A Simple Method for In-Depth Proteome Analysis of Mammalian Cell Culture Conditioned Media Containing Fetal Bovine Serum

Abstract

A conditioned medium of a cell culture is widely used for various biological applications and frequently analyzed to characterize the functional proteins responsible for observed biological functions. However, a large number of abundant proteins in fetal bovine serum (FBS), usually included in the conditioned medium of a mammalian cell culture medium, hampers in-depth proteomic analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS). For a deep proteomic analysis of a conditioned medium by LC-MS/MS, we developed a simple albumin depletion approach coupled with data-independent acquisition (DIA)-mode LC-MS/MS for the conditioned medium of mammalian cells in this study. The results showed that this approach enabled the detection of more than 3700 cell-derived proteins in the cell culture supernatant containing FBS. We further demonstrated the potency of this approach by analyzing proteins in the conditioned media of HeLa cells with and without tumor necrosis factor (TNF) stimulation: >40 differentially accumulated proteins, including four cytokines, upon TNF stimulation were identified in the culture media, which were hardly detected by conventional proteome approaches in the literature.

A Mutant Methionyl-tRNA Synthetase-Based toolkit to assess induced-Mesenchymal Stromal Cell secretome in mixed-culture disease models

“…Another major advantage conferred by the approach described herein is the ability to separate MetRS L274G -expressing cell derived proteins from serum proteins. Highly abundant media components such as albumin and IgG can mask the less abundant, cell-secreted proteins when fetal bovine serum (FBS) or similar supplements are included in the media, but culturing cells in starved conditions is not optimal and is reported to result in a decrease in secreted proteins (29,30). While there are albumindepletion methodologies, such as incubation with Cibacron Blue (31), they are expensive, lack speci city, and typically result in the loss of additional protein beyond the undesired media components.…”

Optimization of Ultrafast Proteomics Using an LC-Quadrupole-Orbitrap Mass Spectrometer with Data-Independent Acquisition

“…Cell samples were prepared as described in a previous study. 17 In brief, precipitates were dissolved in 100 mM Tris-HCl (pH 8.5) containing 2% sodium dodecyl sulfate (SDS) using BIORUPTOR BR-II (SONIC BIO Co., Kanagawa, Japan) with settings at “High” and “30 s On/Off” cycle for a duration of 5 min. The extracted proteins were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) at 1000 ng/μL.…”

“…Protein purification and digestion were performed using the sample preparation (SP3) method. 17 , 18 The tryptic digestion was performed using 500 ng/μL Trypsin/Lys-C Mix (Promega, Madison, WI) overnight at 37 °C. Cell digests were purified using GL-Tip SDB (GL Sciences, Tokyo, Japan) according to the manufacturer’s protocol.…”

“…The extracts were reduced with 10 mM dithiothreitol for 30 min at 50 °C, followed by alkylation with 30 mM iodoacetamide for 30 min at 25 °C in the dark. Protein purification and digestion were performed using the sample preparation (SP3) method. , The tryptic digestion was performed using 500 ng/μL Trypsin/Lys-C Mix (Promega, Madison, WI) overnight at 37 °C. Cell digests were purified using GL-Tip SDB (GL Sciences, Tokyo, Japan) according to the manufacturer’s protocol.…”

Ultrastructural Assessment and Proteomic Analysis in Myofibrillogenesis in the Heart Primordium After Heartbeat Initiation in Rats

“…Sample preparation was performed as previously described with slight modifications ( Nakamura et al, 2021 ). The sample was precipitated in 4-fold acetone and incubated at −20°C for 2 h. After centrifugation at 15,000 g for 15 min at 4°C, the pellet was extracted in 100 mM Tris-HCL pH 8.5, 2% SDS, pH 8.5 by using a water bath-type sonicator (Bioruptor II, CosmoBio, Tokyo, Japan) on the high setting for 15 min.…”