Christie Hunter - Academia.edu (original) (raw)

Papers by Christie Hunter

Research paper thumbnail of Increasing Protein Identification Using Coupled Chip Based NanoLC Columns

Journal of biomolecular techniques, 2011

Nanoflow liquid chromatography coupled with nano-electrospray (nanoLC-MS) is the method of choice... more Nanoflow liquid chromatography coupled with nano-electrospray (nanoLC-MS) is the method of choice for sensitive peptide and protein analysis for proteomics research. We recently reported on a new microfluidic platform (cHiPLC-nanoflex) for nanoLC-MS applications. In addition to delivering easy-to-use, dead-volume-free connections to microfluidic devices, the platform's cHiPLC columns provide excellent column-to-column separation reproducibility. We have reported previously(1) that while increasing only gradient time or column length can improve peak capacity, using a longer column in combination with increasing gradient time is the most effective way of obtaining higher peak capacity. In this presentation we report on how the dead volume free connections of the cHiPLC Nanoflex platform allow for the coupling of two 15 cm long chip columns for improved resolution. The proposed set-up makes it easy to switch between single and dual column mode depending on application requirements...

Research paper thumbnail of Cross-Laboratory Standardization of Preclinical Lipidomics Using Differential Mobility Spectrometry and Multiple Reaction Monitoring

Modern biomarker and translational research as well as personalized health care studies rely heav... more Modern biomarker and translational research as well as personalized health care studies rely heavily on powerful omics' technologies, including metabolomics and lipidomics. However, to translate metabolomics and lipidomics discoveries into a high-throughput clinical setting, standardization is of utmost importance. Here, we compared and benchmarked a quantitative lipidomics platform. The employed Lipidyzer platform is based on lipid class separation by means of differential mobility spectrometry with subsequent multiple reaction monitoring. Quantitation is achieved by the use of 54 deuterated internal standards and an automated informatics approach. We investigated the platform performance across nine laboratories using NIST SRM 1950−Metabolites in Frozen Human Plasma, and three NIST Candidate Reference Materials 8231−Frozen Human Plasma Suite for Metabolomics (high triglyceride, diabetic, and African-American plasma). In addition, we comparatively analyzed 59 plasma samples from individuals with familial hypercholesterolemia from a clinical cohort study. We provide evidence that the more practical methyl-tert-butyl ether extraction outperforms the classic Bligh and Dyer approach and compare our results with two previously published ring trials. In summary, we present standardized lipidomics protocols, allowing for the highly reproducible analysis of several hundred human plasma lipids, and present detailed molecular information for potentially disease relevant and ethnicity-related materials.

Research paper thumbnail of 200 μm ID Chip Based Columns for Increased Throughput Peptide Quantitation with NanoLC/MS

Journal of biomolecular techniques, 2012

Nanoflow liquid chromatography coupled with mass spectrometry (nanoLC/MS) is the method of choice... more Nanoflow liquid chromatography coupled with mass spectrometry (nanoLC/MS) is the method of choice for sensitive peptide quantitation. We recently reported on a new microfluidic platform (cHiPLC-nanoflex) for nanoLC-MS applications. In addition to delivering easy-to-use, dead-volume-free connections to microfluidic devices, the platform's cHiPLC columns provide excellent column-to-column separation reproducibility. While providing excellent sensitivity, the low flow rates when using 75 μm ID columns reduces sample throughput due to gradient delay in the nanoLC system itself, delay in the autosampler and sample loop, and the delay caused by the connecting tubing. One way to address this is to use a larger inner diameter column at a proportionally higher flow rate. While this will reduce sensitivity when the total sample amount is kept equal, the delay times, however, can be greatly reduced and sample throughput accelerated. Reducing the column and gradient lengths can further redu...

Research paper thumbnail of Statistical control of peptide and protein error rates in large-scale targeted data-independent acquisition analyses

Nature Methods, 2017

Liquid chromatography coupled to tandem mass spectrometry is the main method for highthroughput i... more Liquid chromatography coupled to tandem mass spectrometry is the main method for highthroughput identification and quantification of peptides and inferred proteins. Within this field, data-independent acquisition (DIA) combined with peptide-centric scoring, exemplified by SWATH-MS, emerged as a scalable method to achieve deep and consistent proteome coverage across large-scale datasets. Here we discuss the adaptation of statistical concepts developed for discovery proteomics based on spectrum-centric scoring to large-scale DIA experiments analyzed with peptide-centric scoring strategies and provide guidance on their application. We show that optimal tradeoffs between sensitivity and specificity require careful considerations of the Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Research paper thumbnail of On-line Chip based 2D RP/RP LC-MS/MS Method for Proteomic Analysis

Journal of Biomolecular Techniques Jbt, May 1, 2013

Two-dimensional (2D) liquid chromatography is widely used for proteome identification and quantif... more Two-dimensional (2D) liquid chromatography is widely used for proteome identification and quantification using the advantage of increasing peak capacity. Herein, we developed an simplified chip based 2D-LC workflow using a high pH/RP first dimensional separation and low pH/RP secondary dimension coupled to mass spectrometer for proteomic analysis.The 2D LC separation was performed using ekspert™ nanoLC 425(Eksigent, part of AB SCIEX) system. Digested E. coli cell lysates were first loaded onto a 200μm × 15cm C18 column at pH 9.8 with a flowrate of 1 μL/min. Step gradient was used to sequentially elute peptide fractions, which were diluted to pH 2.5 before being captured by a 200μm × 6mm C18 chip trap column. Each fraction was then separated with a 75 μm × 15cm C18 chip column at 300 nL/min and analyzed with TripleTOF® 5600 (AB SCIEX). Data was processed with ProteinPilot™ Software (AB SCIEX).The preliminary result suggested the on-line 2D RP-RP method as an easy and competitive approach for proteome discovery. The comparison experiments of 1D, 2D-6 fraction and 2D-10 fraction were performed. Using ∼1ug of E. coli digested cell lysates, there are 1.8× and 2.1× increase in the identification numbers for 2D-6 and 2D-10 fractions versus the 1D configuration at the peptide level (5% local FDR). The other advantage of 2D workflow is the larger sample loading capacities on the column. When the loading amount was increased by 10×, the number of detected peptides increased by 3.3× and 4× for the 2D 6 and 10 fraction workflows, respectively, over 1D workflow. The measured retention time of peptides detected in both the 1D 1ug and 2D 10ug experiments showed very good correlation (r2 0.99, slope 1.0). Further optimization of both the first and second dimension is ongoing to further improve the peptide detection rates.

Research paper thumbnail of Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications

Molecular & Cellular Proteomics, 2015

Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology,... more Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. High throughput characterization of histone PTMs is mostly performed by using nano-liquid chromatography coupled to mass spectrometry. However, limitations in speed and stochastic sampling of data dependent acquisition methods in MS lead to incomplete discrimination of isobaric peptides and loss of low abundant species. In this work, we analyzed histone PTMs with a data-independent acquisition method, namely SWATH™ analysis. This approach allows for MS/MS-based quantification of all analytes without upfront assay development and no issues of biased and incomplete sampling. We purified histone proteins from human embryonic stem cells and mouse trophoblast stem cells before and after differentiation, and prepared them for MS analysis using the propionic anhydride protocol. Results on histone H3 peptides verified that sequential window acquisition of all theoretical mass spectra could accurately quantify peptides (<9% average coefficient of variation, CV) over four orders of magnitude, and we could discriminate isobaric and co-eluting peptides (e.g. H3K18ac and H3K23ac) using MS/MS-based quantification. This method provided high sensitivity and precision, supported by the fact that we could find significant differences for remarkably low abundance PTMs such as H3K9me2S10ph (relative abundance <0.02%). We performed relative quantification for few sample peptides using different fragment ions and observed high consistency (CV <15%) between the fragments. This indicated that different fragment ions can be used independently to From the ‡Epigenetics Program,

Research paper thumbnail of Use of proteomics in development of biomarkers and early markers of toxicity

Research paper thumbnail of Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications

PROTEOMICS, 2003

Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for ... more Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications The unique scanning capabilities of a hybrid linear ion trap (Q TRAP) mass spectrometer are described with an emphasis on proteomics applications. The combination of the very selective triple quadrupole based tandem mass spectrometry (MS/MS) scans with the very sensitive ion trap product ion scans allows rapid identification of peptides at low concentrations derived from post-translationally modified proteins on chromatographic time scales. The Q TRAP instrument also offers the opportunity to conduct a variety of ion processing steps prior to performing a mass scan. For example, the enhancement of the multiple-charge ion contents of the ion trap can be performed resulting in a survey mass spectrum dominated by double-and triple-charge peptides. This facilitates the identification of relevant biological species in both separated and unseparated peptide mixtures for further MS/MS experiments.

Research paper thumbnail of Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1

Proceedings of the National Academy of Sciences, 2003

Generation of biological function by chemical methods is potentially of great importance for the ... more Generation of biological function by chemical methods is potentially of great importance for the understanding and targeting of physiological processes. Chemical synthesis of proteins offers the ability to alter the properties of target protein molecules in a tailor-made fashion. In the present work it is demonstrated that this methodology can be expanded to the elucidation of protein–protein interactions as exemplified by the complete chemical synthesis of the protooncogene product H-Ras as well as of the Ras-binding domain (RBD) of its effector c-Raf1. The 166-aa polypeptide chain of H-Ras was synthesized by native chemical ligation of three unprotected peptide segments. Similarly, the 81-aa RBD was prepared by ligation of two peptide segments. Both RBD and Ras displayed functional and spectroscopic properties indistinguishable from their recombinant forms as judged by CD spectroscopy and from transient kinetic measurements of the Ras–RBD interaction as well as from nucleotide rep...

Research paper thumbnail of Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

Proceedings of the National Academy of Sciences, 2003

A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near ... more A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near the heme 6-propionate group by replacing K45 and K63 with glutamyl residues. One-dimensional 1 H NMR spectroscopy indicates that Mn 2+ binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn 2+ is 1.28(4) × 10 4 M −1 (pH 6.96, ionic strength I = 17.2 μM, 25°C). In addition, these substitutions lower the reduction potential of the protein and increase the pK a for the water molecule coordinated to the heme iron of metmyoglobin. The peroxidase [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS, as substrate] and the Mn 2+ -peroxidase activity of the variant are both increased ≈3-fold. In contrast to wild-type Mb, both the affinity for azide and the midpoint potential of the variant are significantly influenced by the addition of Mn 2+ . The structure of the variant has been determined by x-ray ...

Research paper thumbnail of Engineered metalloregulation of azide binding affinity and reduction potential of horse heart myoglobin

Dalton Transactions, 2013

Metal ion binding to a previously reported variant of horse heart myoglobin (Lys45Glu/Lys63Glu) w... more Metal ion binding to a previously reported variant of horse heart myoglobin (Lys45Glu/Lys63Glu) with a metal ion binding site on the surface of the protein that is adjacent to the haem binding site has been shown to influence ligand binding and electrochemical properties of the protein. For example, the K(d) (μM) for binding of azide to this variant decreases from 277 ± 9 to 32 ± 3 following addition of a saturating concentration of Mn(2+) (the value for the wild-type protein under the same conditions is 26 ± 1). Similarly, the midpoint reduction potential E(m) (mV vs. standard hydrogen electrode) increases from 9 to 40 in the presence of a saturating concentration of Mn(2+) (the value for the wild-type protein under the same conditions is 45 ± 2). These results demonstrate the potential value of engineered metal ion binding sites as a means of regulating the functional properties of even simple haem proteins.

Research paper thumbnail of A sensitive fluorescence monitor for the detection of activated Ras: total chemical synthesis of site-specifically labeled Ras binding domain of c-Raf1 immobilized on a surface

Chemistry & Biology, 2001

Background: The RasWGDP^RasWGTP cycle plays a central role in eukaryotic signaling cascades. Muta... more Background: The RasWGDP^RasWGTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras which stabilize activated RasWGTP lead to a continuous stimulation of downstream effectors and ultimately to cell proliferation. Ras mutants which increase the steady-state concentration of RasWGTP are involved in about 30% of all human cancers. It is therefore of great interest to develop a biosensor which is sensitive to RasWGTP but not to RasWGDP. Results : The Ras binding domain (RBD) of c-Raf1 was synthesized from two unprotected peptide segments by native chemical ligation. Two fluorescent amino acids with structures based on the nitrobenz-2-oxa-1,3-diazole and coumaryl chromophores were incorporated at a site which is close to the RBD/ RasWGTP binding surface. Additionally, a C-terminal tag consisting of His 6 was introduced. The K d values for binding of the sitespecifically modified proteins to RasWGTP are comparable to that of wild-type RBD. Immobilization of C-terminal His 6 tagmodified fluorescent RBD onto Ni-NTA-coated surfaces allowed the detection of RasWGTP in the 100 nM range. Likewise, RasWGTP/Q61L (an oncogenic mutant of Ras with very low intrinsic GTP hydrolysis activity) can also be detected in this assay system. RasWGDP does not bind to the immobilized RBD, thus allowing discrimination between inactive and activated Ras. Conclusions: The site-specific incorporation of a fluorescent group at a strategic position in a Ras effector protein allows the detection of activated Ras with high sensitivity. This example illustrates the fact that the chemical synthesis of proteins or protein domains makes it possible to incorporate any kind of natural or unnatural amino acid at the position of choice, thereby enabling the facile preparation of specific biosensors, enhanced detection systems for drug screening, or the synthesis of activated proteins, e.g. phosphorylated proteins involved in signaling pathways, as defined molecular species.

Research paper thumbnail of Native Chemical Ligation of Hydrophobic Peptides in Lipid Bilayer Systems

Bioconjugate Chemistry, 2004

Research paper thumbnail of Accelerated Protein Biomarker Discovery from FFPE tissue samples using Single-shot, Short Gradient Microflow SWATH MS

ABSTRACTWe report and evaluated a microflow, single-shot, short gradient SWATH MS method intended... more ABSTRACTWe report and evaluated a microflow, single-shot, short gradient SWATH MS method intended to accelerate the discovery and verification of protein biomarkers in clinical specimens. The method uses 15-min gradient microflow-LC peptide separation, an optimized SWATH MS window configuration and OpenSWATH software for data analysis.We applied the method to a cohort 204 of FFPE prostate tissue samples from 58 prostate cancer patients and 10 prostatic hyperplasia patients. Altogether we identified 27,976 proteotypic peptides and 4,043 SwissProt proteins from these 204 samples. Compared to a reference SWATH method with 2-hour gradient the accelerated method consumed only 27% instrument time, quantified 80% proteins and showed reduced batch effects. 3,800 proteins were quantified by both methods in two different instruments with relatively high consistency (r = 0.77). 75 proteins detected by the accelerated method with differential abundance between clinical groups were selected for ...

Research paper thumbnail of A sensitive fluorescence monitor for the detection of activated Ras: total chemical synthesis of site-specifically labeled Ras binding domain of c-Raf1 immobilized on a surface

Chemistry & Biology, 2001

The Ras.GDP-Ras.GTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras... more The Ras.GDP-Ras.GTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras which stabilize activated Ras.GTP lead to a continuous stimulation of downstream effectors and ultimately to cell proliferation. Ras mutants which increase the steady-state concentration of Ras.GTP are involved in about 30% of all human cancers. It is therefore of great interest to develop a biosensor which is sensitive to Ras.GTP but not to Ras.GDP. The Ras binding domain (RBD) of c-Raf1 was synthesized from two unprotected peptide segments by native chemical ligation. Two fluorescent amino acids with structures based on the nitrobenz-2-oxa-1,3-diazole and coumaryl chromophores were incorporated at a site which is close to the RBD/Ras.GTP binding surface. Additionally, a C-terminal tag consisting of His6 was introduced. The Kd values for binding of the site-specifically modified proteins to Ras.GTP are comparable to that of wild-type RBD. Immobilization of C-terminal His6 tag-modified fluorescent RBD onto Ni-NTA-coated surfaces allowed the detection of Ras.GTP in the 100 nM range. Likewise, Ras.GTP/Q61L (an oncogenic mutant of Ras with very low intrinsic GTP hydrolysis activity) can also be detected in this assay system. Ras.GDP does not bind to the immobilized RBD, thus allowing discrimination between inactive and activated Ras. The site-specific incorporation of a fluorescent group at a strategic position in a Ras effector protein allows the detection of activated Ras with high sensitivity. This example illustrates the fact that the chemical synthesis of proteins or protein domains makes it possible to incorporate any kind of natural or unnatural amino acid at the position of choice, thereby enabling the facile preparation of specific biosensors, enhanced detection systems for drug screening, or the synthesis of activated proteins, e.g. phosphorylated proteins involved in signaling pathways, as defined molecular species.

Research paper thumbnail of Increasing Protein Identification Using Coupled Chip Based NanoLC Columns

Journal of biomolecular techniques, 2011

Nanoflow liquid chromatography coupled with nano-electrospray (nanoLC-MS) is the method of choice... more Nanoflow liquid chromatography coupled with nano-electrospray (nanoLC-MS) is the method of choice for sensitive peptide and protein analysis for proteomics research. We recently reported on a new microfluidic platform (cHiPLC-nanoflex) for nanoLC-MS applications. In addition to delivering easy-to-use, dead-volume-free connections to microfluidic devices, the platform's cHiPLC columns provide excellent column-to-column separation reproducibility. We have reported previously(1) that while increasing only gradient time or column length can improve peak capacity, using a longer column in combination with increasing gradient time is the most effective way of obtaining higher peak capacity. In this presentation we report on how the dead volume free connections of the cHiPLC Nanoflex platform allow for the coupling of two 15 cm long chip columns for improved resolution. The proposed set-up makes it easy to switch between single and dual column mode depending on application requirements...

Research paper thumbnail of Cross-Laboratory Standardization of Preclinical Lipidomics Using Differential Mobility Spectrometry and Multiple Reaction Monitoring

Modern biomarker and translational research as well as personalized health care studies rely heav... more Modern biomarker and translational research as well as personalized health care studies rely heavily on powerful omics' technologies, including metabolomics and lipidomics. However, to translate metabolomics and lipidomics discoveries into a high-throughput clinical setting, standardization is of utmost importance. Here, we compared and benchmarked a quantitative lipidomics platform. The employed Lipidyzer platform is based on lipid class separation by means of differential mobility spectrometry with subsequent multiple reaction monitoring. Quantitation is achieved by the use of 54 deuterated internal standards and an automated informatics approach. We investigated the platform performance across nine laboratories using NIST SRM 1950−Metabolites in Frozen Human Plasma, and three NIST Candidate Reference Materials 8231−Frozen Human Plasma Suite for Metabolomics (high triglyceride, diabetic, and African-American plasma). In addition, we comparatively analyzed 59 plasma samples from individuals with familial hypercholesterolemia from a clinical cohort study. We provide evidence that the more practical methyl-tert-butyl ether extraction outperforms the classic Bligh and Dyer approach and compare our results with two previously published ring trials. In summary, we present standardized lipidomics protocols, allowing for the highly reproducible analysis of several hundred human plasma lipids, and present detailed molecular information for potentially disease relevant and ethnicity-related materials.

Research paper thumbnail of 200 μm ID Chip Based Columns for Increased Throughput Peptide Quantitation with NanoLC/MS

Journal of biomolecular techniques, 2012

Nanoflow liquid chromatography coupled with mass spectrometry (nanoLC/MS) is the method of choice... more Nanoflow liquid chromatography coupled with mass spectrometry (nanoLC/MS) is the method of choice for sensitive peptide quantitation. We recently reported on a new microfluidic platform (cHiPLC-nanoflex) for nanoLC-MS applications. In addition to delivering easy-to-use, dead-volume-free connections to microfluidic devices, the platform's cHiPLC columns provide excellent column-to-column separation reproducibility. While providing excellent sensitivity, the low flow rates when using 75 μm ID columns reduces sample throughput due to gradient delay in the nanoLC system itself, delay in the autosampler and sample loop, and the delay caused by the connecting tubing. One way to address this is to use a larger inner diameter column at a proportionally higher flow rate. While this will reduce sensitivity when the total sample amount is kept equal, the delay times, however, can be greatly reduced and sample throughput accelerated. Reducing the column and gradient lengths can further redu...

Research paper thumbnail of Statistical control of peptide and protein error rates in large-scale targeted data-independent acquisition analyses

Nature Methods, 2017

Liquid chromatography coupled to tandem mass spectrometry is the main method for highthroughput i... more Liquid chromatography coupled to tandem mass spectrometry is the main method for highthroughput identification and quantification of peptides and inferred proteins. Within this field, data-independent acquisition (DIA) combined with peptide-centric scoring, exemplified by SWATH-MS, emerged as a scalable method to achieve deep and consistent proteome coverage across large-scale datasets. Here we discuss the adaptation of statistical concepts developed for discovery proteomics based on spectrum-centric scoring to large-scale DIA experiments analyzed with peptide-centric scoring strategies and provide guidance on their application. We show that optimal tradeoffs between sensitivity and specificity require careful considerations of the Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Research paper thumbnail of On-line Chip based 2D RP/RP LC-MS/MS Method for Proteomic Analysis

Journal of Biomolecular Techniques Jbt, May 1, 2013

Two-dimensional (2D) liquid chromatography is widely used for proteome identification and quantif... more Two-dimensional (2D) liquid chromatography is widely used for proteome identification and quantification using the advantage of increasing peak capacity. Herein, we developed an simplified chip based 2D-LC workflow using a high pH/RP first dimensional separation and low pH/RP secondary dimension coupled to mass spectrometer for proteomic analysis.The 2D LC separation was performed using ekspert™ nanoLC 425(Eksigent, part of AB SCIEX) system. Digested E. coli cell lysates were first loaded onto a 200μm × 15cm C18 column at pH 9.8 with a flowrate of 1 μL/min. Step gradient was used to sequentially elute peptide fractions, which were diluted to pH 2.5 before being captured by a 200μm × 6mm C18 chip trap column. Each fraction was then separated with a 75 μm × 15cm C18 chip column at 300 nL/min and analyzed with TripleTOF® 5600 (AB SCIEX). Data was processed with ProteinPilot™ Software (AB SCIEX).The preliminary result suggested the on-line 2D RP-RP method as an easy and competitive approach for proteome discovery. The comparison experiments of 1D, 2D-6 fraction and 2D-10 fraction were performed. Using ∼1ug of E. coli digested cell lysates, there are 1.8× and 2.1× increase in the identification numbers for 2D-6 and 2D-10 fractions versus the 1D configuration at the peptide level (5% local FDR). The other advantage of 2D workflow is the larger sample loading capacities on the column. When the loading amount was increased by 10×, the number of detected peptides increased by 3.3× and 4× for the 2D 6 and 10 fraction workflows, respectively, over 1D workflow. The measured retention time of peptides detected in both the 1D 1ug and 2D 10ug experiments showed very good correlation (r2 0.99, slope 1.0). Further optimization of both the first and second dimension is ongoing to further improve the peptide detection rates.

Research paper thumbnail of Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications

Molecular & Cellular Proteomics, 2015

Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology,... more Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. High throughput characterization of histone PTMs is mostly performed by using nano-liquid chromatography coupled to mass spectrometry. However, limitations in speed and stochastic sampling of data dependent acquisition methods in MS lead to incomplete discrimination of isobaric peptides and loss of low abundant species. In this work, we analyzed histone PTMs with a data-independent acquisition method, namely SWATH™ analysis. This approach allows for MS/MS-based quantification of all analytes without upfront assay development and no issues of biased and incomplete sampling. We purified histone proteins from human embryonic stem cells and mouse trophoblast stem cells before and after differentiation, and prepared them for MS analysis using the propionic anhydride protocol. Results on histone H3 peptides verified that sequential window acquisition of all theoretical mass spectra could accurately quantify peptides (<9% average coefficient of variation, CV) over four orders of magnitude, and we could discriminate isobaric and co-eluting peptides (e.g. H3K18ac and H3K23ac) using MS/MS-based quantification. This method provided high sensitivity and precision, supported by the fact that we could find significant differences for remarkably low abundance PTMs such as H3K9me2S10ph (relative abundance <0.02%). We performed relative quantification for few sample peptides using different fragment ions and observed high consistency (CV <15%) between the fragments. This indicated that different fragment ions can be used independently to From the ‡Epigenetics Program,

Research paper thumbnail of Use of proteomics in development of biomarkers and early markers of toxicity

Research paper thumbnail of Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications

PROTEOMICS, 2003

Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for ... more Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications The unique scanning capabilities of a hybrid linear ion trap (Q TRAP) mass spectrometer are described with an emphasis on proteomics applications. The combination of the very selective triple quadrupole based tandem mass spectrometry (MS/MS) scans with the very sensitive ion trap product ion scans allows rapid identification of peptides at low concentrations derived from post-translationally modified proteins on chromatographic time scales. The Q TRAP instrument also offers the opportunity to conduct a variety of ion processing steps prior to performing a mass scan. For example, the enhancement of the multiple-charge ion contents of the ion trap can be performed resulting in a survey mass spectrum dominated by double-and triple-charge peptides. This facilitates the identification of relevant biological species in both separated and unseparated peptide mixtures for further MS/MS experiments.

Research paper thumbnail of Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1

Proceedings of the National Academy of Sciences, 2003

Generation of biological function by chemical methods is potentially of great importance for the ... more Generation of biological function by chemical methods is potentially of great importance for the understanding and targeting of physiological processes. Chemical synthesis of proteins offers the ability to alter the properties of target protein molecules in a tailor-made fashion. In the present work it is demonstrated that this methodology can be expanded to the elucidation of protein–protein interactions as exemplified by the complete chemical synthesis of the protooncogene product H-Ras as well as of the Ras-binding domain (RBD) of its effector c-Raf1. The 166-aa polypeptide chain of H-Ras was synthesized by native chemical ligation of three unprotected peptide segments. Similarly, the 81-aa RBD was prepared by ligation of two peptide segments. Both RBD and Ras displayed functional and spectroscopic properties indistinguishable from their recombinant forms as judged by CD spectroscopy and from transient kinetic measurements of the Ras–RBD interaction as well as from nucleotide rep...

Research paper thumbnail of Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

Proceedings of the National Academy of Sciences, 2003

A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near ... more A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near the heme 6-propionate group by replacing K45 and K63 with glutamyl residues. One-dimensional 1 H NMR spectroscopy indicates that Mn 2+ binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn 2+ is 1.28(4) × 10 4 M −1 (pH 6.96, ionic strength I = 17.2 μM, 25°C). In addition, these substitutions lower the reduction potential of the protein and increase the pK a for the water molecule coordinated to the heme iron of metmyoglobin. The peroxidase [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS, as substrate] and the Mn 2+ -peroxidase activity of the variant are both increased ≈3-fold. In contrast to wild-type Mb, both the affinity for azide and the midpoint potential of the variant are significantly influenced by the addition of Mn 2+ . The structure of the variant has been determined by x-ray ...

Research paper thumbnail of Engineered metalloregulation of azide binding affinity and reduction potential of horse heart myoglobin

Dalton Transactions, 2013

Metal ion binding to a previously reported variant of horse heart myoglobin (Lys45Glu/Lys63Glu) w... more Metal ion binding to a previously reported variant of horse heart myoglobin (Lys45Glu/Lys63Glu) with a metal ion binding site on the surface of the protein that is adjacent to the haem binding site has been shown to influence ligand binding and electrochemical properties of the protein. For example, the K(d) (μM) for binding of azide to this variant decreases from 277 ± 9 to 32 ± 3 following addition of a saturating concentration of Mn(2+) (the value for the wild-type protein under the same conditions is 26 ± 1). Similarly, the midpoint reduction potential E(m) (mV vs. standard hydrogen electrode) increases from 9 to 40 in the presence of a saturating concentration of Mn(2+) (the value for the wild-type protein under the same conditions is 45 ± 2). These results demonstrate the potential value of engineered metal ion binding sites as a means of regulating the functional properties of even simple haem proteins.

Research paper thumbnail of A sensitive fluorescence monitor for the detection of activated Ras: total chemical synthesis of site-specifically labeled Ras binding domain of c-Raf1 immobilized on a surface

Chemistry & Biology, 2001

Background: The RasWGDP^RasWGTP cycle plays a central role in eukaryotic signaling cascades. Muta... more Background: The RasWGDP^RasWGTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras which stabilize activated RasWGTP lead to a continuous stimulation of downstream effectors and ultimately to cell proliferation. Ras mutants which increase the steady-state concentration of RasWGTP are involved in about 30% of all human cancers. It is therefore of great interest to develop a biosensor which is sensitive to RasWGTP but not to RasWGDP. Results : The Ras binding domain (RBD) of c-Raf1 was synthesized from two unprotected peptide segments by native chemical ligation. Two fluorescent amino acids with structures based on the nitrobenz-2-oxa-1,3-diazole and coumaryl chromophores were incorporated at a site which is close to the RBD/ RasWGTP binding surface. Additionally, a C-terminal tag consisting of His 6 was introduced. The K d values for binding of the sitespecifically modified proteins to RasWGTP are comparable to that of wild-type RBD. Immobilization of C-terminal His 6 tagmodified fluorescent RBD onto Ni-NTA-coated surfaces allowed the detection of RasWGTP in the 100 nM range. Likewise, RasWGTP/Q61L (an oncogenic mutant of Ras with very low intrinsic GTP hydrolysis activity) can also be detected in this assay system. RasWGDP does not bind to the immobilized RBD, thus allowing discrimination between inactive and activated Ras. Conclusions: The site-specific incorporation of a fluorescent group at a strategic position in a Ras effector protein allows the detection of activated Ras with high sensitivity. This example illustrates the fact that the chemical synthesis of proteins or protein domains makes it possible to incorporate any kind of natural or unnatural amino acid at the position of choice, thereby enabling the facile preparation of specific biosensors, enhanced detection systems for drug screening, or the synthesis of activated proteins, e.g. phosphorylated proteins involved in signaling pathways, as defined molecular species.

Research paper thumbnail of Native Chemical Ligation of Hydrophobic Peptides in Lipid Bilayer Systems

Bioconjugate Chemistry, 2004

Research paper thumbnail of Accelerated Protein Biomarker Discovery from FFPE tissue samples using Single-shot, Short Gradient Microflow SWATH MS

ABSTRACTWe report and evaluated a microflow, single-shot, short gradient SWATH MS method intended... more ABSTRACTWe report and evaluated a microflow, single-shot, short gradient SWATH MS method intended to accelerate the discovery and verification of protein biomarkers in clinical specimens. The method uses 15-min gradient microflow-LC peptide separation, an optimized SWATH MS window configuration and OpenSWATH software for data analysis.We applied the method to a cohort 204 of FFPE prostate tissue samples from 58 prostate cancer patients and 10 prostatic hyperplasia patients. Altogether we identified 27,976 proteotypic peptides and 4,043 SwissProt proteins from these 204 samples. Compared to a reference SWATH method with 2-hour gradient the accelerated method consumed only 27% instrument time, quantified 80% proteins and showed reduced batch effects. 3,800 proteins were quantified by both methods in two different instruments with relatively high consistency (r = 0.77). 75 proteins detected by the accelerated method with differential abundance between clinical groups were selected for ...

Research paper thumbnail of A sensitive fluorescence monitor for the detection of activated Ras: total chemical synthesis of site-specifically labeled Ras binding domain of c-Raf1 immobilized on a surface

Chemistry & Biology, 2001

The Ras.GDP-Ras.GTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras... more The Ras.GDP-Ras.GTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras which stabilize activated Ras.GTP lead to a continuous stimulation of downstream effectors and ultimately to cell proliferation. Ras mutants which increase the steady-state concentration of Ras.GTP are involved in about 30% of all human cancers. It is therefore of great interest to develop a biosensor which is sensitive to Ras.GTP but not to Ras.GDP. The Ras binding domain (RBD) of c-Raf1 was synthesized from two unprotected peptide segments by native chemical ligation. Two fluorescent amino acids with structures based on the nitrobenz-2-oxa-1,3-diazole and coumaryl chromophores were incorporated at a site which is close to the RBD/Ras.GTP binding surface. Additionally, a C-terminal tag consisting of His6 was introduced. The Kd values for binding of the site-specifically modified proteins to Ras.GTP are comparable to that of wild-type RBD. Immobilization of C-terminal His6 tag-modified fluorescent RBD onto Ni-NTA-coated surfaces allowed the detection of Ras.GTP in the 100 nM range. Likewise, Ras.GTP/Q61L (an oncogenic mutant of Ras with very low intrinsic GTP hydrolysis activity) can also be detected in this assay system. Ras.GDP does not bind to the immobilized RBD, thus allowing discrimination between inactive and activated Ras. The site-specific incorporation of a fluorescent group at a strategic position in a Ras effector protein allows the detection of activated Ras with high sensitivity. This example illustrates the fact that the chemical synthesis of proteins or protein domains makes it possible to incorporate any kind of natural or unnatural amino acid at the position of choice, thereby enabling the facile preparation of specific biosensors, enhanced detection systems for drug screening, or the synthesis of activated proteins, e.g. phosphorylated proteins involved in signaling pathways, as defined molecular species.