QIKS – Quantitative identification of kinase substrates

Morandell, Sandra; Grosstessner-Hain, Karin; Roitinger, Elisabeth; Hudecz, Otto; Lindhorst, Thomas; Teis, David; Wrulich, Oliver A.; Mazanek, Michael; Taus, Thomas; Ueberall, Florian; Mechtler, Karl; Huber, Lukas A.

doi:10.1002/pmic.200900749

Cited by 27 publications

(21 citation statements)

References 35 publications

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“…Elution of peptides was facilitated by increasing pH and salt concentration in parallel. Twenty-five separate fractions were collected, desalted and phosphopeptides were enriched by IMAC as described in Morandell et al (22).…”

Section: Methodsmentioning

confidence: 99%

“…The two samples were differentially labeled with either heavy or light methanol and subsequent desalting was performed as previously described (22). Dried peptide pellets (each containing 2 mg of trypsin-digested cell lysate) were dissolved in 1.5 ml of strong cation exchange (SCX) buffer A (buffer composition and the SCX separation gradient are shown in the Supplementary experimental procedures).…”

Section: Methodsmentioning

confidence: 99%

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Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome

Grosstessner-Hain

Hegemann

Novatchkova

et al. 2011

Molecular & Cellular Proteomics

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Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome

Grosstessner-Hain

Hegemann

Novatchkova

et al. 2011

Molecular & Cellular Proteomics

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“…Mass spectrometry-based proteomics has become a powerful tool and been applied to map protein interaction networks, including kinase/phosphatase-substrate networks (10). Large-scale phosphoproteomics, however, does not typically reveal precise connections between protein kinases and their direct substrates (11,12).…”

mentioning

confidence: 99%

“…An elegant chemical genetic approach was developed by Shokat and coworkers to use kinases engineered to accept bulky ATP analogs that can distinguish between wild-type and mutated or analog-sensitive kinases (16). Recently it has been coupled to quantitative proteomics, termed quantitative identification of kinase substrates (10), to identify substrate proteins of mitogen-activated protein kinase/ Erk kinase. All these methods, however, have been limited to the identification of in vitro kinase substrates.…”

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confidence: 99%

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

Xue¹,

Wang²,

Iliuk³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

121

138

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Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity.P rotein kinases and their substrates represent the largest signaling network that regulates protein-protein interactions, subcellular localization, and ultimately cellular functions (1, 2). Deregulation of the signaling network often leads to disease states such as human malignancies, diabetes, and immune disorders. Although many kinases are excellent therapeutic targets, the precise connection between protein kinases and their direct substrates has not been fully elucidated for a majority of protein kinases. Besides classical genetic and biochemical methods, there have been a number of high throughput approaches for the identification of potential kinase substrates. Common methods include in vitro kinase assays using libraries of synthetic peptides (3), phase expression libraries (4), protein/peptide arrays (5-7), or cell extracts (8, 9), but these methods can often be misleading and provide many false positive results. The discovery of physiological substrates for specific protein kinases has remained challenging, even with recent advances in mass spectrometry.Mass spectrometry-based proteomics has become a powerful tool and been applied to map protein interaction networks, including kinase/phosphatase-substrate networks (10). Large-scale phosphoproteomics, however, does not typically reveal precise connections between protein kinases and their direct substrates (11,12). In recent years, there have been increasing attempts to develop mass spectrometry-based proteomic strategies for the identification of elusive kinase substrates (7,13,1...

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“…As a result, indirect effects caused by contaminating kinases during the in vitro kinase assay are largely eliminated. ASKA has recently been coupled with quantitative proteomics, termed Quantitative Identification of Kinase Substrates (QIKS) (12), to identify substrate proteins of Mek1. Recently, one extension of the ASKA technique is for the analog ATP to carry a ␥-thiophosphate group so that in vitro thiophosphorylated proteins can be isolated for mass spectrometric detection (22)(23)(24).…”

mentioning

confidence: 99%

Identification of Direct Tyrosine Kinase Substrates Based on Protein Kinase Assay-Linked Phosphoproteomics

Xue¹,

Geahlen

Tao

2013

Molecular & Cellular Proteomics

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Protein kinases are implicated in multiple diseases such as cancer, diabetes, cardiovascular diseases, and central nervous system disorders. Identification of kinase substrates is critical to dissecting signaling pathways and to understanding disease pathologies. However, methods and techniques used to identify bona fide kinase substrates have remained elusive. Here we describe a proteomic strategy suitable for identifying kinase specificity and direct substrates in high throughput. This approach includes an in vitro kinase assay-based substrate screening and an endogenous kinase dependent phosphorylation profiling. In the in vitro kinase reaction route, a pool of formerly phosphorylated proteins is directly extracted from whole cell extracts, dephosphorylated by phosphatase treatment, after which the kinase of interest is added. Protein phosphorylation plays a pivotal role in regulating biological events such as protein-protein interactions, signal transduction, subcellular localization, and apoptosis (1). Deregulation of kinase-substrate interactions often leads to disease states such as human malignancies, diabetes, and immune disorders (2). Although a number of kinases are being targeted to develop new drugs, our understanding of the precise relationships between protein kinases and their direct substrates is incomplete for the majority of protein kinases (3). Thus, mapping kinase-substrate relationships is essential for the understanding of biological signaling networks and the discovery and development of drugs for targeted therapies (4). Toward this goal, various in vitro kinase assays using synthetic peptide libraries (5), phage expression libraries (6), protein arrays (7-9), or cell extracts (10, 11) have been explored for the screening of kinase substrates.Besides classical biochemical and genetic methods, mass spectrometry-based high throughput approaches have become increasingly attractive because they are capable of sequencing proteins and localizing phosphorylation sites at the same time. Mass spectrometry-based proteomic methods have been extensively applied to kinase-substrate interaction mapping (12) and global phosphorylation profiling (13-15). Although thousands of phosphorylation events can be inspected simultaneously (16,17), large-scale phosphoproteomics does not typically reveal direct relationships between protein kinases and their substrates.Recently, several mass spectrometry-based proteomic strategies have been introduced for identifying elusive kinase substrates (7,18,19). Taking advantage of recent advances of high speed and high-resolution mass spectrometry, these methods used purified, active kinases to phosphorylate cell extracts in vitro, followed by mass spectrometric analysis to identify phosphoproteins. These approaches commonly face the major challenge of distinguishing phosphorylation events triggered by the kinase reaction from background signals introduced by endogenous kinase activities (20). To dissect the phosphorylation cascade, Shokat and colleagues developed an appro...

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QIKS – Quantitative identification of kinase substrates

Cited by 27 publications

References 35 publications

Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome

Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

Identification of Direct Tyrosine Kinase Substrates Based on Protein Kinase Assay-Linked Phosphoproteomics

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