Monitoring Human Milk β-Casein Phosphorylation and O-Glycosylation Over Lactation Reveals Distinct Differences between the Proteome and Endogenous Peptidome

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant’s best start at a healthy life. One key component of human milk is β-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk’s endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of β-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of β-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in β-casein’s known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of β-casein may be important as it resides on known β-casein-derived antimicrobial peptide sequences.

Site-selective recognition of peptide phosphorylation by a terbium(iii) complex in aqueous solution

A terbium(iii) complex performs, in a Zn(ii)-driven mode, site-selective recognition for proximal diphosphorylation of peptides in both buffer and protein extraction solutions.

Phosphorylation-regulated crosslinking of gold nanoparticles: a new strategy for colorimetric detection of protein kinase activity

A facile colorimetric protein kinase assay has been developed based on the peptide phosphorylation-tuned crosslinking and aggregation of gold nanoparticles.

Effect of IKZF1 Deletions on Signal Transduction Pathways in Philadelphia Chromosome-Negative B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)

Overall survival rates for children with Acute Lymphoblastic Leukemia (ALL), the most common type of leukemia in children, are approaching 90% (Mullighan 2013). In the past 5 years, genome wide approaches, studying DNA copy number alterations in ALL, have increased the list of risk stratifications and included IKZF1 deletions to the list of unfavorable prognostic factors. IKZF1 deletions can be identified in approximately 70% of the Philadelphia chromosome-positive (Ph+) and in 15% of the Philadelphia chromosome-negative (Ph-) children with ALL and are associated with an increased risk on relapse and a decreased overall survival (Mullighan 2009, Kuiper 2010, van der Veer 2013). IKZF1 deletions observed in B-cell precursor ALL (BCP-ALL) are typically mono-allelic, resulting in the expression of a dominant-negative isoform (Mullighan 2008). A unique gene expression signature was revealed in IKZF1 deleted BCP-ALL patients, characterized by the downregulation of genes regulating B-cell lineage development and DNA repair upon DNA damage response genes and upregulation of cell cycle/apoptosis genes, JAK/STAT signaling and stem cell self-renewal (Iacobucci 2012). At the level of signal transduction, western blot analysis showed that IKZF1 deletions resulted in B cell receptor (BCR) signaling defects and upregulation of phospho-STAT5 in 2 and 4 Ph+ ALL patients, respectively (Trageser 2009, Iacobucci 2012). However, effects of IKZF1 deletions on signaling pathways in Ph-ALL have not been extensively studied. Pediatric Ph- BCP-ALL patients (N=46) were screened for IKZF1 deletions by multiplex ligation-dependent probe amplification analysis. A total of 15 patients carried an IKZF1 deletion. We performed a kinase activity profile (IKZF1 deleted N=15, IKZF1 wild type N=31) as well as a human phospho-proteome array (IKZF1 deleted N=11, IKZF1 wild type N=17) to elucidate active signal transduction pathways. Kinase activity profiling is a potent high throughput technique using peptides of 11 amino acids in length representing known human phosphorylation sites. In the obtained kinase activity profiles we studied differences in peptide phosphorylation intensities. 37 peptides were differentially expressed between IKZF1 deleted and wild type pediatric Ph- BCP-ALL patients (P ≤ 0.05, Figure 1). From these 37 peptides we first examined peptides derived from proteins involved in the BCR signaling and STAT5. On the kinome array, peptides derived from Src_Y352, CBL_Y371, SYK_Y526, PLCg2_Y753, PLCg2_Y1217, STAT5a_S780, STAT5a_Y694, and STAT5b_Y679 were present but showed no differences in phosphorylation intensities between IKZF1 deleted and IKZF1 wild type Ph- BCP-ALL samples. Neither could we detect differences in phosphorylation intensities of Fyn_Y420, Lyn_Y397, Src_Y419, STAT5a_Y694, STAT5b_Y699, and STAT5a/b_Y694/Y699 using human phospho-proteome arrays, confirming the kinome profiling results. We did, however observe a distinct kinome profile upon hierarchical clustering of 46 BCP-ALL primary samples, based on the 37 peptides identified by t-test (Figure 1). IKZF1 deleted cases showed high phosphorylation of 14 peptides including peptides derived from Akt1_Y326 and Cav1_Y14 (Figure 1). Loss of IKZF1 has been associated with glucocorticoid resistance. Since Akt inhibition reverses glucocorticoid resistance in T cell ALL (Piovan, 2013) and Caveolin 1 is involved in focal adhesion and chemoresistance (Faggi, 2014) we hypothesize that Akt and Caveolin 1 inhibition might convert glucocorticoid resistance in Ph-IKZF1 deleted pediatric BCP-ALL, which requires further investigation. Together, we conclude that kinome profiling revealed a distinct peptide phosphorylation pattern for IKZF1 deleted pediatric Ph- BCP-ALL including novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Application of Kinase Activity Profiles to Predict Upcoming TKI Resistance In CML-Patients.

Abstract 3425 Background: Major breakthroughs in CML research have revolutionized therapy by the introduction of a specific inhibitor of BCR-ABL activity; first Imatinib and now even more potent TKIs like Nilotinib and Dasatinib are available. Imatinib can induce complete hematologic responses in virtually all patients and complete cytogenetic responses in almost 90% of patients and progression free survival in 84% of patients. Patients that reach a more than 1000-fold reduction of the BCR-ABL transcript level (major molecular response) have durable responses with not a single event of progression. Unfortunately, not all patients attain such a favourable response. Primary and secondary resistance develops in around 20% of cases.Predicting the development this resistance would be of great clinical value. Aim: To establish a classifier based on peptide phosphorylation patterns as fingerprints using samples of peripheral blood cells or bone marrow of CML patients undergoing Imatinib treatment in order to enable prediction of emerging resistance against Imatinib. Methods: Stored (“snapfrozen”) mononuclear cell fractions, isolated by Ficoll-Paque centrifugation of bone marrow or peripheral blood of CML patients under continuous Imatinib treatment and in various stages of their disease were lysed in M-PER buffer supplemented with phosphatase and protease inhibitors. Kinase activity profiles of these lysates were generated with standard Tyrosine Kinase PamChip® Arrays that contain 144 peptides as kinase substrates on their porous surface. Peptide phosphorylation was followed through binding of a fluorescently labelled anti-phosphotyrosine antibody during flow-through cycling of the lysates. Activity profiles were generated with PamGene's BioNavigator software. The resulting data were analysed using the R-based package CMA (“Classification for MicroArrays”) that enables the survey and evaluation of most usual classification methods with double cross-validation procedures. Results: A distinct classifier for Imatinib response prediction could be derived for the bone marrow samples, collected at various intervals after diagnosis (18 Imatinib sensitive versus 19 Imatinib resistant patients, of which the samples were collected 3 months to 1.5 year before resistance emergence). Of the twenty-one classification methods that were studied the SupportVectorMachine (SVM) algorithm resulted in the smallest error rate: this was 16% with a standard error of 0.062 and a sensitivity of 84% and specificity of 82% respectively, meaning 3 misclassified sensitive patients and 3 misclassified resistant patients. This classifier has to be validated in a blinded, independent test set. Conclusions: We demonstrated that with this method it is possible to predict Imatinib resistance. Differences in phosphorylation patterns as detected using PamGene's peptide microarray technology with the aid of multivariate statistical analysis suggest the presence of an ongoing process in CML-patients destined in due time to a relapse in spite of continuous Imatinib treatment. We regard these initial class prediction results to be a basis for further development of this kinase activity based test for Imatinib response prediction in CML patients already at the time of diagnosis. Perspectives: Our approach of using kinase activity profiling for the prediction of Imatinib resistance, could equally well be applied to predict response to various other kinase inhibitors. These response predictions could be combined in the same test by adding these inhibitors in vitro and might direct the optimal drug selection at the time point of diagnosis. Disclosures: Boender: PamGene International BV: Employment. Ruijtenbeek:PamGene International BV: Employment. van den Berg:PamGene Inetrnational BV: Employment. de Wijn:PamGene International BV: Employment.