NIMS surface allowed for Na + cationization of carbohydrates and Ag + cationization of steroids. The reliability of the approach is quantitatively demonstrated with a calibration curve over the physiological range of glucose and cholesterol concentrations in human serum (1 -200 μM). Additionally we illustrate the sensitivity of the method by showing its ability to detect carbohydrates and steroids down to the 800-amol and 100-fmol levels, respectively. The technique developed is well suited for tissue imaging of biologically significant metabolites such as sucrose and cholesterol. To highlight its applicability, we used cation-enhanced NIMS to image the distribution of sucrose in a Gerbera jamesonii flower stem and the distribution of cholesterol in a mouse brain. The flower stem and brain sections were placed directly on the ioncoated NIMS surface without further preparation and analyzed directly. The overall results reported underscore the potential of NIMS to analyze and image chemically diverse compounds that have been traditionally challenging to observe with mass spectrometry-based techniques.
Nanostructure initiator mass spectrometry (NIMS) is a recently introduced matrix-free desorption/... more Nanostructure initiator mass spectrometry (NIMS) is a recently introduced matrix-free desorption/ ionization platform that requires minimal sample preparation. Its application to xenobiotics and endogenous metabolites in tissues is demonstrated, where clozapine and N-desmethylclozapine were observed from mouse and rat brain sections. It has also been applied to direct biofluid analysis where ketamine and norketamine were observed from plasma and urine. Detection of xenobiotics from biofluids was made even more effective using a novel NIMS on-surface extraction method taking advantage of the hydrophobic nature of the initiator. Linear response and limit of detection were also evaluated for xenobiotics such as methamphetamine, codeine, alprazolam, and morphine, revealing that NIMS can be used for quantitative analysis. Overall, our results demonstrate the capacity of NIMS to perform sensitive, simple, and rapid analyses from highly complex biological tissues and fluids.
Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolom... more Mass spectrometry has become an indispensable tool for the global study of metabolites (metabolomics), primarily using electrospray ionization mass spectrometry (ESI-MS). However, many important classes of molecules such as neutral lipids do not ionize well by ESI and go undetected. Chemical derivatization of metabolites can enhance ionization for increased sensitivity and metabolomic coverage. Here we describe the use of tris(2,4,6,-trimethoxyphenyl)phosphonium acetic acid (TMPP-AA) to improve liquid chromatography (LC)/ESI-MS detection of hydroxylated metabolites (i.e. lipids) from serum extracts. Cholesterol which is not normally detected from serum using ESI is observed with attomole sensitivity. This approach was applied to identify four endogenous lipids (hexadecanoyl-sn-glycerol, dihydrotachysterol, octadecanol, and alpha-tocopherol) from human serum. Overall, this approach extends the types of metabolites which can be detected using standard ESI-MS instrumentation and demonstrates the potential for targeted metabolomics analysis. Published in
Journal of The American Society for Mass Spectrometry, 2007
The surface structure of porous silicon used in desorption/ionization on porous silicon (DIOS) ma... more The surface structure of porous silicon used in desorption/ionization on porous silicon (DIOS) mass analysis is known to play a primary role in the desorption/ionization (D/I) process. In this study, mass spectrometry and scanning electron microscopy (SEM) are used to examine the correlation between intact ion generation with surface ablation and surface morphology. The DIOS process is found to be highly laser energy dependent and correlates directly with the appearance of surface ions (Si n ϩ and OSiH ϩ ). A threshold laser energy for DIOS is observed (10 mJ/cm 2 ), which supports that DIOS is driven by surface restructuring and is not a strictly thermal process. In addition, three DIOS regimes are observed that correspond to surface restructuring and melting. These results suggest that higher surface area silicon substrates may enhance DIOS performance. A recent example that fits into this mechanism is the surface of silicon nanowires, which has a high surface energy and concomitantly requires lower laser energy for analyte desorption. (J Am Soc Mass Spectrom 2007, 18, 1945-1949
We demonstrate vibrational cooling of anions via collisions with a background gas in an ion trap ... more We demonstrate vibrational cooling of anions via collisions with a background gas in an ion trap attached to a cryogenically controlled cold head (10-400 K). Photoelectron spectra of vibrationally cold C60- anions, produced by electrospray ionization and cooled in the cold ion trap, have been obtained. Relative to spectra taken at room temperature, vibrational hot bands are completely eliminated, yielding well-resolved vibrational structures and a more accurate electron affinity for neutral C60. The electron affinity of C60 is measured to be 2.683+/-0.008 eV. The cold spectra reveal complicated vibrational structures for the transition to the C60 ground state due to the Jahn-Teller effect in the ground state of C60-. Vibrational excitations in the two Ag modes and eight Hg modes are observed, providing ideal data to assess the vibronic couplings in C60-
The strength of the low-barrier hydrogen bond in hydrogen maleate in the gas phase was investigat... more The strength of the low-barrier hydrogen bond in hydrogen maleate in the gas phase was investigated by low-temperature photoelectron spectroscopy and ab initio calculations. Photoelectron spectra of maleic and fumaric acid monoanions (cis-/trans-HO 2 CCHdCHCO 2 -) were obtained at low temperatures and at 193 nm photon energy. Vibrational structure was observed for trans-HO 2 CCHdCHCO 2due to the OCO bending modes; however, cis-HO 2 CCHdCHCO 2yielded a broad and featureless spectrum. The electron binding energy of cis-HO 2 CCHdCHCO 2is about 1 eV blue-shifted relative to trans-HO 2 CCHdCHCO 2due to the formation of intramolecular hydrogen bond in the cis-isomer. Theoretical calculations (CCSD(T)/ aug-cc-pVTZ and B3LYP/aug-cc-pVTZ) were carried out to estimate the strength of the intramolecular hydrogen bond in cis-HO 2 CCHdCHCO 2 -. Combining experimental and theoretical calculations yields an estimate of 21.5 ( 2.0 kcal/mol for the intramolecular hydrogen bond strength in hydrogen maleate.
Photoelectron spectroscopy of vibrationally cold singly and doubly charged higher fullerenes, C n... more Photoelectron spectroscopy of vibrationally cold singly and doubly charged higher fullerenes, C nand C n 2-(n ) 76, 78, and 84), has been investigated at several photon energies. Vibrationally resolved spectra are obtained for both the singly and doubly charged species, and for n ) 78 and 84, transitions from different isomers are also observed. The electron affinities (EAs) of C 76 , C 78 , and C 84 are accurately determined to be 2.975 ( 0.010 eV for C 76 , 3.20 ( 0.01 eV for C 78 (C 2V ), 3.165 ( 0.010 eV for C 78 (D 3 ), 3.23 ( 0.02 for C 78 (C 2V ′), 3.185 ( 0.010 eV for C 84 (D 2 ), and 3.26 ( 0.02 eV for C 84 (D 2d ). The second EA of the higher fullerenes, which represent the electronic stability of the doubly charged anions, are measured to be 0.325 ( 0.010 eV for C 76 , 0.44 ( 0.02 eV for C 78 (C 2v ), 0.53 ( 0.02 eV for C 78 (D 3 ), 0.60 ( 0.04 eV for C 78 (C 2v ′), 0.615 ( 0.010 eV for C 84 (D 2d ), and 0.82 ( 0.01 eV for C 84 (D 2 ). The spectra of the dianions are observed to be similar to those of the singly charged anions, suggesting that the charging induces relatively small structural changes to the fullerene cages. The onsite Coulomb repulsions in the doubly charged fullerenes are directly measured from the differences of the first and second EAs and reveal strong correlation effects between the two extra electrons. The repulsive Coulomb barriers in the doubly charged fullerenes are estimated from the cutoff in the photoelectron spectra and are found to be consistent with estimates from an electrostatic model.
A detailed understanding of the electronic structures of transition metal bis(dithiolene) centers... more A detailed understanding of the electronic structures of transition metal bis(dithiolene) centers is important in the context of their interesting redox, magnetic, and optical properties. The electronic structures of the series [M(mnt) 2 ] n-(M ) Fe − Zn; mnt ) 1,2-S 2 C 2 (CN) 2 ; n ) 1, 2) were examined by a combination of photodetachment photoelectron spectroscopy and density functional theory calculations, providing insights into changes in electronic structure with variation of the metal center and with oxidation. Significant changes were observed for the dianions [M(mnt) 2 ] 2due to stabilization of the metal 3d levels from Fe to Zn and the transition from square-planar to tetrahedral coordination about the metal center (Fe−Ni, D 2h f Cu D 2 f Zn, D 2d ). Changes with oxidation from [M(mnt) 2 ] 2to [M(mnt) 2 ] 1were largely dependent on the nature of the redox-active orbital in the couple [M(mnt) 2 ] 2-/1-. In particular, the first detachment feature for [Fe(mnt) 2 ] 2originated from a metal-based orbital (Fe II f Fe III ) while that for [Fe(mnt) 2 ] 1originated from a ligand-based orbital, a consequence of stabilization of Fe 3d levels in the latter. In contrast, the first detachment feature for both of [Ni(mnt) 2 ] 2and [Ni(mnt) 2 ] 1originated from the same ligand-based orbital in both cases, a result of occupied Ni 3d levels being stabilized relative those of Fe 3d and occurring below the highest energy occupied ligand-based orbital for both of [Ni(mnt) 2 ] 2and [Ni(mnt) 2 ] 1-. The combined data illustrate the subtle interplay between metal-and ligand-based redox chemistry in these species and demonstrate changes in their electronic structures with variation of metal center, oxidation, and coordination geometry.
The cysteine anion was produced in the gas phase by electrospray ionization and investigated by p... more The cysteine anion was produced in the gas phase by electrospray ionization and investigated by photoelectron spectroscopy at low temperature (70 K). The cysteine anion was found to exhibit the thiolate form [ -SCH 2 CH(NH 2 )CO 2 H], rather than the expected carboxylate form [HSCH 2 CH(NH 2 )CO 2 -]. This observation was confirmed by two control experiments, that is, methyl cysteine [CH 3 SCH 2 CH(NH 2 )CO 2 -] and cysteine methyl ester [ -SCH 2 CH(NH 2 )CO 2 CH 3 ]. The electron binding energy of [ -SCH 2 CH(NH 2 )CO 2 H] was measured to be about 0.7 eV blue-shifted relative to [ -SCH 2 CH(NH 2 )CO 2 CH 3 ] due to the formation of an intramolecular -S -‚‚‚HO 2 C-hydrogen bond in the cysteine thiolate. Theoretical calculations at the CCSD(T)/6-311++G-(2df,p) and B3LYP/6-311++G(2df,p) levels were carried out to estimate the strength of this intramolecular -S -‚‚‚HO 2 C-hydrogen bond. Combining experimental measurements and theoretical calculations yielded an estimated value of 16.4 ( 2.0 kcal/mol for the -S -‚‚‚HO 2 C-intramolecular hydrogen-bond strength.
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Papers by Hin-Koon Woo