Mass Spectrometry-based Methods for Phosphorylation Site Mapping of Hyperphosphorylated Proteins Applied to Net1, a Regulator of Exit from Mitosis in Yeast

Molecular & Cellular Proteomics : MCP. Mar, 2002  |  Pubmed ID: 12096118

Prior to anaphase in Saccharomyces cerevisiae, Cdc14 protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast. During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis. The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dissociation of a recombinant Net1.Cdc14 complex in vitro by phosphorylation of Net1. We report here the phosphorylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine. A variety of chromatographic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatography, alkaline phosphatase treatment, matrix-assisted laser-desorption post-source decay, and a multidimensional electrospray mass spectrometry-based approach. No one approach was able to identify all phosphopeptides in the tryptic digests of these proteins. Most notably, the presence of a basic residue near the phosphorylated residue significantly hampered the ability of alkaline phosphatase to hydrolyze the phosphate moiety. A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states. The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises significant concerns about how feasible it is to map phosphorylation sites throughout the proteome using existing technologies.

Improved Sensitivity for Phosphopeptide Mapping Using Capillary Column HPLC and Microionspray Mass Spectrometry: Comparative Phosphorylation Site Mapping from Gel-derived Proteins

Analytical Chemistry. Jul, 2002  |  Pubmed ID: 12141686

Reversible protein phosphorylation regulates many cellular processes. Understanding how phosphorylation controls a given pathway usually involves specific knowledge of which amino acid residues are phosphorylated on a given protein. This is often a nontrivial task. In addition to the difficulties involved in purifying sufficient amounts of any given protein, most phosphoproteins contain multiple, substoichiometric sites of phosphorylation. In this paper, we describe substantial improvements made to our previously reported multidimensional electrospray MS-based phosphopeptide mapping technique that have resulted in a 20-fold increase in sensitivity for the overall process. Chief among these improvements are the incorporation of capillary chromatography and a microionspray source for the mass spectrometer into the first dimension of the analysis. In the first dimension of the process, phosphopeptides present in the proteolytic digest of a protein are selectively detected and collected into fractions during on-line LC/ESMS, which monitors for phosphopeptide specific marker ions. The phosphopeptide containing fractions are then analyzed in the second dimension by either MALDI-PSD or nano-ES with precursor ion scanning. The relative merits and limitations of these two techniques for phosphopeptide detection are demonstrated. The enhancement in sensitivity of the method under the new experimental conditions makes it suitable for phosphorylation mapping (from selective detection through sequencing) on gel-separated phosphoproteins where the level of phosphorylation at any given site is <200 fmol. Furthermore, this method detects serine, threonine, and tyrosine phosphorylation equally well. We have successfully employed this new configuration to map 11 in vivo sites of phosphorylation on the Saccharomyces cerevisiae protein kinase YAK1. YAK1 peptides containing all five YAK1 PKA consensus sites are phosphorylated, suggesting that YAK1 is an in vivo substrate for PKA. In addition, four peptides containing cdk sites and the autophosphorylation site at Tyr530 were found to be phosphorylated. Because the first dimension of this method generates a phosphorylation profile that can be used for a semiquantitative evaluation of site specific phosphoxylation, we evaluated its ability to detect site-specific changes in the phosphorylation profile of a protein in response to altered cellular conditions. This comparative phosphopeptide mapping strategy allowed us to detect a change in phosphorylation stoichiometry on the motor protein myosin-V in response to treatment with either mitotic or interphase Xenopus egg extracts and to identify the single functionally significant phosphorylation site that regulates myosin-V cargo binding.

N-Terminal Peptide Labeling Strategy for Incorporation of Isotopic Tags: a Method for the Determination of Site-specific Absolute Phosphorylation Stoichiometry

Rapid Communications in Mass Spectrometry : RCM. 2002  |  Pubmed ID: 12478578

Determining the phosphorylation stoichiometry at specific sites in a phosphoprotein is a very challenging task. We describe here a novel mass spectrometry based method that is capable of measuring the absolute phosphorylation stoichiometry at specific sites without the need for specific internal standards, phospho-site antibodies or radioactivity. The method is based on a gentle chemical labeling strategy which specifically and differentially labels the N-terminus of all peptides in a sample with either a D(5)- or D(0)-propionyl group and measures the ratio of the abundance of the D(5)/D(0) peptide pairs simultaneously using mass spectrometry. Using matrix-assisted laser desorption/ionization (MALDI), the method can measure absolute stoichiometry to within at least 10% and can be applied to both in vitro and in vivo phosphorylated peptides and proteins. Furthermore, this method can potentially be applied to the quantitative study of other types of protein post-translational modifications, and the profiling of protein expression on the proteome level.

TULA: an SH3- and UBA-containing Protein That Binds to C-Cbl and Ubiquitin

Oncogene. Jun, 2004  |  Pubmed ID: 15107835

Downregulation of protein tyrosine kinases is a major function of the multidomain protein c-Cbl. This effect of c-Cbl is critical for both negative regulation of normal physiological stimuli and suppression of cellular transformation. In spite of the apparent importance of these effects of c-Cbl, their own regulation is poorly understood. To search for possible novel regulators of c-Cbl, we purified a number of c-Cbl-associated proteins by affinity chromatography and identified them by mass spectrometry. Among them, we identified the UBA- and SH3-containing protein T-cell Ubiquitin LigAnd (TULA), which can also bind to ubiquitin. Functional studies in a model system based on co-expression of TULA, c-Cbl, and EGF receptor in 293T cells demonstrate that TULA is capable of inhibiting c-Cbl-mediated downregulation of EGF receptor. Furthermore, modulation of TULA concentration in Jurkat T-lymphoblastoid cells demonstrates that TULA upregulates the activity of both Zap kinase and NF-AT transcription factor. Therefore, our study indicates that TULA counters the inhibitory effect of c-Cbl on protein tyrosine kinases and, thus, may be involved in the regulation of biological effects of c-Cbl. Finally, our results suggest that TULA-mediated inhibition of the effects of c-Cbl on protein tyrosine kinases is caused by TULA-induced ubiquitylation and degradation of c-Cbl.

Selective Detection of Glycopeptides on Ion Trap Mass Spectrometers

Analytical Chemistry. Jun, 2004  |  Pubmed ID: 15167790

Generation of carbohydrate-specific marker ions during LC-ESMS of digested glycoproteins has been demonstrated to be a highly selective and sensitive approach for detection of glycopeptides. In principle, any mass spectrometer can produce and selectively detect carbohydrate marker ions provided that the instrument is capable of collisional excitation in the region prior to the first mass analyzer sufficient to form abundant oxonium ions. This approach has yet to be demonstrated on 3D ion trap mass spectrometers, which have become widely used for proteomic applications. Here we report the successful development and optimization of carbohydrate marker ion detection on a LCQ Deca 3D ion trap utilizing this scan function. Human alpha-1 acid glycoprotein and a therapeutic monoclonal antibody were chosen to illustrate this methodology. Marker ion detection during LC-ESMS facilitated collection of glycopeptide-containing fractions. Analysis of the glycopeptides in these fractions by MS identified the specific glycosylation sites and enabled the prediction of the family of glycoforms at each attachment site. Using these optimized conditions, marker ion detection and glycopeptide analysis could be achieved with as little as 10 pmol of a glycoprotein.

Phosphorylation by Cyclin B-Cdk Underlies Release of Mitotic Exit Activator Cdc14 from the Nucleolus

Science (New York, N.Y.). Jul, 2004  |  Pubmed ID: 15273393

Budding yeast protein phosphatase Cdc14 is sequestered in the nucleolus in an inactive state during interphase by the anchor protein Net1. Upon entry into anaphase, the Cdc14 early anaphase release (FEAR) network initiates dispersal of active Cdc14 throughout the cell. We report that the FEARnetwork promotes phosphorylation of Net1 by cyclin-dependent kinase (Cdk) complexed with cyclin B1 or cyclin B2. These phosphorylations appear to be required for FEAR and sustain the proper timing of late mitotic events. Thus, a regulatory circuit exists to ensure that the arbiter of the mitotic state, Cdk, sets in motion events that culminate in exit from mitosis.

Place of Pattern in Proteomic Biomarker Discovery

Journal of Proteome Research. Jul-Aug, 2005  |  Pubmed ID: 16083265

The role of pattern in biomarker discovery and clinical diagnosis is examined in its historical context. The use of MS-derived pattern is treated as a logical extension of prior applications of non-MS-derived pattern. Criticisms pertaining to specific technology platforms and analytic methodologies are considered separately from the larger issues of pattern utility and deployment in biomarker discovery. We present a hybrid strategy that marries the desirable attributes of high-information content MS pattern with the capability to obtain identity, and explore the key steps in establishing a data analysis pipeline for pattern-based biomarker discovery.

Mapping Posttranslational Modifications of Proteins by MS-based Selective Detection: Application to Phosphoproteomics

Methods in Enzymology. 2005  |  Pubmed ID: 16413312

This chapter outlines general principals that apply to the analysis of posttranslational modifications of proteins, with an emphasis on phosphoproteins. Mass spectrometry (MS)-based approaches for selective detection and site-specific analysis of posttranslationally modified peptides are described, and an MS-based method that relies on production and detection of fragment ions specific for the modification(s) of interest and that was developed in the authors' laboratory is described in detail. The method is applicable to selective detection of N- and O-linked carbohydrates in glycoproteins, O-linked sulfate, and N- and O-linked lipids. Detailed procedures for application of this strategy to phosphorylation-site mapping are presented here.

Systematic Identification of Human Mitochondrial Disease Genes Through Integrative Genomics

Nature Genetics. May, 2006  |  Pubmed ID: 16582907

The majority of inherited mitochondrial disorders are due to mutations not in the mitochondrial genome (mtDNA) but rather in the nuclear genes encoding proteins targeted to this organelle. Elucidation of the molecular basis for these disorders is limited because only half of the estimated 1,500 mitochondrial proteins have been identified. To systematically expand this catalog, we experimentally and computationally generated eight genome-scale data sets, each designed to provide clues as to mitochondrial localization: targeting sequence prediction, protein domain enrichment, presence of cis-regulatory motifs, yeast homology, ancestry, tandem-mass spectrometry, coexpression and transcriptional induction during mitochondrial biogenesis. Through an integrated analysis we expand the collection to 1,080 genes, which includes 368 novel predictions with a 10% estimated false prediction rate. By combining this expanded inventory with genetic intervals linked to disease, we have identified candidate genes for eight mitochondrial disorders, leading to the discovery of mutations in MPV17 that result in hepatic mtDNA depletion syndrome. The integrative approach promises to better define the role of mitochondria in both rare and common human diseases.

PEPPeR, a Platform for Experimental Proteomic Pattern Recognition

Molecular & Cellular Proteomics : MCP. Oct, 2006  |  Pubmed ID: 16857664

Quantitative proteomics holds considerable promise for elucidation of basic biology and for clinical biomarker discovery. However, it has been difficult to fulfill this promise due to over-reliance on identification-based quantitative methods and problems associated with chromatographic separation reproducibility. Here we describe new algorithms termed "Landmark Matching" and "Peak Matching" that greatly reduce these problems. Landmark Matching performs time base-independent propagation of peptide identities onto accurate mass LC-MS features in a way that leverages historical data derived from disparate data acquisition strategies. Peak Matching builds upon Landmark Matching by recognizing identical molecular species across multiple LC-MS experiments in an identity-independent fashion by clustering. We have bundled these algorithms together with other algorithms, data acquisition strategies, and experimental designs to create a Platform for Experimental Proteomic Pattern Recognition (PEPPeR). These developments enable use of established statistical tools previously limited to microarray analysis for treatment of proteomics data. We demonstrate that the proposed platform can be calibrated across 2.5 orders of magnitude and can perform robust quantification of ratios in both simple and complex mixtures with good precision and error characteristics across multiple sample preparations. We also demonstrate de novo marker discovery based on statistical significance of unidentified accurate mass components that changed between two mixtures. These markers were subsequently identified by accurate mass-driven MS/MS acquisition and demonstrated to be contaminant proteins associated with known proteins whose concentrations were designed to change between the two mixtures. These results have provided a real world validation of the platform for marker discovery.

Protein Biomarker Discovery and Validation: the Long and Uncertain Path to Clinical Utility

Nature Biotechnology. Aug, 2006  |  Pubmed ID: 16900146

Better biomarkers are urgently needed to improve diagnosis, guide molecularly targeted therapy and monitor activity and therapeutic response across a wide spectrum of disease. Proteomics methods based on mass spectrometry hold special promise for the discovery of novel biomarkers that might form the foundation for new clinical blood tests, but to date their contribution to the diagnostic armamentarium has been disappointing. This is due in part to the lack of a coherent pipeline connecting marker discovery with well-established methods for validation. Advances in methods and technology now enable construction of a comprehensive biomarker pipeline from six essential process components: candidate discovery, qualification, verification, research assay optimization, biomarker validation and commercialization. Better understanding of the overall process of biomarker discovery and validation and of the challenges and strategies inherent in each phase should improve experimental study design, in turn increasing the efficiency of biomarker development and facilitating the delivery and deployment of novel clinical tests.

MSin1 is Necessary for Akt/PKB Phosphorylation, and Its Isoforms Define Three Distinct MTORC2s

Current Biology : CB. Sep, 2006  |  Pubmed ID: 16919458

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that participates in at least two distinct multiprotein complexes, mTORC1 and mTORC2 . These complexes play important roles in the regulation of cell growth, proliferation, survival, and metabolism. mTORC2 is a hydrophobic motif kinase for the cell-survival protein Akt/PKB and, here, we identify mSin1 as a component of mTORC2 but not mTORC1. mSin1 is necessary for the assembly of mTORC2 and for its capacity to phosphorylate Akt/PKB. Alternative splicing generates at least five isoforms of the mSin1 protein , three of which assemble into mTORC2 to generate three distinct mTORC2s. Even though all mTORC2s can phosphorylate Akt/PKB in vitro, insulin regulates the activity of only two of them. Thus, we propose that cells contain several mTORC2 flavors that may phosphorylate Akt/PKB in response to different signals.

The Connectivity Map: Using Gene-expression Signatures to Connect Small Molecules, Genes, and Disease

Science (New York, N.Y.). Sep, 2006  |  Pubmed ID: 17008526

To pursue a systematic approach to the discovery of functional connections among diseases, genetic perturbation, and drug action, we have created the first installment of a reference collection of gene-expression profiles from cultured human cells treated with bioactive small molecules, together with pattern-matching software to mine these data. We demonstrate that this "Connectivity Map" resource can be used to find connections among small molecules sharing a mechanism of action, chemicals and physiological processes, and diseases and drugs. These results indicate the feasibility of the approach and suggest the value of a large-scale community Connectivity Map project.

PRAS40 is an Insulin-regulated Inhibitor of the MTORC1 Protein Kinase

Molecular Cell. Mar, 2007  |  Pubmed ID: 17386266

The heterotrimeric mTORC1 protein kinase nucleates a signaling network that promotes cell growth in response to insulin and becomes constitutively active in cells missing the TSC1 or TSC2 tumor suppressors. Insulin stimulates the phosphorylation of S6K1, an mTORC1 substrate, but it is not known how mTORC1 kinase activity is regulated. We identify PRAS40 as a raptor-interacting protein that binds to mTORC1 in insulin-deprived cells and whose in vitro interaction with mTORC1 is disrupted by high salt concentrations. PRAS40 inhibits cell growth, S6K1 phosphorylation, and rheb-induced activation of the mTORC1 pathway, and in vitro it prevents the great increase in mTORC1 kinase activity induced by rheb1-GTP. Insulin stimulates Akt/PKB-mediated phosphorylation of PRAS40, which prevents its inhibition of mTORC1 in cells and in vitro. We propose that the relative strengths of the rheb- and PRAS40-mediated inputs to mTORC1 set overall pathway activity and that insulin activates mTORC1 through the coordinated regulation of both.

Proteomic Screen Defines the Polo-box Domain Interactome and Identifies Rock2 As a Plk1 Substrate

The EMBO Journal. May, 2007  |  Pubmed ID: 17446864

Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine-threonine binding domain, the Polo-box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis-specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rho-associated coiled-coil domain-containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.

Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution

Molecular & Cellular Proteomics : MCP. Dec, 2007  |  Pubmed ID: 17939991

Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.

Accurate Inclusion Mass Screening: a Bridge from Unbiased Discovery to Targeted Assay Development for Biomarker Verification

Molecular & Cellular Proteomics : MCP. Oct, 2008  |  Pubmed ID: 18534968

Verification of candidate biomarker proteins in blood is typically done using multiple reaction monitoring (MRM) of peptides by LC-MS/MS on triple quadrupole MS systems. MRM assay development for each protein requires significant time and cost, much of which is likely to be of little value if the candidate biomarker is below the detection limit in blood or a false positive in the original discovery data. Here we present a new technology, accurate inclusion mass screening (AIMS), designed to provide a bridge from unbiased discovery to MS-based targeted assay development. Masses on the software inclusion list are monitored in each scan on the Orbitrap MS system, and MS/MS spectra for sequence confirmation are acquired only when a peptide from the list is detected with both the correct accurate mass and charge state. The AIMS experiment confirms that a given peptide (and thus the protein from which it is derived) is present in the plasma. Throughput of the method is sufficient to qualify up to a hundred proteins/week. The sensitivity of AIMS is similar to MRM on a triple quadrupole MS system using optimized sample preparation methods (low tens of ng/ml in plasma), and MS/MS data from the AIMS experiments on the Orbitrap can be directly used to configure MRM assays. The method was shown to be at least 4-fold more efficient at detecting peptides of interest than undirected LC-MS/MS experiments using the same instrumentation, and relative quantitation information can be obtained by AIMS in case versus control experiments. Detection by AIMS ensures that a quantitative MRM-based assay can be configured for that protein. The method has the potential to qualify large number of biomarker candidates based on their detection in plasma prior to committing to the time- and resource-intensive steps of establishing a quantitative assay.

A Mitochondrial Protein Compendium Elucidates Complex I Disease Biology

Cell. Jul, 2008  |  Pubmed ID: 18614015

Mitochondria are complex organelles whose dysfunction underlies a broad spectrum of human diseases. Identifying all of the proteins resident in this organelle and understanding how they integrate into pathways represent major challenges in cell biology. Toward this goal, we performed mass spectrometry, GFP tagging, and machine learning to create a mitochondrial compendium of 1098 genes and their protein expression across 14 mouse tissues. We link poorly characterized proteins in this inventory to known mitochondrial pathways by virtue of shared evolutionary history. Using this approach, we predict 19 proteins to be important for the function of complex I (CI) of the electron transport chain. We validate a subset of these predictions using RNAi, including C8orf38, which we further show harbors an inherited mutation in a lethal, infantile CI deficiency. Our results have important implications for understanding CI function and pathogenesis and, more generally, illustrate how our compendium can serve as a foundation for systematic investigations of mitochondria.

Metabolic Profiling of the Human Response to a Glucose Challenge Reveals Distinct Axes of Insulin Sensitivity

Molecular Systems Biology. 2008  |  Pubmed ID: 18682704

Glucose ingestion after an overnight fast triggers an insulin-dependent, homeostatic program that is altered in diabetes. The full spectrum of biochemical changes associated with this transition is currently unknown. We have developed a mass spectrometry-based strategy to simultaneously measure 191 metabolites following glucose ingestion. In two groups of healthy individuals (n=22 and 25), 18 plasma metabolites changed reproducibly, including bile acids, urea cycle intermediates, and purine degradation products, none of which were previously linked to glucose homeostasis. The metabolite dynamics also revealed insulin's known actions along four key axes--proteolysis, lipolysis, ketogenesis, and glycolysis--reflecting a switch from catabolism to anabolism. In pre-diabetics (n=25), we observed a blunted response in all four axes that correlated with insulin resistance. Multivariate analysis revealed that declines in glycerol and leucine/isoleucine (markers of lipolysis and proteolysis, respectively) jointly provide the strongest predictor of insulin sensitivity. This observation indicates that some humans are selectively resistant to insulin's suppression of proteolysis, whereas others, to insulin's suppression of lipolysis. Our findings lay the groundwork for using metabolic profiling to define an individual's 'insulin response profile', which could have value in predicting diabetes, its complications, and in guiding therapy.

Metabolite Profiling of Blood from Individuals Undergoing Planned Myocardial Infarction Reveals Early Markers of Myocardial Injury

The Journal of Clinical Investigation. Oct, 2008  |  Pubmed ID: 18769631

Emerging metabolomic tools have created the opportunity to establish metabolic signatures of myocardial injury. We applied a mass spectrometry-based metabolite profiling platform to 36 patients undergoing alcohol septal ablation treatment for hypertrophic obstructive cardiomyopathy, a human model of planned myocardial infarction (PMI). Serial blood samples were obtained before and at various intervals after PMI, with patients undergoing elective diagnostic coronary angiography and patients with spontaneous myocardial infarction (SMI) serving as negative and positive controls, respectively. We identified changes in circulating levels of metabolites participating in pyrimidine metabolism, the tricarboxylic acid cycle and its upstream contributors, and the pentose phosphate pathway. Alterations in levels of multiple metabolites were detected as early as 10 minutes after PMI in an initial derivation group and were validated in a second, independent group of PMI patients. A PMI-derived metabolic signature consisting of aconitic acid, hypoxanthine, trimethylamine N-oxide, and threonine differentiated patients with SMI from those undergoing diagnostic coronary angiography with high accuracy, and coronary sinus sampling distinguished cardiac-derived from peripheral metabolic changes. Our results identify a role for metabolic profiling in the early detection of myocardial injury and suggest that similar approaches may be used for detection or prediction of other disease states.

Protein Quantitation Through Targeted Mass Spectrometry: the Way out of Biomarker Purgatory?

Clinical Chemistry. Nov, 2008  |  Pubmed ID: 18957555

Bead-based Profiling of Tyrosine Kinase Phosphorylation Identifies SRC As a Potential Target for Glioblastoma Therapy

Nature Biotechnology. Jan, 2009  |  Pubmed ID: 19098899

The aberrant activation of tyrosine kinases represents an important oncogenic mechanism, and yet the majority of such events remain undiscovered. Here we describe a bead-based method for detecting phosphorylation of both wild-type and mutant tyrosine kinases in a multiplexed, high-throughput and low-cost manner. With the aim of establishing a tyrosine kinase-activation catalog, we used this method to profile 130 human cancer lines. Follow-up experiments on the finding that SRC is frequently phosphorylated in glioblastoma cell lines showed that SRC is also activated in primary glioblastoma patient samples and that the SRC inhibitor dasatinib (Sprycel) inhibits viability and cell migration in vitro and tumor growth in vivo. Testing of dasatinib-resistant tyrosine kinase alleles confirmed that SRC is indeed the relevant target of dasatinib, which inhibits many tyrosine kinases. These studies establish the feasibility of tyrosine kinome-wide phosphorylation profiling and point to SRC as a possible therapeutic target in glioblastoma.

A Human Proteome Detection and Quantitation Project

Molecular & Cellular Proteomics : MCP. May, 2009  |  Pubmed ID: 19131327

The lack of sensitive, specific, multiplexable assays for most human proteins is the major technical barrier impeding development of candidate biomarkers into clinically useful tests. Recent progress in mass spectrometry-based assays for proteotypic peptides, particularly those with specific affinity peptide enrichment, offers a systematic and economical path to comprehensive quantitative coverage of the human proteome. A complete suite of assays, e.g. two peptides from the protein product of each of the approximately 20,500 human genes (here termed the human Proteome Detection and Quantitation project), would enable rapid and systematic verification of candidate biomarkers and lay a quantitative foundation for subsequent efforts to define the larger universe of splice variants, post-translational modifications, protein-protein interactions, and tissue localization.

Prediction of High-responding Peptides for Targeted Protein Assays by Mass Spectrometry

Nature Biotechnology. Feb, 2009  |  Pubmed ID: 19169245

Protein biomarker discovery produces lengthy lists of candidates that must subsequently be verified in blood or other accessible biofluids. Use of targeted mass spectrometry (MS) to verify disease- or therapy-related changes in protein levels requires the selection of peptides that are quantifiable surrogates for proteins of interest. Peptides that produce the highest ion-current response (high-responding peptides) are likely to provide the best detection sensitivity. Identification of the most effective signature peptides, particularly in the absence of experimental data, remains a major resource constraint in developing targeted MS-based assays. Here we describe a computational method that uses protein physicochemical properties to select high-responding peptides and demonstrate its utility in identifying signature peptides in plasma, a complex proteome with a wide range of protein concentrations. Our method, which employs a Random Forest classifier, facilitates the development of targeted MS-based assays for biomarker verification or any application where protein levels need to be measured.

Identifying the Proteins to Which Small-molecule Probes and Drugs Bind in Cells

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2009  |  Pubmed ID: 19255428

Most small-molecule probes and drugs alter cell circuitry by interacting with 1 or more proteins. A complete understanding of the interacting proteins and their associated protein complexes, whether the compounds are discovered by cell-based phenotypic or target-based screens, is extremely rare. Such a capability is expected to be highly illuminating--providing strong clues to the mechanisms used by small-molecules to achieve their recognized actions and suggesting potential unrecognized actions. We describe a powerful method combining quantitative proteomics (SILAC) with affinity enrichment to provide unbiased, robust and comprehensive identification of the proteins that bind to small-molecule probes and drugs. The method is scalable and general, requiring little optimization across different compound classes, and has already had a transformative effect on our studies of small-molecule probes. Here, we describe in full detail the application of the method to identify targets of kinase inhibitors and immunophilin binders.

Directed Sample Interrogation Utilizing an Accurate Mass Exclusion-based Data-dependent Acquisition Strategy (AMEx)

Journal of Proteome Research. Jun, 2009  |  Pubmed ID: 19344186

The ability to perform thorough sampling is of critical importance when using mass spectrometry to characterize complex proteomic mixtures. A common approach is to reinterrogate a sample multiple times by LC-MS/MS. However, the conventional data-dependent acquisition methods that are typically used in proteomics studies will often redundantly sample high-intensity precursor ions while failing to sample low-intensity precursors entirely. We describe a method wherein the masses of successfully identified peptides are used to generate an accurate mass exclusion list such that those precursors are not selected for sequencing during subsequent analyses. We performed multiple concatenated analytical runs to sample a complex cell lysate, using either accurate mass exclusion-based data-dependent acquisition (AMEx) or standard data-dependent acquisition, and found that utilization of AMEx on an ESI-Orbitrap instrument significantly increases the total number of validated peptide identifications relative to a standard DDA approach. The additional identified peptides represent precursor ions that exhibit low signal intensity in the sample. Increasing the total number of peptide identifications augmented the number of proteins identified, as well as improved the sequence coverage of those proteins. Together, these data indicate that using AMEx is an effective strategy to improve the characterization of complex proteomic mixtures.

Developing Multiplexed Assays for Troponin I and Interleukin-33 in Plasma by Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry

Clinical Chemistry. Jun, 2009  |  Pubmed ID: 19372185

Protein biomarker candidates from discovery proteomics must be quantitatively verified in patient samples before they can progress to clinical validation. Here we demonstrate that peptide immunoaffinity enrichment coupled with stable isotope dilution mass spectrometry (SISCAPA-MRM) can be used to configure assays with performance suitable for candidate biomarker verification. As proof of principle, we configured SISCAPA assays for troponin I (cTnI), an established biomarker of cardiac injury, and interleukin 33 (IL-33), an emerging immunological and cardiovascular marker for which robust immunoassays are currently not available.

Plk1 Self-organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells

PLoS Biology. May, 2009  |  Pubmed ID: 19468302

Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space.

Multi-site Assessment of the Precision and Reproducibility of Multiple Reaction Monitoring-based Measurements of Proteins in Plasma

Nature Biotechnology. Jul, 2009  |  Pubmed ID: 19561596

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low mug/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.

Quantification of Cardiovascular Biomarkers in Patient Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution

Molecular & Cellular Proteomics : MCP. Oct, 2009  |  Pubmed ID: 19596694

Verification of candidate biomarkers requires specific assays to selectively detect and quantify target proteins in accessible biofluids. The primary objective of verification is to screen potential biomarkers to ensure that only the highest quality candidates from the discovery phase are taken forward into preclinical validation. Because antibody reagents for a clinical grade immunoassay often exist for a small number of candidates, alternative methodologies are required to credential new and unproven candidates in a statistically viable number of serum or plasma samples. Using multiple reaction monitoring coupled with stable isotope dilution MS, we developed quantitative, multiplexed assays in plasma for six proteins of clinical relevance to cardiac injury. The process described does not require antibodies for immunoaffinity enrichment of either proteins or peptides. Limits of detection and quantitation for each signature peptide used as surrogates for the target proteins were determined by the method of standard addition using synthetic peptides and plasma from a healthy donor. Limits of quantitation ranged from 2 to 15 ng/ml for most of the target proteins. Quantitative measurements were obtained for one to two signature peptides derived from each target protein, including low abundance protein markers of cardiac injury in the nanogram/milliliter range such as the cardiac troponins. Intra- and interassay coefficients of variation were predominantly <10 and 25%, respectively. The configured multiplex assay was then used to measure levels of these proteins across three time points in six patients undergoing alcohol septal ablation for hypertrophic obstructive cardiomyopathy. These results are the first demonstration of a multiplexed, MS-based assay for detection and quantification of changes in concentration of proteins associated with cardiac injury in the low nanogram/milliliter range. Our results also demonstrate that these assays retain the necessary precision, reproducibility, and sensitivity to be applied to novel and uncharacterized candidate biomarkers for verification of proteins in blood.

Proteomic and Genetic Approaches Identify Syk As an AML Target

Cancer Cell. Oct, 2009  |  Pubmed ID: 19800574

Cell-based screening can facilitate the rapid identification of compounds inducing complex cellular phenotypes. Advancing a compound toward the clinic, however, generally requires the identification of precise mechanisms of action. We previously found that epidermal growth factor receptor (EGFR) inhibitors induce acute myeloid leukemia (AML) differentiation via a non-EGFR mechanism. In this report, we integrated proteomic and RNAi-based strategies to identify their off-target, anti-AML mechanism. These orthogonal approaches identified Syk as a target in AML. Genetic and pharmacological inactivation of Syk with a drug in clinical trial for other indications promoted differentiation of AML cells and attenuated leukemia growth in vivo. These results demonstrate the power of integrating diverse chemical, proteomic, and genomic screening approaches to identify therapeutic strategies for cancer.

Empirical Bayes Analysis of Quantitative Proteomics Experiments

PloS One. 2009  |  Pubmed ID: 19829701

Advances in mass spectrometry-based proteomics have enabled the incorporation of proteomic data into systems approaches to biology. However, development of analytical methods has lagged behind. Here we describe an empirical Bayes framework for quantitative proteomics data analysis. The method provides a statistical description of each experiment, including the number of proteins that differ in abundance between 2 samples, the experiment's statistical power to detect them, and the false-positive probability of each protein.

ZBED6, a Novel Transcription Factor Derived from a Domesticated DNA Transposon Regulates IGF2 Expression and Muscle Growth

PLoS Biology. Dec, 2009  |  Pubmed ID: 20016685

A single nucleotide substitution in intron 3 of IGF2 in pigs abrogates a binding site for a repressor and leads to a 3-fold up-regulation of IGF2 in skeletal muscle. The mutation has major effects on muscle growth, size of the heart, and fat deposition. Here, we have identified the repressor and find that the protein, named ZBED6, is previously unknown, specific for placental mammals, and derived from an exapted DNA transposon. Silencing of Zbed6 in mouse C2C12 myoblasts affected Igf2 expression, cell proliferation, wound healing, and myotube formation. Chromatin immunoprecipitation (ChIP) sequencing using C2C12 cells identified about 2,500 ZBED6 binding sites in the genome, and the deduced consensus motif gave a perfect match with the established binding site in Igf2. Genes associated with ZBED6 binding sites showed a highly significant enrichment for certain Gene Ontology classifications, including development and transcriptional regulation. The phenotypic effects in mutant pigs and ZBED6-silenced C2C12 myoblasts, the extreme sequence conservation, its nucleolar localization, the broad tissue distribution, and the many target genes with essential biological functions suggest that ZBED6 is an important transcription factor in placental mammals, affecting development, cell proliferation, and growth.

Automated Detection of Inaccurate and Imprecise Transitions in Peptide Quantification by Multiple Reaction Monitoring Mass Spectrometry

Clinical Chemistry. Feb, 2010  |  Pubmed ID: 20022980

Multiple reaction monitoring mass spectrometry (MRM-MS) of peptides with stable isotope-labeled internal standards (SISs) is increasingly being used to develop quantitative assays for proteins in complex biological matrices. These assays can be highly precise and quantitative, but the frequent occurrence of interferences requires that MRM-MS data be manually reviewed, a time-intensive process subject to human error. We developed an algorithm that identifies inaccurate transition data based on the presence of interfering signal or inconsistent recovery among replicate samples.

Integration of Proteomic-based Tools for Improved Biomarkers of Myocardial Injury

Clinical Chemistry. Feb, 2010  |  Pubmed ID: 20022985

Given the mounting evidence in favor of early pharmacologic and catheter-based interventions for patients across the spectrum of acute coronary syndromes, discovering novel diagnostically sensitive and specific biomarkers that provide biochemical proof of early or reversible myocardial injury could have a substantial positive impact on patient care.

A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G(2)/M DNA Damage Checkpoint

PLoS Biology. Jan, 2010  |  Pubmed ID: 20126263

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.

Optimizing Performance of Glycopeptide Capture for Plasma Proteomics

Journal of Proteome Research. Apr, 2010  |  Pubmed ID: 20235580

Selective capture of glycopolypeptides followed by release and analysis of the former glycosylation-site peptides has been shown to have promise for reducing the complexity of body fluids such as blood for biomarker discovery. In this work, a protocol based on capture of polypeptides containing a N-linked carbohydrate from human plasma using commercially available magnetic beads coupled with hydrazide chemistry was optimized and partially automated through the use of a KingFisher magnetic particle processor. Comparison of bead-based glycocapture at the protein-level vs the peptide-level revealed differences in the specificity, reproducibility, and absolute number of former glycosylation-site peptides detected. Evaluation of a range of capture and elution conditions led to an optimized protocol with a 24% intraday and 30% interday CV and a glycopeptide capture specificity of 99%. Depleting the plasma of 14 high abundance proteins improved detection sensitivity by approximately 1 order of magnitude compared to nondepleted plasma and resulted in an increase of 24% in the number of identified glycoproteins. The sensitivity of SPEG for detection of glycoproteins in depleted, non-fractionated plasma was found to be in the 10-100 pmol/mL range corresponding to glycoprotein levels ranging from 100's of nanograms/mL to 10's of micrograms/mL. Despite high capture specificity, the total number of glycoproteins detected and the sensitivity of SPEG in plasma is surprisingly limited.

Rictor Phosphorylation on the Thr-1135 Site Does Not Require Mammalian Target of Rapamycin Complex 2

Molecular Cancer Research : MCR. Jun, 2010  |  Pubmed ID: 20501647

In animal cells, growth factors coordinate cell proliferation and survival by regulating the phosphoinositide 3-kinase/Akt signaling pathway. Deregulation of this signaling pathway is common in a variety of human cancers. The PI3K-dependent signaling kinase complex defined as mammalian target of rapamycin complex 2 (mTORC2) functions as a regulatory Ser-473 kinase of Akt. We find that activation of mTORC2 by growth factor signaling is linked to the specific phosphorylation of its component rictor on Thr-1135. The phosphorylation of this site is induced by the growth factor stimulation and expression of the oncogenic forms of ras or PI3K. Rictor phosphorylation is sensitive to the inhibition of PI3K, mTOR, or expression of integrin-linked kinase. The substitution of wild-type rictor with its specific phospho-mutants in rictor null mouse embryonic fibroblasts did not alter the growth factor-dependent phosphorylation of Akt, indicating that the rictor Thr-1135 phosphorylation is not critical in the regulation of the mTORC2 kinase activity. We found that this rictor phosphorylation takes place in the mTORC2-deficient cells, suggesting that this modification might play a role in the regulation of not only mTORC2 but also the mTORC2-independent function of rictor.

Metabolic Signatures of Exercise in Human Plasma

Science Translational Medicine. May, 2010  |  Pubmed ID: 20505214

Exercise provides numerous salutary effects, but our understanding of how these occur is limited. To gain a clearer picture of exercise-induced metabolic responses, we have developed comprehensive plasma metabolite signatures by using mass spectrometry to measure >200 metabolites before and after exercise. We identified plasma indicators of glycogenolysis (glucose-6-phosphate), tricarboxylic acid cycle span 2 expansion (succinate, malate, and fumarate), and lipolysis (glycerol), as well as modulators of insulin sensitivity (niacinamide) and fatty acid oxidation (pantothenic acid). Metabolites that were highly correlated with fitness parameters were found in subjects undergoing acute exercise testing and marathon running and in 302 subjects from a longitudinal cohort study. Exercise-induced increases in glycerol were strongly related to fitness levels in normal individuals and were attenuated in subjects with myocardial ischemia. A combination of metabolites that increased in plasma in response to exercise (glycerol, niacinamide, glucose-6-phosphate, pantothenate, and succinate) up-regulated the expression of nur77, a transcriptional regulator of glucose utilization and lipid metabolism genes in skeletal muscle in vitro. Plasma metabolic profiles obtained during exercise provide signatures of exercise performance and cardiovascular disease susceptibility, in addition to highlighting molecular pathways that may modulate the salutary effects of exercise.

Chemical Genetic Strategy Identifies Histone Deacetylase 1 (HDAC1) and HDAC2 As Therapeutic Targets in Sickle Cell Disease

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2010  |  Pubmed ID: 20616024

The worldwide burden of sickle cell disease is enormous, with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a unique bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease.

DNA Damage Activates a Spatially Distinct Late Cytoplasmic Cell-cycle Checkpoint Network Controlled by MK2-mediated RNA Stabilization

Molecular Cell. Oct, 2010  |  Pubmed ID: 20932473

Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells.

Effect of Collision Energy Optimization on the Measurement of Peptides by Selected Reaction Monitoring (SRM) Mass Spectrometry

Analytical Chemistry. Dec, 2010  |  Pubmed ID: 21090646

Proteomics experiments based on Selected Reaction Monitoring (SRM, also referred to as Multiple Reaction Monitoring or MRM) are being used to target large numbers of protein candidates in complex mixtures. At present, instrument parameters are often optimized for each peptide, a time and resource intensive process. Large SRM experiments are greatly facilitated by having the ability to predict MS instrument parameters that work well with the broad diversity of peptides they target. For this reason, we investigated the impact of using simple linear equations to predict the collision energy (CE) on peptide signal intensity and compared it with the empirical optimization of the CE for each peptide and transition individually. Using optimized linear equations, the difference between predicted and empirically derived CE values was found to be an average gain of only 7.8% of total peak area. We also found that existing commonly used linear equations fall short of their potential, and should be recalculated for each charge state and when introducing new instrument platforms. We provide a fully automated pipeline for calculating these equations and individually optimizing CE of each transition on SRM instruments from Agilent, Applied Biosystems, Thermo-Scientific and Waters in the open source Skyline software tool ( http://proteome.gs.washington.edu/software/skyline ).

Overview of Peptide and Protein Analysis by Mass Spectrometry

Current Protocols in Protein Science / Editorial Board, John E. Coligan ... [et Al.]. Nov, 2010  |  Pubmed ID: 21104985

Mass spectrometry is an indispensable tool for peptide and protein analysis owing to its speed, sensitivity, and versatility. It can be used to determine amino acid sequences of peptides, and to characterize a wide variety of post-translational modifications such as phosphorylation and glycosylation. Mass spectrometry can also be used to determine absolute and relative protein quantities, and can identify and quantify thousands of proteins from complex samples, which makes it an extremely powerful tool for systems biology studies. The main goals of this unit are to familiarize peptide and protein chemists and biologists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide.

Performance Metrics for Liquid Chromatography-tandem Mass Spectrometry Systems in Proteomics Analyses

Molecular & Cellular Proteomics : MCP. Feb, 2010  |  Pubmed ID: 19837981

A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.

Interlaboratory Study Characterizing a Yeast Performance Standard for Benchmarking LC-MS Platform Performance

Molecular & Cellular Proteomics : MCP. Feb, 2010  |  Pubmed ID: 19858499

Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize pre-analytical and analytical variation in comparative proteomics experiments.

Introduction: Advances in Protein Analysis for the Clinical Laboratory

Clinical Chemistry. Feb, 2010  |  Pubmed ID: 19910505

Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography-tandem Mass Spectrometry

Journal of Proteome Research. Feb, 2010  |  Pubmed ID: 19921851

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.

Protein-based Multiplex Assays: Mock Presubmissions to the US Food and Drug Administration

Clinical Chemistry. Feb, 2010  |  Pubmed ID: 20007858

As a part of ongoing efforts of the NCI-FDA Interagency Oncology Task Force subcommittee on molecular diagnostics, members of the Clinical Proteomic Technology Assessment for Cancer program of the National Cancer Institute have submitted 2 protein-based multiplex assay descriptions to the Office of In Vitro Diagnostic Device Evaluation and Safety, US Food and Drug Administration. The objective was to evaluate the analytical measurement criteria and studies needed to validate protein-based multiplex assays. Each submission described a different protein-based platform: a multiplex immunoaffinity mass spectrometry platform for protein quantification, and an immunological array platform quantifying glycoprotein isoforms. Submissions provided a mutually beneficial way for members of the proteomics and regulatory communities to identify the analytical issues that the field should address when developing protein-based multiplex clinical assays.

Analytical Validation of Protein-based Multiplex Assays: a Workshop Report by the NCI-FDA Interagency Oncology Task Force on Molecular Diagnostics

Clinical Chemistry. Feb, 2010  |  Pubmed ID: 20007859

Clinical proteomics has the potential to enable the early detection of cancer through the development of multiplex assays that can inform clinical decisions. However, there has been some uncertainty among translational researchers and developers as to the specific analytical measurement criteria needed to validate protein-based multiplex assays. To begin to address the causes of this uncertainty, a day-long workshop titled "Interagency Oncology Task Force Molecular Diagnostics Workshop" was held in which members of the proteomics and regulatory communities discussed many of the analytical evaluation issues that the field should address in development of protein-based multiplex assays for clinical use. This meeting report explores the issues raised at the workshop and details the recommendations that came out of the day's discussions, such as a workshop summary discussing the analytical evaluation issues that specific proteomic technologies should address when seeking US Food and Drug Administration approval.

Evaluation of Large Scale Quantitative Proteomic Assay Development Using Peptide Affinity-based Mass Spectrometry

Molecular & Cellular Proteomics : MCP. Apr, 2011  |  Pubmed ID: 21245105

Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays.

Lipid Profiling Identifies a Triacylglycerol Signature of Insulin Resistance and Improves Diabetes Prediction in Humans

The Journal of Clinical Investigation. Apr, 2011  |  Pubmed ID: 21403394

Dyslipidemia is an independent risk factor for type 2 diabetes, although exactly which of the many plasma lipids contribute to this remains unclear. We therefore investigated whether lipid profiling can inform diabetes prediction by performing liquid chromatography/mass spectrometry-based lipid profiling in 189 individuals who developed type 2 diabetes and 189 matched disease-free individuals, with over 12 years of follow up in the Framingham Heart Study. We found that lipids of lower carbon number and double bond content were associated with an increased risk of diabetes, whereas lipids of higher carbon number and double bond content were associated with decreased risk. This pattern was strongest for triacylglycerols (TAGs) and persisted after multivariable adjustment for age, sex, BMI, fasting glucose, fasting insulin, total triglycerides, and HDL cholesterol. A combination of 2 TAGs further improved diabetes prediction. To explore potential mechanisms that modulate the distribution of plasma lipids, we performed lipid profiling during oral glucose tolerance testing, pharmacologic interventions, and acute exercise testing. Levels of TAGs associated with increased risk for diabetes decreased in response to insulin action and were elevated in the setting of insulin resistance. Conversely, levels of TAGs associated with decreased diabetes risk rose in response to insulin and were poorly correlated with insulin resistance. These studies identify a relationship between lipid acyl chain content and diabetes risk and demonstrate how lipid profiling could aid in clinical risk assessment.

A Pipeline That Integrates the Discovery and Verification of Plasma Protein Biomarkers Reveals Candidate Markers for Cardiovascular Disease

Nature Biotechnology. Jul, 2011  |  Pubmed ID: 21685905

We developed a pipeline to integrate the proteomic technologies used from the discovery to the verification stages of plasma biomarker identification and applied it to identify early biomarkers of cardiac injury from the blood of patients undergoing a therapeutic, planned myocardial infarction (PMI) for treatment of hypertrophic cardiomyopathy. Sampling of blood directly from patient hearts before, during and after controlled myocardial injury ensured enrichment for candidate biomarkers and allowed patients to serve as their own biological controls. LC-MS/MS analyses detected 121 highly differentially expressed proteins, including previously credentialed markers of cardiovascular disease and >100 novel candidate biomarkers for myocardial infarction (MI). Accurate inclusion mass screening (AIMS) qualified a subset of the candidates based on highly specific, targeted detection in peripheral plasma, including some markers unlikely to have been identified without this step. Analyses of peripheral plasma from controls and patients with PMI or spontaneous MI by quantitative multiple reaction monitoring mass spectrometry or immunoassays suggest that the candidate biomarkers may be specific to MI. This study demonstrates that modern proteomic technologies, when coherently integrated, can yield novel cardiovascular biomarkers meriting further evaluation in large, heterogeneous cohorts.

Exoplasmic Cysteine Cys384 of the HDL Receptor SR-BI is Critical for Its Sensitivity to a Small-molecule Inhibitor and Normal Lipid Transport Activity

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2011  |  Pubmed ID: 21746906

The HDL receptor, scavenger receptor, class B, type I (SR-BI), is a homooligomeric cell surface glycoprotein that controls HDL structure and metabolism by mediating the cellular selective uptake of lipids, mainly cholesteryl esters, from HDL. The mechanism underlying SR-BI-mediated lipid transfer, which differs from classic receptor-mediated endocytosis, involves a two-step process (binding followed by lipid transport) that is poorly understood. Our previous structure/activity analysis of the small-molecule inhibitor blocker of lipid transport 1 (BLT-1), which potently (IC(50) ∼ 50 nM) blocks SR-BI-mediated lipid transport, established that the sulfur in BLT-1's thiosemicarbazone moiety was essential for activity. Here we show that BLT-1 is an irreversible inhibitor of SR-BI, raising the possibility that cysteine(s) in SR-BI interact with BLT-1. Mass spectrometric analysis of purified SR-BI showed two of its six exoplasmic cysteines have free thiol groups (Cys251 and Cys384). Converting Cys384 (but not Cys251) to serine resulted in complete BLT-1 insensitivity, establishing that the unique molecular target of BLT-1 inhibition of cellular SR-BI dependent lipid transport is SR-BI itself. The C384S substitution reduced the receptor's intrinsic lipid uptake activity by approximately 60% without dramatically altering its surface expression, homooligomerization, or HDL binding. Thus, a small-molecule screening approach identified a key residue in SR-BI involved in lipid transport, providing a powerful springboard into the analyses of the structure and mechanism of SR-BI, and highlighting the power of this approach for such analyses.

Selective Killing of Cancer Cells by a Small Molecule Targeting the Stress Response to ROS

Nature. Jul, 2011  |  Pubmed ID: 21753854

Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage). Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress.

AAK1 Identified As an Inhibitor of Neuregulin-1/ErbB4-dependent Neurotrophic Factor Signaling Using Integrative Chemical Genomics and Proteomics

Chemistry & Biology. Jul, 2011  |  Pubmed ID: 21802010

Target identification remains challenging for the field of chemical biology. We describe an integrative chemical genomic and proteomic approach combining the use of differentially active analogs of small molecule probes with stable isotope labeling by amino acids in cell culture-mediated affinity enrichment, followed by subsequent testing of candidate targets using RNA interference-mediated gene silencing. We applied this approach to characterizing the natural product K252a and its ability to potentiate neuregulin-1 (Nrg1)/ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4)-dependent neurotrophic factor signaling and neuritogenesis. We show that AAK1 (adaptor-associated kinase 1) is a relevant target of K252a, and that the loss of AAK1 alters ErbB4 trafficking and expression levels, providing evidence for a previously unrecognized role for AAK1 in Nrg1-mediated neurotrophic factor signaling. Similar strategies should lead to the discovery of novel targets for therapeutic development.

Status and Prospects for Discovery and Verification of New Biomarkers of Cardiovascular Disease by Proteomics

Circulation Research. Aug, 2011  |  Pubmed ID: 21817166

Despite unmet needs for cardiovascular biomarkers, few new protein markers have been approved by the US Food and Drug Administration for the diagnosis or screening of cardiovascular diseases. Mass spectrometry-based proteomics technologies are capable of identifying hundreds to thousands of proteins in cells, tissues, and biofluids. Proteomics may therefore provide the opportunity to elucidate new biomarkers and pathways without a prior known association with cardiovascular disease; however, important obstacles remain. In this review, we focus on emerging techniques that may form a coherently integrated pipeline to overcome present limitations to both the discovery and validation processes.

Mutations in MTFMT Underlie a Human Disorder of Formylation Causing Impaired Mitochondrial Translation

Cell Metabolism. Sep, 2011  |  Pubmed ID: 21907147

The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)), which is used for translation initiation; however, the requirement of formylation for initiation in human mitochondria is still under debate. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified compound heterozygous mutations in MTFMT in two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-tRNA(Met) levels and an abnormal formylation profile of mitochondrially translated COX1. Our findings demonstrate that MTFMT is critical for efficient human mitochondrial translation and reveal a human disorder of Met-tRNA(Met) formylation.

Cdk5 is Required for Memory Function and Hippocampal Plasticity Via the CAMP Signaling Pathway

PloS One. 2011  |  Pubmed ID: 21984943

Memory formation is modulated by pre- and post-synaptic signaling events in neurons. The neuronal protein kinase Cyclin-Dependent Kinase 5 (Cdk5) phosphorylates a variety of synaptic substrates and is implicated in memory formation. It has also been shown to play a role in homeostatic regulation of synaptic plasticity in cultured neurons. Surprisingly, we found that Cdk5 loss of function in hippocampal circuits results in severe impairments in memory formation and retrieval. Moreover, Cdk5 loss of function in the hippocampus disrupts cAMP signaling due to an aberrant increase in phosphodiesterase (PDE) proteins. Dysregulation of cAMP is associated with defective CREB phosphorylation and disrupted composition of synaptic proteins in Cdk5-deficient mice. Rolipram, a PDE4 inhibitor that prevents cAMP depletion, restores synaptic plasticity and memory formation in Cdk5-deficient mice. Collectively, our results demonstrate a critical role for Cdk5 in the regulation of cAMP-mediated hippocampal functions essential for synaptic plasticity and memory formation.

Systematic Discovery of TLR Signaling Components Delineates Viral-sensing Circuits

Cell. Nov, 2011  |  Pubmed ID: 22078882

Deciphering the signaling networks that underlie normal and disease processes remains a major challenge. Here, we report the discovery of signaling components involved in the Toll-like receptor (TLR) response of immune dendritic cells (DCs), including a previously unkown pathway shared across mammalian antiviral responses. By combining transcriptional profiling, genetic and small-molecule perturbations, and phosphoproteomics, we uncover 35 signaling regulators, including 16 known regulators, involved in TLR signaling. In particular, we find that Polo-like kinases (Plk) 2 and 4 are essential components of antiviral pathways in vitro and in vivo and activate a signaling branch involving a dozen proteins, among which is Tnfaip2, a gene associated with autoimmune diseases but whose role was unknown. Our study illustrates the power of combining systematic measurements and perturbations to elucidate complex signaling circuits and discover potential therapeutic targets.

The Matrisome: in Silico Definition and in Vivo Characterization by Proteomics of Normal and Tumor Extracellular Matrices

Molecular & Cellular Proteomics : MCP. Dec, 2011  |  Pubmed ID: 22159717

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins providing both biophysical and biochemical cues that are important regulators of cell proliferation, survival, differentiation and migration. We present here a proteomic strategy developed to characterize the in vivo ECM composition of normal tissues and tumors using enrichment of protein extracts for ECM components and subsequent analysis by mass spectrometry. In parallel, we have developed a bioinformatic approach to predict the in silico ″matrisome″ defined as the ensemble of ECM proteins and associated factors. We report the characterization of the extracellular matrices of murine lung and colon, each comprising more than 100 ECM proteins and each presenting a characteristic signature. Moreover, using human tumor xenografts in mice, we show that both tumor cells and stromal cells contribute to the production of the tumor matrix and that tumors of differing metastatic potential differ in both the tumor- and the stroma-derived ECM components. The strategy we describe and illustrate here can be broadly applied and, to facilitate application of these methods by others, we provide resources including laboratory protocols, inventories of ECM domains and proteins, and instructions for bioinformatically deriving the human and mouse matrisome.

Inter-laboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

Molecular & Cellular Proteomics : MCP. Dec, 2011  |  Pubmed ID: 22199228

The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to MRM-MS produces immuno-MRM assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the inter-laboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay CV, LOD, LOQ and recovery was evaluated. Limits of detection were at or below 1 ng/mL for the assayed proteins in 30 uL of plasma. Assay reproducibility was acceptable for verification studies, with median intra- and inter-laboratory CVs above the LOQ of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope labeled protein as an internal standard instead of stable isotope labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-MRM-MS can be made reproducible across independent laboratories and has the potential to be adopted widely for assaying proteins in matrices as complex as plasma.

ITRAQ Labeling is Superior to MTRAQ for Quantitative Global Proteomics and Phosphoproteomics

Molecular & Cellular Proteomics : MCP. Dec, 2011  |  Pubmed ID: 22210691

Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ or TMT allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, non-isobaric labeling with mTRAQ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly 3-fold more phosphopeptides (12,129 vs. 4,448) and nearly 2-fold more proteins (2,699 vs. 1,597) than mTRAQ labeling. While most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are co-fragmented in the same precursor isolation window, dampening observed ratios toward unity. However, due to tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of non-regulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification.

STK33 Kinase Inhibitor BRD-8899 Has No Effect on KRAS-dependent Cancer Cell Viability

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2012  |  Pubmed ID: 22323609

Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33's kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33's kinase activity does not represent a promising anti-KRAS therapeutic strategy.

Identifying Cellular Targets of Small-molecule Probes and Drugs with Biochemical Enrichment and SILAC

Methods in Molecular Biology (Clifton, N.J.). 2012  |  Pubmed ID: 22065222

Sequencing of the human genome in the last decade has not yet led to a concomitant increase in the numbers of novel drug targets. While the pharmaceutical industry has invested heavily in improving drugs for existing protein targets, it has not tended toward a similar investment in experimental approaches to identify cellular targets of drugs. It is striking that the targets of numerous widely used FDA-approved drugs remain unknown. The development of robust, unbiased methods for target identification would greatly enhance our understanding the mechanisms-of-action of small molecules. Cell-based phenotypic screens followed by unbiased target identification have the potential to identify novel combinations of small molecules and their protein targets, shed light on drug polypharmacology, and enable unbiased screening approaches to drug discovery. Classical biochemical enrichment with immobilized small molecules has been used for over four decades but has been limited by issues concerning specificity and sensitivity. The application of mass spectrometry-based quantitative proteomics in combination with these affinity reagents has proven to be especially useful in addressing these common issues in affinity purification experiments. We describe the use of SILAC in identifying proteins that bind small-molecule probes and drugs in a cellular context.