Proceedings of The National Academy of Sciences, 2008
We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which... more We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorousphase interactions. This ''soft'' immobilization allows efficient desorption/ionization while also enabling the use of surfacewashing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of the Nimzyme assay to analyze complex mixtures is illustrated by identifying and directly characterizing -1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65°C and 5.5, respectively, and the activity was inhibited by both phenylethyl--D-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced ␥-proteobacterium. In general, this assay provides an efficient method for detection and characterization of enzymatic activities in complex biological mixtures prior to sequencing or cloning efforts. More generally, this approach may have important applications for screening both enzymatic and inhibitor libraries, constructing and screening glycan microarrays, and complementing fluorous-phase organic synthesis.
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
Background: Rapidly characterizing the operational interrelationships among all genes in a given ... more Background: Rapidly characterizing the operational interrelationships among all genes in a given organism is a critical bottleneck to significantly advancing our understanding of thousands of newly sequenced microbial and eukaryotic species. While evolving technologies for global profiling of transcripts, proteins, and metabolites are making it possible to comprehensively survey cellular physiology in newly sequenced organisms, these experimental techniques have not kept pace with sequencing efforts. Compounding these technological challenges is the fact that individual experiments typically only stimulate relatively small-scale cellular responses, thus requiring numerous expensive experiments to survey the operational relationships among nearly all genetic elements. Therefore, a relatively quick and inexpensive strategy for observing changes in large fractions of the genetic elements is highly desirable.
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.
IEEE Transactions on Neural Systems and Rehabilitation Engineering, 2009
This paper reports a 128-channel neural recording integrated circuit (IC) with on-the-fly spike f... more This paper reports a 128-channel neural recording integrated circuit (IC) with on-the-fly spike feature extraction and wireless telemetry. The chip consists of eight 16-channel front-end recording blocks, spike detection and feature extraction digital signal processor (DSP), ultra wideband (UWB) transmitter, and on-chip bias generators. Each recording channel has amplifiers with programmable gain and bandwidth to accommodate different types of biological signals. An analog-to-digital converter (ADC) shared by 16 amplifiers through time-multiplexing results in a balanced trade-off between the power consumption and chip area. A nonlinear energy operator (NEO) based spike detector is implemented for identifying spikes, which are further processed by a digital frequency-shaping filter. The computationally efficient spike detection and feature extraction algorithms attribute to an auspicious DSP implementation on-chip. UWB telemetry is designed to wirelessly transfer raw data from 128 recording channels at a data rate of 90 Mbit/s. The chip is realized in 0.35 m complementary metal-oxide-semiconductor (CMOS) process with an area of 8.8 7.2 mm 2 and consumes 6 mW by employing a sequential turn-on architecture that selectively powers off idle analog circuit blocks. The chip has been tested for electrical specifications and verified in an ex vivo biological environment.
An emerging class of multi-channel neural recording systems aims to simultaneously monitor the ac... more An emerging class of multi-channel neural recording systems aims to simultaneously monitor the activity of many neurons by miniaturizing and increasing the number of recording channels. Vast volume of data from the recording systems, however, presents a challenge for processing and transmitting wirelessly. An on-chip neural signal processor is needed for filtering uninterested recording samples and performing spike sorting. This paper presents a VLSI architecture of a neural signal processor that can reliably detect spike via a nonlinear energy operator, enhance spike signal over noise ratio by a noise shaping filter, and select meaningful recording samples for clustering by using informative samples. The architecture is implemented in 90-nm CMOS process, occupies 0.2 mm2, and consumes 0.5 mW of power.
Future retinal prosthesis requires more stimulation sites to restore vision to the level of indep... more Future retinal prosthesis requires more stimulation sites to restore vision to the level of independent mobility, large print reading and facial recognition. This paper presents a modular design of a 1024 pixel retinal stimulator. The individual module consists of 64 drivers, chosen to optimize the system. The circuit design and the layout of the 64-driver module are presented. The chip is currently under fabrication in 0.35-mum CMOS. The digital controller block for the chip is currently being designed and will be integrated with this driver chip in the future
AbstractThis paper reports an integrated 256-channel epiretinal prosthesis integrated circuit (I... more AbstractThis paper reports an integrated 256-channel epiretinal prosthesis integrated circuit (IC). This epiretinal pros-thesis system consists of a power telemetry subsystem to deliver 100 mW power, a data telemetry subsystem to transfer 2 Mbps data, digital controllers to ...
This article describes a study on neural noise and neural signal feature extraction, targeting re... more This article describes a study on neural noise and neural signal feature extraction, targeting real-time spike sorting with miniaturized microchip implementation. Neuronal signature, noise shaping, and adaptive bandpass filtering are reported as the techniques to enhance the signal-to-noise ratio (SNR). A subset of informative samples of the waveforms is extracted as features for classification. Quantitative and comparative experiments with both synthesized and animal data are included to evaluate different feature extraction approaches. In addition, a preliminary hardware implementation has been realized using an integrated circuit.
To promote studies of cytochrome c (Cyt c) ranging from apoptosis to protein folding, a system fo... more To promote studies of cytochrome c (Cyt c) ranging from apoptosis to protein folding, a system for facile mutagenesis and high-level expression is desirable. This work used a generally applicable strategy for improving yields of heterologously expressed protein in Escherichia coli. Starting with the yeast Cyt c plus heme lyase construct of Pollock et al. [Pollock, W. B., Rosell, F. I., Twitchett, M. B., Dumont, M. E., and Mauk, A. G. (1998) Biochemistry 37, 6124-6131]
The alkaline transition of cytochrome c is a model for protein structural switching in which the ... more The alkaline transition of cytochrome c is a model for protein structural switching in which the normal heme ligand is replaced by another group. Stopped flow data following a jump to high pH detect two slow kinetic phases, suggesting two rate-limiting structure changes. Results described here indicate that these events are controlled by the same structural unfolding reactions that account for the first two steps in the reversible unfolding pathway of cytochrome c. These and other results show that the cooperative folding-unfolding behavior of protein foldons can account for a variety of functional activities in addition to determining folding pathways.
Proceedings of The National Academy of Sciences, 2001
Magnetic relaxation has been used extensively to study and characterize biological tissues. In pa... more Magnetic relaxation has been used extensively to study and characterize biological tissues. In particular, spin-lattice relaxation in the rotating frame (T 1) of water in protein solutions has been demonstrated to be sensitive to macromolecular weight and composition. However, the nature of the contribution from low frequency processes to water relaxation remains unclear. We have examined this problem by studying the water T1 dispersion in peptide solutions ( 14 N-and 15 N-labeled), glycosaminoglycan solutions, and samples of bovine articular cartilage before and after proteoglycan degradation. We find in model systems and tissue that hydrogen exchange from NH and OH groups to water dominates the low frequency water T 1 dispersion, in the context of the model used to interpret the relaxation data. Further, low frequency dispersion changes are correlated with loss of proteoglycan from the extra-cellular matrix of articular cartilage. This finding has significance for the noninvasive detection of matrix degradation.
The measurement of amino acid-resolved hydrogen exchange (HX) has provided the most detailed info... more The measurement of amino acid-resolved hydrogen exchange (HX) has provided the most detailed information so far available on the structure and properties of protein folding intermediates. Direct HX measurements can define the structure of tenuous molten globule forms that are generally inaccessible to the usual crystallographic and NMR methods (C. Redfield review in this issue). HX pulse labeling methods can specify the structure, stability and kinetics of folding intermediates that exist for less than 1 s during kinetic folding. Native state HX methods can detect and characterize folding intermediates that exist as infinitesimally populated high energy excited state forms under native conditions. The results obtained in these ways suggest principles that appear to explain the properties of partially folded intermediates and how they are organized into folding pathways. The application of these methods is detailed here.
Proceedings of The National Academy of Sciences, 2008
We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which... more We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorousphase interactions. This ''soft'' immobilization allows efficient desorption/ionization while also enabling the use of surfacewashing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of the Nimzyme assay to analyze complex mixtures is illustrated by identifying and directly characterizing -1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65°C and 5.5, respectively, and the activity was inhibited by both phenylethyl--D-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced ␥-proteobacterium. In general, this assay provides an efficient method for detection and characterization of enzymatic activities in complex biological mixtures prior to sequencing or cloning efforts. More generally, this approach may have important applications for screening both enzymatic and inhibitor libraries, constructing and screening glycan microarrays, and complementing fluorous-phase organic synthesis.
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
Background: Rapidly characterizing the operational interrelationships among all genes in a given ... more Background: Rapidly characterizing the operational interrelationships among all genes in a given organism is a critical bottleneck to significantly advancing our understanding of thousands of newly sequenced microbial and eukaryotic species. While evolving technologies for global profiling of transcripts, proteins, and metabolites are making it possible to comprehensively survey cellular physiology in newly sequenced organisms, these experimental techniques have not kept pace with sequencing efforts. Compounding these technological challenges is the fact that individual experiments typically only stimulate relatively small-scale cellular responses, thus requiring numerous expensive experiments to survey the operational relationships among nearly all genetic elements. Therefore, a relatively quick and inexpensive strategy for observing changes in large fractions of the genetic elements is highly desirable.
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.
IEEE Transactions on Neural Systems and Rehabilitation Engineering, 2009
This paper reports a 128-channel neural recording integrated circuit (IC) with on-the-fly spike f... more This paper reports a 128-channel neural recording integrated circuit (IC) with on-the-fly spike feature extraction and wireless telemetry. The chip consists of eight 16-channel front-end recording blocks, spike detection and feature extraction digital signal processor (DSP), ultra wideband (UWB) transmitter, and on-chip bias generators. Each recording channel has amplifiers with programmable gain and bandwidth to accommodate different types of biological signals. An analog-to-digital converter (ADC) shared by 16 amplifiers through time-multiplexing results in a balanced trade-off between the power consumption and chip area. A nonlinear energy operator (NEO) based spike detector is implemented for identifying spikes, which are further processed by a digital frequency-shaping filter. The computationally efficient spike detection and feature extraction algorithms attribute to an auspicious DSP implementation on-chip. UWB telemetry is designed to wirelessly transfer raw data from 128 recording channels at a data rate of 90 Mbit/s. The chip is realized in 0.35 m complementary metal-oxide-semiconductor (CMOS) process with an area of 8.8 7.2 mm 2 and consumes 6 mW by employing a sequential turn-on architecture that selectively powers off idle analog circuit blocks. The chip has been tested for electrical specifications and verified in an ex vivo biological environment.
An emerging class of multi-channel neural recording systems aims to simultaneously monitor the ac... more An emerging class of multi-channel neural recording systems aims to simultaneously monitor the activity of many neurons by miniaturizing and increasing the number of recording channels. Vast volume of data from the recording systems, however, presents a challenge for processing and transmitting wirelessly. An on-chip neural signal processor is needed for filtering uninterested recording samples and performing spike sorting. This paper presents a VLSI architecture of a neural signal processor that can reliably detect spike via a nonlinear energy operator, enhance spike signal over noise ratio by a noise shaping filter, and select meaningful recording samples for clustering by using informative samples. The architecture is implemented in 90-nm CMOS process, occupies 0.2 mm2, and consumes 0.5 mW of power.
Future retinal prosthesis requires more stimulation sites to restore vision to the level of indep... more Future retinal prosthesis requires more stimulation sites to restore vision to the level of independent mobility, large print reading and facial recognition. This paper presents a modular design of a 1024 pixel retinal stimulator. The individual module consists of 64 drivers, chosen to optimize the system. The circuit design and the layout of the 64-driver module are presented. The chip is currently under fabrication in 0.35-mum CMOS. The digital controller block for the chip is currently being designed and will be integrated with this driver chip in the future
AbstractThis paper reports an integrated 256-channel epiretinal prosthesis integrated circuit (I... more AbstractThis paper reports an integrated 256-channel epiretinal prosthesis integrated circuit (IC). This epiretinal pros-thesis system consists of a power telemetry subsystem to deliver 100 mW power, a data telemetry subsystem to transfer 2 Mbps data, digital controllers to ...
This article describes a study on neural noise and neural signal feature extraction, targeting re... more This article describes a study on neural noise and neural signal feature extraction, targeting real-time spike sorting with miniaturized microchip implementation. Neuronal signature, noise shaping, and adaptive bandpass filtering are reported as the techniques to enhance the signal-to-noise ratio (SNR). A subset of informative samples of the waveforms is extracted as features for classification. Quantitative and comparative experiments with both synthesized and animal data are included to evaluate different feature extraction approaches. In addition, a preliminary hardware implementation has been realized using an integrated circuit.
To promote studies of cytochrome c (Cyt c) ranging from apoptosis to protein folding, a system fo... more To promote studies of cytochrome c (Cyt c) ranging from apoptosis to protein folding, a system for facile mutagenesis and high-level expression is desirable. This work used a generally applicable strategy for improving yields of heterologously expressed protein in Escherichia coli. Starting with the yeast Cyt c plus heme lyase construct of Pollock et al. [Pollock, W. B., Rosell, F. I., Twitchett, M. B., Dumont, M. E., and Mauk, A. G. (1998) Biochemistry 37, 6124-6131]
The alkaline transition of cytochrome c is a model for protein structural switching in which the ... more The alkaline transition of cytochrome c is a model for protein structural switching in which the normal heme ligand is replaced by another group. Stopped flow data following a jump to high pH detect two slow kinetic phases, suggesting two rate-limiting structure changes. Results described here indicate that these events are controlled by the same structural unfolding reactions that account for the first two steps in the reversible unfolding pathway of cytochrome c. These and other results show that the cooperative folding-unfolding behavior of protein foldons can account for a variety of functional activities in addition to determining folding pathways.
Proceedings of The National Academy of Sciences, 2001
Magnetic relaxation has been used extensively to study and characterize biological tissues. In pa... more Magnetic relaxation has been used extensively to study and characterize biological tissues. In particular, spin-lattice relaxation in the rotating frame (T 1) of water in protein solutions has been demonstrated to be sensitive to macromolecular weight and composition. However, the nature of the contribution from low frequency processes to water relaxation remains unclear. We have examined this problem by studying the water T1 dispersion in peptide solutions ( 14 N-and 15 N-labeled), glycosaminoglycan solutions, and samples of bovine articular cartilage before and after proteoglycan degradation. We find in model systems and tissue that hydrogen exchange from NH and OH groups to water dominates the low frequency water T 1 dispersion, in the context of the model used to interpret the relaxation data. Further, low frequency dispersion changes are correlated with loss of proteoglycan from the extra-cellular matrix of articular cartilage. This finding has significance for the noninvasive detection of matrix degradation.
The measurement of amino acid-resolved hydrogen exchange (HX) has provided the most detailed info... more The measurement of amino acid-resolved hydrogen exchange (HX) has provided the most detailed information so far available on the structure and properties of protein folding intermediates. Direct HX measurements can define the structure of tenuous molten globule forms that are generally inaccessible to the usual crystallographic and NMR methods (C. Redfield review in this issue). HX pulse labeling methods can specify the structure, stability and kinetics of folding intermediates that exist for less than 1 s during kinetic folding. Native state HX methods can detect and characterize folding intermediates that exist as infinitesimally populated high energy excited state forms under native conditions. The results obtained in these ways suggest principles that appear to explain the properties of partially folded intermediates and how they are organized into folding pathways. The application of these methods is detailed here.
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