ABSTRACT
Unlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted.
Subject(s)
Immunity, Cellular , Mitogen-Activated Protein Kinase 7/metabolism , Animals , Biomarkers , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Cytokines/genetics , Cytokines/metabolism , Enzyme Activation , Gene Expression , Gene Expression Profiling , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , HeLa Cells , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Immunity, Cellular/drug effects , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Inflammation Mediators/metabolism , Inhibitory Concentration 50 , Mice , Mitogen-Activated Protein Kinase 7/antagonists & inhibitors , Mitogen-Activated Protein Kinase 7/genetics , Molecular Structure , Phosphorylation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Structure-Activity Relationship , TranscriptomeABSTRACT
Palbociclib is a cyclin-dependent kinase (CDK) 4/CDK6 inhibitor approved for breast cancer that is estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative. We profiled palbociclib in cells either sensitive or resistant to the drug using an ATP/ADP probe-based chemoproteomics platform. Palbociclib only engaged CDK4 or CDK6 in sensitive cells. In resistant cells, no inhibition of CDK4 or CDK6 was observed, although the off-target profiles were similar in both cell types. Prolonged incubation of sensitive cells with the compound (24 h) resulted in the downregulation of additional kinases, including kinases critical for cell cycle progression. This downregulation is consistent with cell cycle arrest caused by palbociclib treatment. Both the direct and indirect targets were also observed in a human tumor xenograft study using the COLO-205 cell line in which phosphorylation of the retinoblastoma protein was tracked as the pharmacodyanamic marker. Together, these results suggest that this probe-based approach could be an important strategy toward predicting patient responsiveness to palbociclib.
Subject(s)
Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Neoplasms/pathology , Piperazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Proteomics , Pyridines/pharmacology , Animals , Cell Line, Tumor , Humans , Mice , Neoplasms/enzymology , Xenograft Model Antitumor AssaysABSTRACT
Hsp90 is an ATP-dependent chaperone of widespread interest as a drug target. Here, using an LC-MS/MS chemoproteomics platform based on a lysine-reactive ATP acyl phosphate probe, several Hsp90 inhibitors were profiled in native cell lysates. Inhibitor specificities for all four human paralogs of Hsp90 were simultaneously monitored at their endogenous relative abundances. Equipotent inhibition of probe labeling in each paralog occurred at sites both proximal to and distal from bound ATP observed in Hsp90 cocrystal structures, suggesting that the ATP probe is assaying a native conformation not predicted by available structures. Inhibitor profiling against a comprehensive panel of protein kinases and other ATP-binding proteins detected in native cell lysates identified PMS2, a member of the GHKL ATPase superfamily as an off-target of NVP-AUY922 and radicicol. Because of the endogenously high levels of Hsp90 paralogs in typical cell lysates, the measured potency of inhibitors was weaker than published IC50 values. Significant inhibition of Hsp90 required inhibitor concentrations above a threshold where off-target activity was detectable. Direct on- and off-target engagement was measured by profiling lysates derived from cells treated with Hsp90 inhibitors. These studies also assessed the downstream cellular pathway effects of Hsp90 inhibition, including the down regulation of several known Hsp90 client proteins and some previously unknown client proteins. Overall, the ATP probe-based assay methodology enabled a broad characterization of Hsp90 inhibitor activity and specificity in native cell lysates.
Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Adenosine Triphosphate/metabolism , Cell Line , HSP90 Heat-Shock Proteins/chemistry , Humans , Signal Transduction , Tandem Mass SpectrometryABSTRACT
KIAA1363 is a serine hydrolase whose activity has been shown to be positively associated with tumor cell invasiveness. Thus, inhibitors of KIAA1363 represent a novel targeted therapy approach towards cancer. AX11890 ((1-bromo-2-naphthyl) N,N-dimethylcarbamate) was identified as a KIAA1363 inhibitor with an IC(50) value of 1.2 µM and was shown using ESI-MS to carbamylate the catalytic residue Ser(191). SAR studies explored both substitution of the 1-bromo group and derivatization of the 6-position. Activity-based protein profiling demonstrated AX13057 inhibited tumor-localized KIAA1363 in SK-OV-3 xenograft-bearing mice.
Subject(s)
Carbamates/chemistry , Carbamates/pharmacology , Carboxylic Ester Hydrolases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Sterol Esterase/antagonists & inhibitors , Animals , Carbamates/chemical synthesis , Carbamates/therapeutic use , Carboxylic Ester Hydrolases/metabolism , Cell Line, Tumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/therapeutic use , Female , Humans , Mice , Mice, SCID , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Sterol Esterase/metabolism , Structure-Activity RelationshipABSTRACT
The synthesis, GSK-3ß inhibitory activity, and anti-microbial activity of bicyclic and tricyclic derivatives of the 5,7-diamino-6-fluoro-4-quinolone-3-carboxylic acid scaffold were studied. Kinase selectivity profiling indicated that members of this class were potent and highly selective GSK-3 inhibitors.
Subject(s)
Glycogen Synthase Kinase 3/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , 4-Quinolones/chemistry , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Clinical Trials as Topic , Drug Design , Drug Evaluation, Preclinical , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , HL-60 Cells , High-Throughput Screening Assays , Humans , Inhibitory Concentration 50 , Isoenzymes , Molecular Targeted Therapy , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Substrate SpecificityABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0152934.].
ABSTRACT
Polo-like kinases (PLKs) play critical roles throughout mitosis. Here, we report that wortmannin, which was previously thought to be a highly selective inhibitor of phosphoinositide (PI) 3-kinases, is a potent inhibitor of mammalian PLK1. Observation of the wortmannin-PLK1 interaction was enabled by a tetramethylrhodamine-wortmannin conjugate (AX7503) that permits rapid detection of PLK1 activity and expression in complex proteomes. Importantly, we show that wortmannin inhibits PLK1 activity in an in vitro kinase assay with an IC(50) of 24 nM and when incubated with intact cells. Taken together, our results indicate that, at the concentrations of wortmannin commonly used to inhibit PI 3-kinases, PLK1 is also significantly inhibited.
Subject(s)
Androstadienes/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Jurkat Cells , Molecular Conformation , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Rhodamines/chemical synthesis , Rhodamines/chemistry , Rhodamines/pharmacology , Wortmannin , Polo-Like Kinase 1ABSTRACT
We describe the identification of a novel, tumor-specific missense mutation in the active site of casein kinase 1α (CSNK1A1) using activity-based proteomics. Matched normal and tumor colon samples were analyzed using an ATP acyl phosphate probe in a kinase-targeted LC-MS2 platform. An anomaly in the active-site peptide from CSNK1A1 was observed in a tumor sample that was consistent with an altered catalytic aspartic acid. Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1. Genomic sequencing of the colon tumor samples confirmed the presence of a missense mutation in the catalytic aspartic acid of CSNK1A1 (GACâAAC). To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics. The tumorigenic potential of this mutation remains to be determined.
Subject(s)
Adenocarcinoma/genetics , Casein Kinase Ialpha/genetics , Colonic Neoplasms/genetics , Mutation, Missense , Adenocarcinoma/pathology , Base Sequence , Casein Kinase Ialpha/chemistry , Catalytic Domain , Colon/pathology , Colonic Neoplasms/pathology , HEK293 Cells , Humans , Molecular Sequence Data , Proteomics/methodsABSTRACT
Much attention has recently been given to a class of proteases that cleave proteins and peptides after proline residues. This class includes dipeptidyl peptidase IV (DPP IV; also termed CD26), fibroblast activation protein alpha (FAP; seprase), DPP7 (DPP II; quiescent cell proline dipeptidase), DPP8, DPP9, and prolyl carboxypeptidase (PCP; angiotensinase C). More distant members include prolyl oligopeptidase (POP; post proline cleaving enzyme) and acylaminoacylpeptidase (AAP; acylpeptide hydrolase). The DPPs and related proteins contain both membrane-bound and soluble members and span a broad range of expression patterns, tissue distributions and compartmentalization. These proteins have important roles in regulation of signaling by peptide hormones, and are emerging targets for diabetes, oncology and other indications.
Subject(s)
Dipeptidases/metabolism , Gelatinases , Serine Endopeptidases/metabolism , Animals , Carboxypeptidases/metabolism , Diabetes Mellitus/drug therapy , Dipeptidases/antagonists & inhibitors , Dipeptidases/chemistry , Dipeptidyl Peptidase 4/chemistry , Dipeptidyl Peptidase 4/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Drug Delivery Systems , Endopeptidases , Humans , Membrane Proteins/metabolism , Peptide Hormones/metabolism , Prolyl Oligopeptidases , Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/therapeutic use , Substrate SpecificityABSTRACT
The largest mammalian enzyme family is the kinases. Kinases and other nucleotide-binding proteins are key regulators of signal transduction pathways and the mutation or overexpression of these proteins is often the difference between health and disease. As a result, a massive research effort has focused on understanding how these proteins function and how to inhibit them for therapeutic benefit. Recent advances in chemical biological tools have enabled functional interrogation of these enzymes to provide a deeper understanding of their physiological roles. In addition, these innovative platforms have paved the way for a new generation of drugs whose properties have been guided by functional profiling.
Subject(s)
Phosphotransferases/physiology , Adenosine Triphosphate/metabolism , Animals , Enzyme Assays , Humans , Phosphorylation , Phosphotransferases/chemistry , Protein Interaction Maps , Protein Processing, Post-Translational , ProteomicsABSTRACT
Dipeptidyl peptidases (DPPs) are proteolytic enzymes that regulate many physiological systems by degrading signaling peptides. DPP8 and DPP9 are distinct from DPP4 in sequence, cellular localization and expression levels, thus implying distinct functions. However, DPP8 and DPP9 expression needs further delineation. We evaluated DPP4, DPP8 and DPP9 expression using three independent methods at the mRNA, protein, and functional levels to better understand the local physiological contribution of each enzyme. Sprague Dawley rats and cynomolgus monkeys were selected for DPP4, DPP8 and DPP9 expression profiling to represent animal species commonly utilized for drug preclinical safety evaluation. A novel Xhibit assay of DPP protease activity was applied in addition to newly available antibodies for immunohistochemical localization. This combined approach can facilitate a functional evaluation of protease expression, which is important for understanding physiological relevance. Few inter-species differences were observed. Tissue mRNA and protein levels generally correlated to functional DPP4 and DPP8/9 enzymatic activity. All three proteins were seen in epithelial cells, lymphoid cells and some endothelial and vascular smooth muscle cells. Combined DPP8/DPP9 enzymatic activity was uniformly intracellular across tissues at approximately 10-fold lower levels than non-renal DPP4. Consistent levels of each DPP were detected among most non-renal tissues in rats and monkeys. DPP4 was ubiquitous, principally detected on cell membranes of epithelial and endothelial cells and was greatest in the kidney. The expression patterns suggest that DPP8 and DPP9 may act similarly across tissues, and that their actions might in part overlap with DPP4.
Subject(s)
Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Kidney/enzymology , Amino Acid Sequence , Animals , Dipeptidyl Peptidase 4/genetics , Dipeptidyl Peptidase 4/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Female , Gene Expression , Macaca fascicularis , Male , Molecular Sequence Data , Organ Specificity , Pancreas/enzymology , Rats , Rats, Sprague-Dawley , Species SpecificityABSTRACT
The Karyopherin (Kap) family of nuclear transport receptors enables trafficking of proteins to and from the nucleus in a precise, regulated manner. Individual members function in overlapping pathways, while simultaneously being very specific for their main cargoes. The details of this apparent contradiction and rules governing pathway preference remain to be further elucidated. S. cerevisiae Lhp1 is an abundant protein that functions as an RNA chaperone in a variety of biologically important processes. It localizes almost exclusively to the nucleus and is imported by Kap108. We show that mutation of 3 of the 275 residues in Lhp1 alters its import pathway to a Kap121-dependent process. This mutant does not retain wild-type function and is bound by several chaperones. We propose that Kap121 also acts as a chaperone, one that can act as a genetic buffer by transporting mutated proteins to the nucleus.
Subject(s)
Cell Nucleus/metabolism , Membrane Transport Proteins/metabolism , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Point Mutation , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Active Transport, Cell Nucleus , Amino Acid Sequence , Models, Molecular , Mutant Proteins/genetics , Protein Folding , Protein Stability , Protein Structure, Tertiary , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Substrate SpecificityABSTRACT
INTRODUCTION: Increase of serum alanine aminotransferase (ALT) activity is widely used as a surrogate marker for tissue damage. Two ALT isoforms, ALT1 and ALT2, have been cloned recently in mammals. The study investigated the source of elevated ALT activity in serum of dogs treated with a hepatotoxic compound. METHODS: ALT activity was measured by enzyme assay. Immunoblot analysis was performed using generated specific peptide antibodies against dog ALTs. LC-MS/MS-based proteomics analysis was conducted to independently identify dog ALT peptides. Serum samples immunodepleted of major serum components by Seppro IgY-D11 microbead spin column were evaluated by the immunoblot analysis, and compared with those of the ALT activity. RESULTS: Involvement of ALT enzyme(s) is consistent with the following observations: 1) all the substrates (L-alanine and alpha-ketoglutarate) were required for serum ALT activity as purified porcine ALT1 needed for activity, 2) serum ALT activity was inhibited by L-cycloserine, a known ALT inhibitor, and 3) apparent Km value for the ALT reaction catalyzed by the serum, liver, and skeletal muscle was roughly similar. Immunoblot analysis showed that ALT1 was detected in liver and both ALTs were detected in the skeletal muscle. The relative expression level of ALTs was -: liver ALT1>skeletal muscle ALT1>skeletal muscle ALT2. LC-MS/MS-based proteomics analysis gave similar results. Immunoblot analysis of the depleted serum samples revealed the presence of ALT1 in compound-treated dogs. Intensity of the ALT1 band detected in the sera correlated well with the ALT activity measured by the enzyme assay. DISCUSSION: Based on these findings, we conclude that the elevation of serum ALT activity in dogs with liver injury is attributed to elevation of ALT1 protein level in serum. The methodology to directly detect ALT proteins in serum could be a tool to facilitate our understanding of biological and toxicological significance of the ALT isoenzymes.
Subject(s)
Alanine Transaminase/blood , Disease Models, Animal , Liver Diseases/blood , Liver Diseases/enzymology , Reperfusion Injury/blood , Reperfusion Injury/enzymology , Alanine Transaminase/classification , Alanine Transaminase/metabolism , Amino Acid Sequence , Animals , Biomarkers/blood , Biomarkers/metabolism , Dogs , Enzyme Activation/physiology , Humans , Isoenzymes/blood , Isoenzymes/metabolism , Liver Diseases/pathology , Male , Molecular Sequence Data , Muscle, Skeletal/enzymology , Reperfusion Injury/pathology , Substrate Specificity/physiology , SwineABSTRACT
A cell permeable DPP II [also known as DPP2, DPP7, and quiescent cell proline dipeptidase (QPP)] inhibitor has been synthesized. The azabicyclo[3.3.0]octane-based inhibitor is potent and selective and elicits very similar quiescent lymphocyte death to previously characterized inhibitors that are not as selective.
Subject(s)
Aza Compounds/chemical synthesis , Aza Compounds/pharmacology , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/antagonists & inhibitors , Octanes/chemical synthesis , Octanes/pharmacology , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Apoptosis/drug effects , Cell Death/drug effects , Humans , In Vitro Techniques , Jurkat Cells , Kinetics , Lymphocytes/drug effectsABSTRACT
Polo-like kinases play crucial roles throughout mitosis. We previously reported that wortmannin potently inhibits Polo-like kinase 1 (Plk1). In this study, we show that wortmannin also strongly inhibits Polo-like kinase 3 (Plk3). To further characterize this inhibition, we identified the sites of labeling on Plk1 and Plk3 targeted by AX7503, a tetramethylrhodamine-wortmannin conjugate. AX7503 labeling on Plk1 and Plk3 was found to occur on a conserved ATP binding site residue. In addition, we show that wortmannin inhibits Plk3 activity in live cells at concentrations commonly used to inhibit the more well known targets of wortmannin, the phosphoinositide 3-kinases. Importantly, we found that inhibition of Plk3 by wortmannin lead to a decrease in phosphorylation of p53 on serine 20 induced by DNA damage, demonstrating the effect of wortmannin on a downstream Plk3 target. Taken together, our results suggest that wortmannin can affect multiple functions of Plk3 in cell cycle progression and at the DNA damage check point. The identification of the labeling sites of Plk1 and Plk3 by AX7503 may be useful in designing more effective compounds to target Polo-like kinases for cancer treatment and also may be useful for the structural study of Plk domains.
Subject(s)
Androstadienes/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Amino Acid Sequence , Androstadienes/chemistry , Binding Sites , Cell Line , DNA Damage , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Lysine , Molecular Sequence Data , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Rhodamines/chemistry , Rhodamines/pharmacology , Sequence Alignment , Substrate Specificity , Tumor Suppressor Proteins , WortmanninABSTRACT
Dipeptide-based inhibitors with C-substituted (alkyl or aminoalkyl) alpha-amino acids in the P2 position and boro-norleucine (boro-Nle) in the P1 position were synthesized. Relative to boro-proline, boro-Nle as a P1 residue was shown able to significantly dial out DPP4, FAP, DPP8, and DPP9 activity. Dab-boro-Nle (4g) proved to be the most selective and potent DPP7 inhibitor with a DPP7 IC50 value of 480 pM.
Subject(s)
Boronic Acids , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/antagonists & inhibitors , Norleucine/analogs & derivatives , Dipeptides/chemical synthesis , Dipeptides/pharmacology , Drug Design , Humans , Inhibitory Concentration 50 , Norleucine/pharmacology , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The structure-activity relationship of various N-alkyl Gly-boro-Pro derivatives against three dipeptidyl peptidases (DPPs) was studied. In a series of N-cycloalkyl analogs, DPP4 and fibroblast activation protein-alpha (FAP) optimally preferred N-cycloheptyl whereas DPP7 tolerated even larger cycloalkyl rings. Gly alpha-carbon derivatization of N-cyclohexyl or N-(2-adamantyl) Gly-boro-Pro resulted in a significant decrease in potency against all the three DPPs.
Subject(s)
Dipeptides/chemical synthesis , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/antagonists & inhibitors , Adenosine Deaminase Inhibitors , Antigens, Neoplasm , Biomarkers, Tumor/antagonists & inhibitors , Boronic Acids , Dipeptides/pharmacology , Dipeptidyl Peptidase 4 , Endopeptidases , Gelatinases , Glycoproteins/antagonists & inhibitors , Humans , Immunologic Factors/chemical synthesis , Immunologic Factors/pharmacology , Inhibitory Concentration 50 , Membrane Proteins , Serine Endopeptidases , Structure-Activity RelationshipABSTRACT
An analysis of the structurally and catalytically diverse serine hydrolase protein family in the Saccharomyces cerevisiae proteome was undertaken using two independent but complementary, large-scale approaches. The first approach is based on computational analysis of serine hydrolase active site structures; the second utilizes the chemical reactivity of the serine hydrolase active site in complex mixtures. These proteomics approaches share the ability to fractionate the complex proteome into functional subsets. Each method identified a significant number of sequences, but 15 proteins were identified by both methods. Eight of these were unannotated in the Saccharomyces Genome Database at the time of this study and are thus novel serine hydrolase identifications. Three of the previously uncharacterized proteins are members of a eukaryotic serine hydrolase family, designated as Fsh (family of serine hydrolase), identified here for the first time. OVCA2, a potential human tumor suppressor, and DYR-SCHPO, a dihydrofolate reductase from Schizosaccharomyces pombe, are members of this family. Comparing the combined results to results of other proteomic methods showed that only four of the 15 proteins were identified in a recent large-scale, "shotgun" proteomic analysis and eight were identified using a related, but similar, approach (neither identifies function). Only 10 of the 15 were annotated using alternate motif-based computational tools. The results demonstrate the precision derived from combining complementary, function-based approaches to extract biological information from complex proteomes. The chemical proteomics technology indicates that a functional protein is being expressed in the cell, while the computational proteomics technology adds details about the specific type of function and residue that is likely being labeled. The combination of synergistic methods facilitates analysis, enriches true positive results, and increases confidence in novel identifications. This work also highlights the risks inherent in annotation transfer and the use of scoring functions for determination of correct annotations.