Judith Steen | Harvard Medical School (original) (raw)
Papers by Judith Steen
Tryptic peptides are the analytes of choice for mass spectrometric analysis of protein and peptid... more Tryptic peptides are the analytes of choice for mass spectrometric analysis of protein and peptide as they display a favorable fragmentation pattern due to the presence of a C-terminal basic amino acid residue. Clean y fragment ion series is most commonly observed for these species. In contrast, non-tryptic peptides with undefined locations of basic amino acid residues give rise to a mixture of b and y fragment ions, often preventing unambiguous assignment of fragment ion types, which in turn impedes the interpretation of the product ion spectra. Here we report that the fragmentation pattern of multiply charged non-tryptic peptides can be modulated by fragmenting the monosodiated multiply charged species instead of the multiply protonated species. Even when b fragment ions dominate the product ion spectrum of the protonated species due to the presence of a charge sequestering basic residue at the N-terminus, mainly singly charged sodium cationized y fragment ions [yn + Na]+ are obse...
Molecular Biology of the Cell, 2012
Methods in molecular biology (Clifton, N.J.), 2012
A protein molecule exists as a heterogeneous population of posttranslationally modified forms, wh... more A protein molecule exists as a heterogeneous population of posttranslationally modified forms, which are of potential interest to biologists. However, due to detection or methodology limitations, they remain uncharacterized. When a protein does become a prioritized interest in a laboratory, workflows aimed for its purification and characterization are implemented. Inherent in these workflows is the enrichment of the protein from the biological lysate, rendering it an ideal sample for mass spectrometry (MS), as detection of several peptides is greatly increased. In order to capitalize on this enhanced detection of the protein of interest, we have developed a full-length expressed protein quantification standard (FLEXIQuant standard) that is in vitro synthesized, devoid of posttranslational modifications (PTMs), and implemented into the purification workflow of the endogenous counterpart-as such it serves as an internal MS standard. FLEXIQuantification allows for the unbiased identifi...
Methods in molecular biology (Clifton, N.J.), 2012
Many cancers have been associated with the deregulation of kinases, and thus, kinases have become... more Many cancers have been associated with the deregulation of kinases, and thus, kinases have become a prime target for the development of cancer treatments. This focus on kinases has resulted in the approval of several small-molecule kinase inhibitors for cancer treatments. Further, the use of these inhibitors as tools to study cancer has provided valuable information about biological mechanisms. However, to date, not much is known about the global effects of kinases on the proteome or phosphoproteome. In this protocol, we describe methodology to study the impact of kinase inhibitors on the proteome and phosphoproteome using mass spectrometry-based quantitative proteomics. More specifically, we focus on the effects of Aurora B kinase inhibitors on the proteome, cytoskeleton proteome, the phosphoproteome, and the cytoskeleton phosphoproteome during cell cycle. This methodology is easily extended to other biological studies whose aim is to study the global proteomic effects of a kinase ...
Molecular & Cellular Proteomics, 2013
Molecular & Cellular Proteomics, 2012
Molecular & Cellular Proteomics, 2013
This paper proposes a novel, automated method for evaluating sets of proteins identified using ma... more This paper proposes a novel, automated method for evaluating sets of proteins identified using mass spectrometry. The remaining peptide-spectrum match score distributions of protein sets are compared to an empirical absent peptide-spectrum match score distribution, and a Bayesian non-parametric method reminiscent of the Dirichlet process is presented to accurately perform this comparison. Thus, for a given protein set, the process computes the likelihood that the proteins identified are correctly identified. First, the method is used to evaluate protein sets chosen using different protein-level false discovery rate (FDR) thresholds, assigning each protein set a likelihood. The protein set assigned the highest likelihood is used to choose a non-arbitrary protein-level FDR threshold. Because the method can be used to evaluate any protein identification strategy (and is not limited to mere comparisons of different FDR thresholds), we subsequently use the method to compare and evaluate multiple simple methods for merging peptide evidence over replicate experiments. The general statistical approach can be applied to other types of data (e.g. RNA sequencing) and generalizes to multivariate problems.
Journal of Proteome Research, 2013
Arbitrary cutoffs are ubiquitous in quantitative computational proteomics: maximum acceptable MS/... more Arbitrary cutoffs are ubiquitous in quantitative computational proteomics: maximum acceptable MS/MS PSM or peptide q value, minimum ion intensity to calculate a fold change, the minimum number of peptides that must be available to trust the estimated protein fold change (or the minimum number of PSMs that must be available to trust the estimated peptide fold change), and the "significant" fold change cutoff. Here we introduce a novel experimental setup and nonparametric Bayesian algorithm for determining the statistical quality of a proposed differential set of proteins or peptides. By comparing putatively nonchanging case-control evidence to an empirical null distribution derived from a control-control experiment, we successfully avoid some of these common parameters. We then apply our method to evaluating different fold-change rules and find that for our data a 1.2-fold change is the most permissive of the plausible fold-change rules.
Journal of Biological Chemistry, 2013
Although Tau accumulation is a feature of several neurodegenerative conditions, treatment options... more Although Tau accumulation is a feature of several neurodegenerative conditions, treatment options for these conditions are nonexistent. Targeting Tau kinases represents a potential therapeutic approach. Small molecules in the diaminothiazole class are potent Tau kinase inhibitors that target CDK5 and GSK3β. Lead compounds from the series have IC50 values toward CDK5/p25 and GSK3β in the low nanomolar range and no observed toxicity in the therapeutic dose range. Neuronal protective effects and decreased PHF-1 immunoreactivity were observed in two animal models, 3×Tg-AD and CK-p25. Treatment nearly eliminated Sarkosyl-insoluble Tau with the most prominent effect on the phosphorylation at Ser-404. Treatment also induced the recovery of memory in a fear conditioning assay. Given the contribution of both CDK5/p25 and GSK3β to Tau phosphorylation, effective treatment of tauopathies may require dual kinase targeting.
Proceedings of the National Academy of Sciences of the United States of America, Jan 21, 2011
Spinal muscular atrophy (SMA), caused by the deletion of the SMN1 gene, is the leading genetic ca... more Spinal muscular atrophy (SMA), caused by the deletion of the SMN1 gene, is the leading genetic cause of infant mortality. SMN protein is present at high levels in both axons and growth cones, and loss of its function disrupts axonal extension and pathfinding. SMN is known to associate with the RNA-binding protein hnRNP-R, and together they are responsible for the transport and/or local translation of β-actin mRNA in the growth cones of motor neurons. However, the full complement of SMN-interacting proteins in neurons remains unknown. Here we used mass spectrometry to identify HuD as a novel neuronal SMN-interacting partner. HuD is a neuron-specific RNA-binding protein that interacts with mRNAs, including candidate plasticity-related gene 15 (cpg15). We show that SMN and HuD form a complex in spinal motor axons, and that both interact with cpg15 mRNA in neurons. CPG15 is highly expressed in the developing ventral spinal cord and can promote motor axon branching and neuromuscular syna...
PloS one, 2011
We measured the expression of 187 miRNAs using quantitative real time PCR in the hippocampal CA1 ... more We measured the expression of 187 miRNAs using quantitative real time PCR in the hippocampal CA1 region of contextually conditioned mice and cultured embryonic rat hippocampal neurons after neuronal stimulation with either NMDA or bicuculline. Many of the changes in miRNA expression after these three types of stimulation were similar. Surprisingly, the expression level of half of the 187 measured miRNAs was changed in response to contextual conditioning in an NMDA receptor-dependent manner. Genes that control miRNA biogenesis and components of the RISC also exhibited activity induced expression changes and are likely to contribute to the widespread changes in the miRNA profile. The widespread changes in miRNA expression are consistent with the finding that genes up-regulated by contextual conditioning have longer 3' UTRs and more predicted binding sites for miRNAs. Among the miRNAs that changed their expression after contextual conditioning, several inhibit inhibitors of the mT...
Tauopathies, including Alzheimer’s disease (AD), are associated with the aggregation of modified ... more Tauopathies, including Alzheimer’s disease (AD), are associated with the aggregation of modified microtubule associated protein tau. This pathological state of tau is often referred to as “hyperphosphorylated”. Due to limitations in technology, an accurate quantitative description of this state is lacking. Here, a mass spectrometry-based assay, FLEXITau, is presented to measure phosphorylation stoichiometry and provide an unbiased quantitative view of the tau post-translational modification (PTM) landscape. The power of this assay is demonstrated by measuring the state of hyperphosphorylation from tau in a cellular model for AD pathology, mapping, and calculating site occupancies for over 20 phosphorylations. We further employ FLEXITau to define the tau PTM landscape present in AD post-mortem brain. As shown in this study, the application of this assay provides mechanistic understanding of tau pathology that could lead to novel therapeutics, and we envision its further use in prognostic and diagnostic approaches for tauopathies.
Proteomics is the study of the abundance, function and dynamics of all proteins present in a livi... more Proteomics is the study of the abundance, function and dynamics of all proteins present in a living organism, and mass spectrometry (MS) has become its most important tool due to its unmatched sensitivity, resolution and potential for high-throughput experimentation. A frequently used variant of mass spectrometry is coupled with liquid chromatography (LC) and is denoted as "LC/MS". It produces two-dimensional
Functional and Structural Proteomics of Glycoproteins, 2010
... Glucose GlcNAc N-acetylglucosamine JJ Steen (B) FM Kirby Center for Neurobiology, Proteomics ... more ... Glucose GlcNAc N-acetylglucosamine JJ Steen (B) FM Kirby Center for Neurobiology, Proteomics Center at Children's Hospital Boston, Boston, MA 02115, USA e-mail:judith.steen@childrens.harvard.edu 103 RJ Owens, JE ...
Cell reports, Jan 25, 2014
Poly(ADP-ribose) polymerases (PARPs) catalyze poly(ADP-ribose) addition onto proteins, an importa... more Poly(ADP-ribose) polymerases (PARPs) catalyze poly(ADP-ribose) addition onto proteins, an important posttranslational modification involved in transcription, DNA damage repair, and stem cell identity. Previous studies established the activation of PARP1 in response to DNA damage, but little is known about PARP1 regulation outside of DNA repair. We developed an assay for measuring PARP activity in cell lysates and found that the basal activity of PARP1 was highly variable across breast cancer cell lines, independent of DNA damage. Sucrose gradient fractionation demonstrated that PARP1 existed in at least three biochemically distinct states in both high- and low-activity lines. A discovered complex containing the NuA4 chromatin-remodeling complex and PARP1 was responsible for high basal PARP1 activity, and NuA4 subunits were required for this activity. These findings present a pathway for PARP1 activation and a direct link between PARP1 and chromatin remodeling outside of the DNA dama...
Proceedings of the National Academy of Sciences of the United States of America, Jan 22, 2008
The anaphase promoting complex (APC) controls the degradation of proteins during exit from mitosi... more The anaphase promoting complex (APC) controls the degradation of proteins during exit from mitosis and entry into S-phase. The activity of the APC is regulated by phosphorylation during mitosis. Because the phosphorylation pattern provides insights into the complexity of regulation of the APC, we studied in detail the phosphorylation patterns at a single mitotic state of arrest generated by various antimitotic drugs. We examined the phosphorylation patterns of the APC in HeLa S3 cells after they were arrested in prometaphase with taxol, nocodazole, vincristine, or monastrol. There were 71 phosphorylation sites on nine of the APC subunits. Despite the common state of arrest, the various antimitotic drug treatments resulted in differences in the phosphorylation patterns and phosphorylation stoichiometries. The relative phosphorylation stoichiometries were determined by using a method adapted from the isotope-free quantitation of the extent of modification (iQEM). We could show that du...
The EMBO journal, Jan 13, 2015
The cell surface is the cellular compartment responsible for communication with the environment. ... more The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis...
Wiley Interdisciplinary Reviews: Systems Biology and Medicine, 2009
Tryptic peptides are the analytes of choice for mass spectrometric analysis of protein and peptid... more Tryptic peptides are the analytes of choice for mass spectrometric analysis of protein and peptide as they display a favorable fragmentation pattern due to the presence of a C-terminal basic amino acid residue. Clean y fragment ion series is most commonly observed for these species. In contrast, non-tryptic peptides with undefined locations of basic amino acid residues give rise to a mixture of b and y fragment ions, often preventing unambiguous assignment of fragment ion types, which in turn impedes the interpretation of the product ion spectra. Here we report that the fragmentation pattern of multiply charged non-tryptic peptides can be modulated by fragmenting the monosodiated multiply charged species instead of the multiply protonated species. Even when b fragment ions dominate the product ion spectrum of the protonated species due to the presence of a charge sequestering basic residue at the N-terminus, mainly singly charged sodium cationized y fragment ions [yn + Na]+ are obse...
Molecular Biology of the Cell, 2012
Methods in molecular biology (Clifton, N.J.), 2012
A protein molecule exists as a heterogeneous population of posttranslationally modified forms, wh... more A protein molecule exists as a heterogeneous population of posttranslationally modified forms, which are of potential interest to biologists. However, due to detection or methodology limitations, they remain uncharacterized. When a protein does become a prioritized interest in a laboratory, workflows aimed for its purification and characterization are implemented. Inherent in these workflows is the enrichment of the protein from the biological lysate, rendering it an ideal sample for mass spectrometry (MS), as detection of several peptides is greatly increased. In order to capitalize on this enhanced detection of the protein of interest, we have developed a full-length expressed protein quantification standard (FLEXIQuant standard) that is in vitro synthesized, devoid of posttranslational modifications (PTMs), and implemented into the purification workflow of the endogenous counterpart-as such it serves as an internal MS standard. FLEXIQuantification allows for the unbiased identifi...
Methods in molecular biology (Clifton, N.J.), 2012
Many cancers have been associated with the deregulation of kinases, and thus, kinases have become... more Many cancers have been associated with the deregulation of kinases, and thus, kinases have become a prime target for the development of cancer treatments. This focus on kinases has resulted in the approval of several small-molecule kinase inhibitors for cancer treatments. Further, the use of these inhibitors as tools to study cancer has provided valuable information about biological mechanisms. However, to date, not much is known about the global effects of kinases on the proteome or phosphoproteome. In this protocol, we describe methodology to study the impact of kinase inhibitors on the proteome and phosphoproteome using mass spectrometry-based quantitative proteomics. More specifically, we focus on the effects of Aurora B kinase inhibitors on the proteome, cytoskeleton proteome, the phosphoproteome, and the cytoskeleton phosphoproteome during cell cycle. This methodology is easily extended to other biological studies whose aim is to study the global proteomic effects of a kinase ...
Molecular & Cellular Proteomics, 2013
Molecular & Cellular Proteomics, 2012
Molecular & Cellular Proteomics, 2013
This paper proposes a novel, automated method for evaluating sets of proteins identified using ma... more This paper proposes a novel, automated method for evaluating sets of proteins identified using mass spectrometry. The remaining peptide-spectrum match score distributions of protein sets are compared to an empirical absent peptide-spectrum match score distribution, and a Bayesian non-parametric method reminiscent of the Dirichlet process is presented to accurately perform this comparison. Thus, for a given protein set, the process computes the likelihood that the proteins identified are correctly identified. First, the method is used to evaluate protein sets chosen using different protein-level false discovery rate (FDR) thresholds, assigning each protein set a likelihood. The protein set assigned the highest likelihood is used to choose a non-arbitrary protein-level FDR threshold. Because the method can be used to evaluate any protein identification strategy (and is not limited to mere comparisons of different FDR thresholds), we subsequently use the method to compare and evaluate multiple simple methods for merging peptide evidence over replicate experiments. The general statistical approach can be applied to other types of data (e.g. RNA sequencing) and generalizes to multivariate problems.
Journal of Proteome Research, 2013
Arbitrary cutoffs are ubiquitous in quantitative computational proteomics: maximum acceptable MS/... more Arbitrary cutoffs are ubiquitous in quantitative computational proteomics: maximum acceptable MS/MS PSM or peptide q value, minimum ion intensity to calculate a fold change, the minimum number of peptides that must be available to trust the estimated protein fold change (or the minimum number of PSMs that must be available to trust the estimated peptide fold change), and the "significant" fold change cutoff. Here we introduce a novel experimental setup and nonparametric Bayesian algorithm for determining the statistical quality of a proposed differential set of proteins or peptides. By comparing putatively nonchanging case-control evidence to an empirical null distribution derived from a control-control experiment, we successfully avoid some of these common parameters. We then apply our method to evaluating different fold-change rules and find that for our data a 1.2-fold change is the most permissive of the plausible fold-change rules.
Journal of Biological Chemistry, 2013
Although Tau accumulation is a feature of several neurodegenerative conditions, treatment options... more Although Tau accumulation is a feature of several neurodegenerative conditions, treatment options for these conditions are nonexistent. Targeting Tau kinases represents a potential therapeutic approach. Small molecules in the diaminothiazole class are potent Tau kinase inhibitors that target CDK5 and GSK3β. Lead compounds from the series have IC50 values toward CDK5/p25 and GSK3β in the low nanomolar range and no observed toxicity in the therapeutic dose range. Neuronal protective effects and decreased PHF-1 immunoreactivity were observed in two animal models, 3×Tg-AD and CK-p25. Treatment nearly eliminated Sarkosyl-insoluble Tau with the most prominent effect on the phosphorylation at Ser-404. Treatment also induced the recovery of memory in a fear conditioning assay. Given the contribution of both CDK5/p25 and GSK3β to Tau phosphorylation, effective treatment of tauopathies may require dual kinase targeting.
Proceedings of the National Academy of Sciences of the United States of America, Jan 21, 2011
Spinal muscular atrophy (SMA), caused by the deletion of the SMN1 gene, is the leading genetic ca... more Spinal muscular atrophy (SMA), caused by the deletion of the SMN1 gene, is the leading genetic cause of infant mortality. SMN protein is present at high levels in both axons and growth cones, and loss of its function disrupts axonal extension and pathfinding. SMN is known to associate with the RNA-binding protein hnRNP-R, and together they are responsible for the transport and/or local translation of β-actin mRNA in the growth cones of motor neurons. However, the full complement of SMN-interacting proteins in neurons remains unknown. Here we used mass spectrometry to identify HuD as a novel neuronal SMN-interacting partner. HuD is a neuron-specific RNA-binding protein that interacts with mRNAs, including candidate plasticity-related gene 15 (cpg15). We show that SMN and HuD form a complex in spinal motor axons, and that both interact with cpg15 mRNA in neurons. CPG15 is highly expressed in the developing ventral spinal cord and can promote motor axon branching and neuromuscular syna...
PloS one, 2011
We measured the expression of 187 miRNAs using quantitative real time PCR in the hippocampal CA1 ... more We measured the expression of 187 miRNAs using quantitative real time PCR in the hippocampal CA1 region of contextually conditioned mice and cultured embryonic rat hippocampal neurons after neuronal stimulation with either NMDA or bicuculline. Many of the changes in miRNA expression after these three types of stimulation were similar. Surprisingly, the expression level of half of the 187 measured miRNAs was changed in response to contextual conditioning in an NMDA receptor-dependent manner. Genes that control miRNA biogenesis and components of the RISC also exhibited activity induced expression changes and are likely to contribute to the widespread changes in the miRNA profile. The widespread changes in miRNA expression are consistent with the finding that genes up-regulated by contextual conditioning have longer 3' UTRs and more predicted binding sites for miRNAs. Among the miRNAs that changed their expression after contextual conditioning, several inhibit inhibitors of the mT...
Tauopathies, including Alzheimer’s disease (AD), are associated with the aggregation of modified ... more Tauopathies, including Alzheimer’s disease (AD), are associated with the aggregation of modified microtubule associated protein tau. This pathological state of tau is often referred to as “hyperphosphorylated”. Due to limitations in technology, an accurate quantitative description of this state is lacking. Here, a mass spectrometry-based assay, FLEXITau, is presented to measure phosphorylation stoichiometry and provide an unbiased quantitative view of the tau post-translational modification (PTM) landscape. The power of this assay is demonstrated by measuring the state of hyperphosphorylation from tau in a cellular model for AD pathology, mapping, and calculating site occupancies for over 20 phosphorylations. We further employ FLEXITau to define the tau PTM landscape present in AD post-mortem brain. As shown in this study, the application of this assay provides mechanistic understanding of tau pathology that could lead to novel therapeutics, and we envision its further use in prognostic and diagnostic approaches for tauopathies.
Proteomics is the study of the abundance, function and dynamics of all proteins present in a livi... more Proteomics is the study of the abundance, function and dynamics of all proteins present in a living organism, and mass spectrometry (MS) has become its most important tool due to its unmatched sensitivity, resolution and potential for high-throughput experimentation. A frequently used variant of mass spectrometry is coupled with liquid chromatography (LC) and is denoted as "LC/MS". It produces two-dimensional
Functional and Structural Proteomics of Glycoproteins, 2010
... Glucose GlcNAc N-acetylglucosamine JJ Steen (B) FM Kirby Center for Neurobiology, Proteomics ... more ... Glucose GlcNAc N-acetylglucosamine JJ Steen (B) FM Kirby Center for Neurobiology, Proteomics Center at Children's Hospital Boston, Boston, MA 02115, USA e-mail:judith.steen@childrens.harvard.edu 103 RJ Owens, JE ...
Cell reports, Jan 25, 2014
Poly(ADP-ribose) polymerases (PARPs) catalyze poly(ADP-ribose) addition onto proteins, an importa... more Poly(ADP-ribose) polymerases (PARPs) catalyze poly(ADP-ribose) addition onto proteins, an important posttranslational modification involved in transcription, DNA damage repair, and stem cell identity. Previous studies established the activation of PARP1 in response to DNA damage, but little is known about PARP1 regulation outside of DNA repair. We developed an assay for measuring PARP activity in cell lysates and found that the basal activity of PARP1 was highly variable across breast cancer cell lines, independent of DNA damage. Sucrose gradient fractionation demonstrated that PARP1 existed in at least three biochemically distinct states in both high- and low-activity lines. A discovered complex containing the NuA4 chromatin-remodeling complex and PARP1 was responsible for high basal PARP1 activity, and NuA4 subunits were required for this activity. These findings present a pathway for PARP1 activation and a direct link between PARP1 and chromatin remodeling outside of the DNA dama...
Proceedings of the National Academy of Sciences of the United States of America, Jan 22, 2008
The anaphase promoting complex (APC) controls the degradation of proteins during exit from mitosi... more The anaphase promoting complex (APC) controls the degradation of proteins during exit from mitosis and entry into S-phase. The activity of the APC is regulated by phosphorylation during mitosis. Because the phosphorylation pattern provides insights into the complexity of regulation of the APC, we studied in detail the phosphorylation patterns at a single mitotic state of arrest generated by various antimitotic drugs. We examined the phosphorylation patterns of the APC in HeLa S3 cells after they were arrested in prometaphase with taxol, nocodazole, vincristine, or monastrol. There were 71 phosphorylation sites on nine of the APC subunits. Despite the common state of arrest, the various antimitotic drug treatments resulted in differences in the phosphorylation patterns and phosphorylation stoichiometries. The relative phosphorylation stoichiometries were determined by using a method adapted from the isotope-free quantitation of the extent of modification (iQEM). We could show that du...
The EMBO journal, Jan 13, 2015
The cell surface is the cellular compartment responsible for communication with the environment. ... more The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis...
Wiley Interdisciplinary Reviews: Systems Biology and Medicine, 2009