Abstract A67: An LC-MRM method to simultaneously measure abundance and activation of Src family tyrosine kinases in a B cell line model of IL-6 signaling
Authors: Aliancy Joel , Meads Mark B. , Wood Elizabeth , Chen Yi , Koomen John M. , Dalton William S. , Shain Kenneth H. 
Published: December 2010

Abstract
Introduction: Interleukin-6 (IL-6) is a pleiotropic cytokine important to tumor biology in epithelial and hematopoietic malignancies. IL-6 signaling through gp130 mediates cell survival, proliferation, and migration via multiple downstream signaling pathways. Preliminary data has demonstrated that point mutations within a discrete acidic region of the cytoplasmic domain of gp130 attenuate both IL-6-mediated Src family tyrosine kinase (SFK) activation and cell migration in a pro-B cell line. These data suggest that SFK activation via this domain is integral in modulating the adherent potential and migratory properties of IL-6 stimulated B cells. Importantly, the role of SFKs in IL-6-mediated cell migration has not been described previously. Nine SFKs are differentially expressed in various tissues; seven are specific to hematopoietic cells (Src, Fyn, Lyn, Hck, Lck, Blk, and Fgr). Identification of the relevant SFKs in this model will enable us to fully characterize the role of these effectors in relevant biological endpoints.
Methods: Currently, the measurement of specific SFK member expression and activation is limited by the ability of available antibodies to distinguish between highly homologous family members. Therefore, we are developing a mass spectrometry-based method that utilizes Liquid Chromatography-Multiple Reaction Monitoring, (LC-MRM) to provide simultaneous quantification of expression and post-translational modification of SFKs.
Results: Src family members have been screened using SDS-PAGE coupled to LC-MRM to identify specific peptides that can be used to monitor the expression of each individual protein (e.g. Src peptides containing amino acids 164-172 and 472-480: LLLNAENPR and EVLDQVER). The activation of each SFK will also be assessed using a ratio of tyrosine phosphorylated and unmodified peptides. Assays have already been developed to measure the following tryptic peptides containing a tyrosine phosphorylation site (underlined residue): LIEDNEYTAR (Src, Fyn, Yes, Lck) and VIEDNEYTAR (Lyn, Hck). Further, we have also identified a longer LysC peptide corresponding to amino acids 383-404 of Src (IADFGLARLIEDNEYTARQGAK) that is unique among each of seven SFKs expressed in hematopoietic cells. This peptide and the corresponding sequences in other SFKs may be used as an alternative method to simultaneously quantify tyrosine phosphorylation of individual SFKs.
Conclusions: This work will establish a new LC-MRM peptide based method to simultaneously measure both the activity and abundance of specific SFKs in very small sample sizes that are not amenable to analysis by other methods. As such, this technique will have a broad range of experimental applications and will be particularly useful in future studies that validate results from cell line models with patient specimens, which inherently contain a very low number of cells. This presenting student is supported by an ARRA supplement to the NCI Cancer Center Support Grant awarded to Moffitt (3P30 CA076292-11S6 PI WS Dalton) to create a training program for underrepresented undergraduate students in clinical proteomics, Project LINK (Leaders In New Knowledge-Emerging Technologies), that also emphasizes education in health disparities and community outreach.
Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A67.