Universal Quantitative Kinase Assay Based on Diagonal SCX Chromatography and Stable Isotope Dimethyl Labeling Provides High-definition Kinase Consensus Motifs for PKA and Human Mps1

In order to understand cellular signaling, a clear understanding of kinase–substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates <i>in vitro</i> and <i>in vivo</i>. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome

Universal Quantitative Kinase Assay Based on Diagonal SCX Chromatography and Stable Isotope Dimethyl Labeling Provides High-definition Kinase Consensus Motifs for PKA and Human Mps1

In order to understand cellular signaling, a clear understanding of kinase–substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates <i>in vitro</i> and <i>in vivo</i>. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome

Universal Quantitative Kinase Assay Based on Diagonal SCX Chromatography and Stable Isotope Dimethyl Labeling Provides High-definition Kinase Consensus Motifs for PKA and Human Mps1

In order to understand cellular signaling, a clear understanding of kinase–substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates <i>in vitro</i> and <i>in vivo</i>. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome

Universal Quantitative Kinase Assay Based on Diagonal SCX Chromatography and Stable Isotope Dimethyl Labeling Provides High-definition Kinase Consensus Motifs for PKA and Human Mps1

In order to understand cellular signaling, a clear understanding of kinase–substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates <i>in vitro</i> and <i>in vivo</i>. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome

Universal Quantitative Kinase Assay Based on Diagonal SCX Chromatography and Stable Isotope Dimethyl Labeling Provides High-definition Kinase Consensus Motifs for PKA and Human Mps1

In order to understand cellular signaling, a clear understanding of kinase–substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates <i>in vitro</i> and <i>in vivo</i>. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome

Implementation of Ultraviolet Photodissociation on a Benchtop Q Exactive Mass Spectrometer and Its Application to Phosphoproteomics

Proteomics applications performed on the popular benchtop Q Exactive Orbitrap mass spectrometer have so far relied exclusively on higher collision-energy dissociation (HCD) fragmentation for peptide sequencing. While this fragmentation technique is applicable to a wide range of biological questions, it also has limitations, and all questions cannot be addressed equally well. Here, we demonstrate that the fragmentation capabilities of the Q Exactive mass spectrometer can be extended with ultraviolet photodissociation (UVPD) fragmentation, complete with synchronization triggering to make it compatible with liquid chromatography (LC)/tandem mass spectrometry (MS/MS) workflows. We show that UVPD not only is directly compatible with LC/MS workflows but also, when combined with these workflows, can result in higher database scores and increased identification rates for complex samples as compared to HCD methods. UVPD as a fragmentation technique offers prompt, high-energy fragmentation, which can potentially lead to improved analyses of labile post-translational modifications. Techniques like HCD result in substantial amounts of modification losses, competing with fragmentation pathways that provide information-rich ion fragments. We investigate here the utility of UVPD for identification of phosphorylated peptides and find that UVPD fragmentation reduces the extent of labile modification loss by up to ∼60%. Collectively, when integrated into a complete workflow on the Q Exactive Orbitrap, UVPD provides distinct advantages to the analysis of post-translational modifications and is a powerful and complementary addition to the proteomic toolbox

Interferon-related protein expression ratios of stimulated monocytes.

<p>The protein expression ratios (median and range of three biological replicates) of indicated interferon-related proteins normalized to medium control (1.0) after 24 and 48 hours of Al(OH)₃ or DTaP stimulation are depicted. Significance of difference is determined with a <i>t</i>-test with a two stage setup method of Benjamini, <i>p</i>-Values <0.05 are denoted as: * when upregulated compared to the other stimulation condition.</p

Venn diagram and protein network analysis of regulated proteins.

<p>Venn diagrams indicate numbers of sharing and upregulated (A) and downregulated (B) proteins in DTaP <i>versus</i> Al(OH)₃ adjuvant-stimulated monocytes after 24 hours and 48 hours. Purple represents Al(OH)₃ 24 hours, yellow represents DTaP 24 hours, green represents Al(OH)₃ 48 hours and red represents DTaP 48 hours. All proteins depicted in these Venn diagrams were regulated by a factor of at least 1.5 in at least two out of three donors. Up and downregulated proteins, identified per stimulation condition and per time point by mass spectrometric analysis, were assessed based on Gene Ontology biological process enrichment (GO terms). An overview of the main enriched pathways is depicted in a protein network (C). The yellow circles represent the stimulation conditions, the purple squares represent the processes. Green lines from a condition towards a process represent a downregulated pathways. Red lines from a condition towards a process represent an enrichment of these processes in the upregulated protein sets. The width of the line represents the significance of the enrichment factor: the thicker the line the more significantly enriched the process; the thinnest lines represent a <i>p</i>-value <0.05, the medium lines represent a <i>p</i>-value <0.01 and the thickest lines represent a <i>p</i>-value <0.001. The black arrows connect daughter terms with the mother term. All terms are at least enriched with a False Discovery Rate <i>p</i> value of <0.05. Bar graphs of the number of proteins found to be regulated upon the stimulation conditions after 24 hours (D) and 48 hours (E), in which red represents that process was enriched in the upregulated proteins set and green represents that the process is enriched in the downregulated protein set. The * depicts the significantly regulated pathways.</p

Overview of all data.

<p>The different effects of Al(OH)₃ and DTaP stimulation on monocyte functions are summarized. The red arrows from a stimulation DTaP and FHA (A) or Al(OH)₃ (B) represent an upregulation, the green arrows represent a down regulation. Red arrows towards a box indicate that the genes/proteins in the box are upregulated. The title in the box represents the process regulated. For interferon secretion, individual arrows indicate if genes or processes are upregulated. The dashed arrows represent connections based on the literature. The circles represent measurements at the gene expression level while the rounded boxes represent measurements at the protein level. The green and orange rectangles represent consequences of an inhibition or activation by one of the stimulations.</p

Fold changes of interferon-related genes relative to control after 48 hours of stimulation.

<p>Fold changes of interferon-related genes relative to control after 48 hours of stimulation.</p