De Novo Proteome Analysis of Genetically Modified Tumor Cells By a Metabolic Labeling/Azide-alkyne Cycloaddition Approach

Abstract

Activin receptor type II (ACVR2) is a member of the transforming growth factor type II receptor family and controls cell growth and differentiation, thereby acting as a tumor suppressor. ACVR2 inactivation is known to drive colorectal tumorigenesis. We used an ACVR2-deficient microsatellite unstable colon cancer cell line (HCT116) to set up a novel experimental design for comprehensive analysis of proteomic changes associated with such functional loss of a tumor suppressor. To this end we combined two existing technologies. First, the ACVR2 gene was reconstituted in an ACVR2-deficient colorectal cancer (CRC) cell line by means of recombinase-mediated cassette exchange, resulting in the generation of an inducible expression system that allowed the regulation of ACVR2 gene expression in a doxycycline-dependent manner. Functional expression in the induced cells was explicitly proven. Second, we used the methionine analog azidohomoalanine for metabolic labeling of newly synthesized proteins in our cell line model. Labeled proteins were tagged with biotin via a Click-iT chemistry approach enabling specific extraction of labeled proteins by streptavidin-coated beads. Tryptic on-bead digestion of captured proteins and subsequent ultra-high-performance LC coupled to LTQ Orbitrap XL mass spectrometry identified 513 proteins, with 25 of them differentially expressed between ACVR2-deficient and -proficient cells. Among these, several candidates that had already been linked to colorectal cancer or were known to play a key role in cell growth or apoptosis control were identified, proving the utility of the presented experimental approach. In principle, this strategy can be adapted to analyze any gene of interest and its effect on the cellular de novo proteome. Molecular & Cellular

Reconstitution of TGFBR2-Mediated Signaling Causes Upregulation of GDF-15 in HCT116 Colorectal Cancer Cells

“…After click-it-mediated biotinylation, labelled proteins were captured by streptavidin-coated magnetic beads and analyzed by mass spectrometry. In order to account for proteins that show unspecific binding to the beads or proteins that reside a priori on the commercially available streptavidin-coated beads, the same experiment was performed in the absence of the labeling reagent (-AHA) [ 14 ]. At least 480 de novo synthesized proteins were detected on average in both cell clones ( S1 Table ) and under both conditions (-Dox: TGFBR2-deficient; +Dox: TGFBR2-proficient) upon stimulation with TGF-ß1 und hence remained unaffected by the TGFBR2 expression status ( Table 1 ).…”

“…After metabolic AHA labeling (C10102; Life Technologies, Karlsruhe, Germany), cell pellets were lysed with 100 μl of 1% SDS in 50 mM Tris-HCl, pH 8, supplemented with protease inhibitor cocktail, sonicated for 30 s and incubated at least 30 min at 4°C while rotating as described previously [ 14 ]. After centrifugation at 4°C for 30 min at 12,000 g, protein concentration was determined by Bradford Assay (Bio-Rad Laboratories GmbH, Munich, Germany).…”

“…For mass spectrometry, samples were prepared and analyzed as described previously [ 14 ]. Briefly, protein-loaded beads were reduced, alkylated and trypsinysed at 37°C overnight.…”

“…Using this HCT116-TGFBR2 model cell line in combination with a click chemistry approach for specific isolation of the de novo proteome [ 14 ] and subsequent mass spectrometry (MS) analysis, we identified a limited number of proteins that turned out to be newly expressed or whose synthesis was abolished upon TGFBR2 expression. One of these candidate targets, the Growth and differentiation factor-15 (GDF-15) showed a TGFBR2-dependent transcriptional upregulation that correlated with increased levels of intracellular and secreted protein.…”

Comprehensive Proteomic Characterization of the Human Colorectal Carcinoma Reveals Signature Proteins and Perturbed Pathways

“…This study reinforced the importance of measuring the protein abundance alteration in CRC. Although many studies have focused on measuring the protein changes associated with CRC789, a comprehensive characterization of the CRC proteome has not been accomplished. We thus analyzed proteomes of 44 samples (22 paired tumors and adjacent normal tissues) using a standardized quantitative proteomics workflow including pre-fractionation of protein samples by SDS-gel for maximizing the coverage, evaluation of the completeness of proteomic profiles using ten groups of well-defined “housekeeping” protein complexes for ensuring data quality, spectral counting-based quantification using the unit of parts per million (ppm) for describing the protein abundance, and a novel pathway analysis strategy for analyzing the biological consequence of the changes of the proteome.…”