Complementary PTM Profiling of Drug Response in Human Gastric Carcinoma by Immunoaffinity and IMAC Methods with Total Proteome Analysis

Abstract

Gaining insight into normal cellular signaling and disease biology is a critical goal of proteomic analyses. The ability to perform these studies successfully to extract the maximum value and discovery of biologically relevant candidate biomarkers is therefore of primary importance. Many successful studies in the past have focused on total proteome analysis (changes at the protein level) combined with phosphorylation analysis by metal affinity enrichment (changes at the PTM level). Here, we use the gastric carcinoma cell line MKN-45 treated with the c-Met inhibitor SU11274 and PKC inhibitor staurosporine to investigate the most efficient and most comprehensive strategies for both total protein and PTM analysis. Under the conditions used, total protein analysis yielded few changes in response to either compound, while analysis of phosphorylation identified thousands of sites that changed differentially between the two treatments. Both metal affinity and antibody-based enrichments were used to assess phosphopeptide changes, and the data generated by the two methods was largely complementary (non-overlapping). Label-free quantitation of peptide peak abundances was used to accurately determine fold-changes between control and treated samples. Protein interaction network analysis allowed the data to be placed in a biologically relevant context, and follow-up validation of selected findings confirmed the accuracy of the proteomic data. Together, this study provides a framework for start-to-finish proteomic analysis of any experimental system under investigation to maximize the value of the proteomic study and yield the best chance for uncovering actionable target candidates.

Affinity-Bead Assisted Mass Spectrometry (Affi-BAMS): A Multiplexed Microarray Platform for Targeted Proteomics

“…Consistent with previous reports, upon ST treatment we observed a decrease in phosphorylation with phospho-C-MET (pY1234 and pY1235) and a more significant decrease upon SU treatment. Total C-MET levels, in contrast, were consistent throughout the control and treatment conditions (as observed by Stokes and coworkers) [18]. After confirming consistency between control Western blots and the previous work, we combined protein lysates from the light and heavy SILAC pairs (1:1, by total protein mass) and prepared the material for Affi-BAMS assay to monitor phospho-C-MET (pY1234 and pY1235) levels in response to treatment with ST or SU inhibition.…”

“…As SIS peptides may not always be readily customized to perform quantitative experiments on targets of interest for a highly multiplexed Affi-BAMS assays using a cell line model, we demonstrate that a classical light and heavy Stable Isotope Labeling by/with Amino Acids in Cell Culture (SILAC) experiment is well suited for quantitative Affi-BAMS analyses [45]. To demonstrate this application, we cultured MKN45 (a C-MET driven cancer cell line) under light and heavy SILAC conditions (light = kinase inhibitor, heavy = DMSO; K+8 and R+10) and treated cells with either 0.2 µM Staurosporine (ST, protein kinase C [PKC] inhibitor) or 1.0 µM SU11274 (SU, C-MET inhibitor) for 2 h (see Methods) as described by Stokes and colleagues [18]. We performed Western blots for both total C-MET and phospho-C-MET (pY1234 and pY1235) to confirm inhibition by both kinase inhibitors prior to performing subsequent Affi-BAMS assays ( Figure 7A,B).…”

“…LC-MS/MS can additionally require relatively large amounts of starting sample material to identify and accurately quantify low abundant protein variants (e.g., specific sites of phosphorylation, acetylation, methylation, point mutations). Furthermore, sample preparation workflows can require multi-dimensional separation strategies and/or targeted depletions [18][19][20][21], while subsequent bioinformatic analysis can require deconstruction of chimeric spectra [22] to effectively quantify specific proteins of interest.…”

Proteomic profiling of FBXW7‐mutant serous endometrial cancer cells reveals upregulation of PADI2, a potential therapeutic target

“…Cellular extracts were sonicated, centrifuged, reduced with dithiothreitol, and alkylated with iodoacetamide. For phosphorylated protein analysis, 500 μg total protein was digested with trypsin, purified over C18 columns (Waters), and used for immobilized metal affinity chromatography (IMAC) enrichment with Fe‐NTA magnetic beads (CST ® ) as previously described 11 . For total protein analysis, 100 μg of each sample was digested with LysC/trypsin, labeled with TMT10plex TM reagent (Thermo Fisher Scientific), bRP fractionated (96 fractions concatenated nonsequentially to 12), and C18 purified as previously described 12 .…”

Sirtuin 5 is required for mouse survival in response to cardiac pressure overload

“…Samples were analyzed using the PTMScan method as described previously (49,50). Cellular extracts were prepared in urea lysis buffer, sonicated, centrifuged, reduced with DTT, and alkylated with iodoacetamide.…”