Stoichiometric Quantification of Akt Phosphorylation Using LC-MS/MS

The Ptdlns-3-kinase (PI3-K) signaling pathway plays a vital role in cell survival, proliferation, apoptosis and differentiation in normal cells, as well as in diseases such as cancer and diabetes. Quantification of phospho-Akt is a standard way of assessing the activity of the PI3-K signaling pathway in both cells and tumors. This measurement is traditionally performed semiquantitatively using immunoassays such as Western blot. Here we report an LC-MS method to accurately measure the stoichiometry of Akt phosphorylation in biological samples. The procedure includes immunoprecipitation, gel electrophoresis, in-gel digestion, addition of isotopicaly labeled internal standards and LC-MS/MS. Two proteolytic enzymes, chymotrypsin and trypsin, were used to generate suitable peptide fragments for measuring Thr308 and Ser473 phosphorylation, respectively. The interday imprecision was estimated to be 3.8% and 2.3% for Thr308 and Ser473, respectively. This method has been tested on human T-cells grown in presence and absence of pervanadate and with or without a PI3-K inhibitor and on human glioblastoma cells (U-87 MG) grown in presence and absence of wortmannin (PI3-K inhibitor).The results of T cells suggest that the levels of Akt phosphorylation in untreated cells were below 1% for both phosphorylation sites. Pervanadate treatment provoked an 18-fold increase in phosphorylation of Thr308 and the PI3-K inhibitor partially reversed the increase. A comparison between LC-MS/MS and Western blotting suggests that the LC-MS based method is of comparable sensitivity and provides a more accurate phosphorylation stoichiometry, a wider dynamic range and more in-depth information. The application of the new method and its utility to providing predictive markers of response to targeted therapies is discussed

Hierarchical clustering of canine and human datasets using exclusively the expression levels of the NF-κB target genes (probesets).

<p>Hierarchical clustering of the canine (A) and human (B) datasets using exclusively the expression levels of the NF-κB target gene set. The samples are arranged in the columns (blue squares denote healthy and red squares denote DLBCL) and the probesets are in the rows. The dendrograms are drawn using Euclidean distances with average linkage method. (A) In the canine dataset (GSE30881), the 199 NF-κB target probesets separate the dataset into three top-level clusters. While the first and the third clusters have exclusively of DLBCL samples, the second cluster has all the healthy samples with two DLBCL samples. (B) In the human dataset (GSE12195), the 259 NF-κB target probesets separate the dataset into two top-level clusters. The first cluster has 12 samples that include all the healthy samples and two DLBCL samples, while the second cluster is solely of 43 DLBCL samples. The numbers above each column refer to sample identification numbers.</p

Comparison of enrichment of KEGG pathways in the co-expressed clusters of canine DLBCL and human DLBCL.

<p>The highly connected gene clusters identified in the co-expression networks of canine DLBCL and human DLBCL were analysed for enrichment of KEGG pathways using DAVID functional annotation tool. The results from the analysis of each cluster are compiled and the <i>p-values</i> of the enrichment score computed by Fisher's exact test are represented graphically as coloured icons.</p

The effect of doxorubicin and IKK inhibitor VII on the activation of NF-κB in human B cell lymphoma cell lines and canine B cell lymphoma cell lines.

<p>Nuclear protein from human B (RL, JM1, Pfeiffer) cell lymphoma lines as well as canine B (3132) cell lymphoma line treated with doxorubicin (A) and/or IKK inhibitor VII (B), were extracted. EMSAs were performed on these samples using non-radioactive DIG-labelled NF-κB consensus oligonucleotide probes in binding reactions. Samples were then subjected to electrophoresis in DNA retardation gels, before transfer onto nylon membranes and chemiluminescence detection of the DIG-labels. Bands are indicated by arrows and annotated. ‘+’ and ‘−’ refer to components being added or omitted respectively, to standard gel shift binding reactions. Expression of classical pathway NF-κB subunits in 3132 nuclear extracts when 3132 cells are treated with doxorubicin and/or IKK inhibitor VII at IC₅₀ doses was detected by western blotting of whole cell lysates (B).</p

NF-κB target genes in the differentially expressed gene set of the canine DLBCL.

<p>NF-κB target genes in the differentially expressed gene set of the canine DLBCL.</p

Comparison of differentially expressed probesets in canine and human DLBCLs.

<p>Venn diagrams comparing the NF-κB target genes in the differentially expressed probesets (between DLBCL and healthy) of canine DLBCL and human DLBCL and comparison of the number of the differentially expressed probesets of canine DLBCL and human DLBCL. The differentially expressed probesets in the datasets were identified by one-way ANOVA (DLBCL Vs. healthy) of the expression values; selecting probesets with log₂ fold change over 2 with FDR adjusted <i>p-value</i> less than 0.05. (A) 25 NF-κB target probsets (17 NF-κB target genes out of the 120 genes) present in the differentially expressed probesets of canine DLBCL. (B) 101 NF-κB target probsets (54 NF-κB target genes out of the 120 genes) present in the differentially expressed probesets of human DLBCL. (C) Comparison of canine array probesets converted to orthologous human array probesets. (D) Comparison of human array probesets converted to orthologous canine array probesets.</p

In vivo and in vitro Validation of NF-κB Status in Canine and Human DLBCL.

<p>NF-κB expression and activation in human T and B cell lymphoma cell lines and canine B cell lymphoma cell line. Whole cell lysates from Jurkat, JM1, Pfeiffer, RL and 3132 cells were eletrophoresed and immunoblotted for the detection of expression of signaling components of the NF-κB pathway (A). Nuclear protein from human T (Jurkat) and B (RL, JM1, Pfeiffer) cell lymphoma lines as well as canine B (3132) cell lymphoma line, were extracted. EMSAs were performed on these samples using non-radioactive DIG-labeled NF-κB consensus oligonucleotide probes in binding reactions. Specificity was tested by competition using unlabeled (cold) specific and non-specific probes and “supershift” using specific antibody for NF-κB p65/RelA and a non-specific antibody (p53). Samples were then subjected to electrophoresis in DNA retardation gels, before transfer onto nylon membranes and chemiluminescence detection of the DIG-labels. EMSAs were performed using nuclear extracts of canine (B) and human (C) lymphoma cell lines. Bands are indicated by arrows and annotated. ‘+’ and ‘−‘ refer to components being added or omitted respectively, to standard gel shift binding reactions.</p

Top 20 Gene Ontology (BP) Enrichment in the differentially expressed probesets in canine DLBCL and human DLBCL (ranking based on FDR in the DAVID functional annotation chart).

<p>Top 20 Gene Ontology (BP) Enrichment in the differentially expressed probesets in canine DLBCL and human DLBCL (ranking based on FDR in the DAVID functional annotation chart).</p

Illustrative photomicrographs showing different nuclear staining intensities for p65 and p52.

<p>p65 staining in human DLBCL; weak (a), strong (b). p65 staining in canine DLBCL weak (c), strong (d). p52 staining in human DLBCL; weak (e), strong (f). p52 staining in canine DLBCL; weak (g), strong (h).</p

PCA of canine and human datasets using exclusively the expression levels of the NF-κB target genes (probesets).

<p>Three dimensional plots of the PCA of canine and human datasets using the first three principal components of the expression levels of the NF-κB target genes (probesets) show clear separation of DLBCL samples from the healthy samples in both the datasets. Blue spheres denote the healthy samples and red spheres denote the DLBCL samples. The ellipsoids drawn around the clusters mark the limit of the 2 standard deviations from the centre in 3-dimensional space. (A) In the canine dataset (GSE30881), the amounts of variance captured by the first three principal components are 23.6%, 14% and 8.52% respectively. (B) In the human dataset (GSE12195), the amounts of variance captured by the first three principal components are 31.6%, 7.87% and 6.54% respectively.</p