Stable Isotope Dynamic Labeling of Secretomes (SIDLS) Identifies Authentic Secretory Proteins Released by Cancer and Stromal Cells

Abstract

Analysis of secretomes critically underpins the capacity to understand the mechanisms determining interactions between cells and between cells and their environment. In the context of cancer cell micro-environments, the relevant interactions are recognized to be an important determinant of tumor progression. Global proteomic analyses of secretomes are often performed at a single time point and frequently identify both classical secreted proteins (possessing an N-terminal signal sequence), as well as many intracellular proteins, the release of which is of uncertain biological significance. Here, we describe a mass spectrometry-based method for stable isotope dynamic labeling of secretomes (SIDLS) that, by dynamic SILAC, discriminates the secretion kinetics of classical secretory proteins and intracellular proteins released from cancer and stromal cells in culture. SIDLS is a robust classifier of the different cellular origins of proteins within the secretome and should be broadly applicable to nonproliferating cells and cells grown in short term culture.

Novel sites of CCK2R expression

“…For Western blots and proteomic analysis, media were concentrated using StrataClean resin (Agilent Technologies) as describe previously (Holmberg et al 2012, Hammond et al 2018. To avoid protein degradation samples were handled on ice during the whole process.…”

“…Cells were further incubated for 6 h at 37°C at 5% CO2. Media were collected and processed as described before (Kristensen et al 2012, Hammond et al 2018. In brief, supernatants were centrifuged at 800x g for 4 min to remove any remaining cell debris.…”

“…Culture media of melanoma cells treated with gastrin for 24 and 6 hours were collected and concentrated using StrataClean resin as describe previously in section 2.13 (Holmberg et al 2012, Hammond et al 2018.…”

“…Putative gastrin targets in Skmel-2 and G361 melanoma secretomes were identified using Stable Isotope Dynamic Labelling of Secretomes (SIDLS) technique previously described in section 2.14 (Kristensen et al 2012, Hammond et al 2018. Briefly, cells were incubated with hG17 (10 nmol/L) for 24 h, the last 6h containing either 12 C6 lysine (light label), or 13 C6 lysine (heavy label) to mark secreted proteins.…”

“…Given the fact that melanoma cells express a functional CCK2 receptor (Mathieu et al 2005), and that in vitro studies revealed increased invasion and migration of melanoma cells as a response to gastrin stimuli (see Chapter 5), we collected conditioned media for a proteomic analysis to identify proteins potentially involved in mediating gastrin regulated cancer progression. We applied a method based on Stable Isotope Dynamic Labelling of Secretomes (SIDLS) as described previously by Hammond et al to specifically profile secretome proteins by taking advantage of the kinetic differences between classically secreted proteins and intracellular ones (Kristensen et al 2012, Hammond et al 2018.…”