SILAC-Based Quantification of TGFBR2-Regulated Protein Expression in Extracellular Vesicles of Microsatellite Unstable Colorectal Cancers

Abstract

Microsatellite unstable (MSI) colorectal cancers (CRCs) are characterized by mutational inactivation of Transforming Growth Factor Beta Receptor Type 2 (TGFBR2). TGFBR2-deficient CRCs present altered target gene and protein expression. Such cellular alterations modulate the content of CRC-derived extracellular vesicles (EVs). EVs function as couriers of proteins, nucleic acids, and lipids in intercellular communication. At a qualitative level, we have previously shown that TGFBR2 deficiency causes overall alterations in the EV protein content. To deepen the basic understanding of altered protein dynamics, this work aimed to determine TGFBR2-dependent EV protein signatures in a quantitative manner. Using a stable isotope labeling with amino acids in cell culture (SILAC) approach for mass spectrometry-based quantification, 48 TGFBR2-regulated proteins were identified in MSI CRC-derived EVs. Overall, TGFBR2 deficiency caused upregulation of several EV proteins related to the extracellular matrix and nucleosome as well as downregulation of proteasome-associated proteins. The present study emphasizes the general overlap of proteins between EVs and their parental CRC cells but also highlights the impact of TGFBR2 deficiency on EV protein composition. From a clinical perspective, TGFBR2-regulated quantitative differences of protein expression in EVs might nominate novel biomarkers for liquid biopsy-based MSI typing in the future.

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

“…Similarly, iTRAQ labelling was also used to quantify human seminal EV proteins [ 138 ]. A stable isotope labelling with amino acids in cell culture (SILAC) combined with high-resolution MS was also applied to perform a comprehensive analysis of proteins in microsatellite unstable colorectal cancer-derived EVs [ 139 ]. Moreover, a PROMIS-Quan (PROteomics of MIcroparticles using Super-SILAC Quantification), which is based on SILAC quantification, was described by Harel et al for valid plasma microparticle extraction, followed by SILAC-based relative and absolute quantification [ 140 ].…”

Transforming Growth Factor-β Signaling Pathway in Colorectal Cancer and Its Tumor Microenvironment

“…TGFBR2 mutation in CRC can also cause changes in the components secreted by cancer cells. For example, in vitro experiments with MSI-H TGFBR2-deficient HCT116 cells revealed that extracellular matrix and nucleosome-related proteins were upregulated, while proteasome-associated proteins in the extracellular vesicles were downregulated [52,53]. Clinical implications of these cell line-based experiments are, however, not fully understood.…”

(Phospho)proteomic Profiling of Microsatellite Unstable CRC Cells Reveals Alterations in Nuclear Signaling and Cholesterol Metabolism Caused by Frameshift Mutation of NMD Regulator UPF3A

“…Peptides from tryptic digestion were separated using the Dionex UltiMate 3000 nanoUPLC system as described before [ 72 ]. Peptides were trapped on an Acclaim Pepmap 100 column (100 μm × 20 mm, particle size 5 μm).…”

“…Peptides after phosphopeptide enrichment and peptides for incorporation analysis were analyzed by a linear ion trap quadrupole LTQ Orbitrap-XL mass spectrometer (Thermo Fisher Scientific) coupled to a nanoAcquity ultra high-performance liquid chromatography (UPLC) system (Waters) as described in [ 64 , 72 ]. Peptides were separated on a BEH C18 (100 μm × 100 mm, particle size 1.7 µm), analytical column at a constant flow of 0.4 μL/min using a 3h stepped linear gradient of solvent C (98.9% water, 1% acetonitrile, 0.1% formic acid) and solvent D (99.9% acetonitrile and 0.1% formic acid) in the following sequence from 0 to 4% D in 1 min, from 4 to 25% D in 139 min, from 25 to 40% D in 15 min, from 40 to 85% D in 10 min, 5 min at 85% D, from 85 to 4% D in 2 min, and 15 min at 4% D. The Orbitrap was operated with the following parameters: ESI voltage 2000 V, capillary temperature 200 °C, normalized collision energy 35 V. Data were acquired using XCalibur (version 2.0.7; Thermo Fisher Scientific) by scan cycles of one FTMS scan with a resolution of 60,000 at m/z 400 and a range from 300 to 2000 m/z in parallel with six MS/MS scans in the ion trap of the most abundant precursor ions.…”