MCAM: Multiple Clustering Analysis Methodology for Deriving Hypotheses and Insights from High-Throughput Proteomic Datasets

Advances in proteomic technologies continue to substantially accelerate capability for generating experimental data on protein levels, states, and activities in biological samples. For example, studies on receptor tyrosine kinase signaling networks can now capture the phosphorylation state of hundreds to thousands of proteins across multiple conditions. However, little is known about the function of many of these protein modifications, or the enzymes responsible for modifying them. To address this challenge, we have developed an approach that enhances the power of clustering techniques to infer functional and regulatory meaning of protein states in cell signaling networks. We have created a new computational framework for applying clustering to biological data in order to overcome the typical dependence on specific a priori assumptions and expert knowledge concerning the technical aspects of clustering. Multiple clustering analysis methodology (‘MCAM’) employs an array of diverse data transformations, distance metrics, set sizes, and clustering algorithms, in a combinatorial fashion, to create a suite of clustering sets. These sets are then evaluated based on their ability to produce biological insights through statistical enrichment of metadata relating to knowledge concerning protein functions, kinase substrates, and sequence motifs. We applied MCAM to a set of dynamic phosphorylation measurements of the ERRB network to explore the relationships between algorithmic parameters and the biological meaning that could be inferred and report on interesting biological predictions. Further, we applied MCAM to multiple phosphoproteomic datasets for the ERBB network, which allowed us to compare independent and incomplete overlapping measurements of phosphorylation sites in the network. We report specific and global differences of the ERBB network stimulated with different ligands and with changes in HER2 expression. Overall, we offer MCAM as a broadly-applicable approach for analysis of proteomic data which may help increase the current understanding of molecular networks in a variety of biological problems.National Institutes of Health (U.S.) (NIH-U54-CA112967 )National Institutes of Health (U.S.) (NIH-R01-CA096504

Multiplexed phosphospecific flow cytometry enables large-scale signaling profiling and drug screening in blood platelets

Background; Dissecting the signaling events that contribute to platelet activation will increase our understanding of platelet function and aid in the development of new antiplatelet agents. However, high-throughput methodology for the quantitative analysis of platelet signaling events is still lacking. Objective; To develop a high-throughput assay for the analysis of platelet signaling events in whole blood. Methods and Results; We developed a fluorescent barcoding protocol to facilitate multiplexing and enable large-scale signaling profiling in platelets in whole blood. The methodology allowed simultaneous staining and acquisition of 24–96 samples in a single analysis tube with a standard flow cytometer. This approach significantly reduced experimental numbers, data acquisition time, and antibody consumption, while providing automated statistically rich quantitative data on signaling events. Using vasodilator-stimulated phosphoprotein (VASP), an established marker of platelet inhibition and antiplatelet drug therapy, we demonstrated that the assay could detect subtle changes in phosphoVASP-Ser157/239 in response to cAMP-elevating agents of varying potency and known modulators of the cAMP signaling cascade. The assay could be used with washed platelets or whole blood, analyzed immediately or frozen, without any significant change in assay performance. To demonstrate the usefulness of the assay as a drug discovery platform, we examined a prostaglandin screening library. Our screen of 70 prostaglandin derivatives revealed three previously uncharacterized lipids that stimulated phosphorylation of VASP-Ser157. Follow-up analyses demonstrated that these agents elevated intraplatelet cAMP and inhibited collagen-induced platelet aggregation. Conclusions;This novel method enables rapid, large-scale quantitative signalling profiling and compound screening in human platelets present in whole blood

Kinome analysis of receptor-induced phosphorylation in human natural killer cells.

BACKGROUND: Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation. RESULTS: A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. CONCLUSIONS: The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses