Dataset Information

In-depth proteome analysis of Hela cell culture conditioned media containing fetal bovine serum.

ABSTRACT: We performed DIA-MS based proteomic analysis of Hela cells conditioned media containing fetal bovine serum by albumin (Alb)-depletion and non-depletion. In addition, we attempted to enrich secreted proteins by reducing the volume of culture medium (10mL, 5mL and 2mL). The supernatants of each culture condition were subjected to albumin depletion followed by DIA-MS. We further analyzed proteins in the conditioned media of HeLa cells with and without tumor necrosis factor (TNF) stimulation.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yusuke Kawashima

PROVIDER: PXD024272 | JPOST Repository | Tue Feb 22 00:00:00 GMT 2022

REPOSITORIES: jPOST

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Publications

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

Nakamura Ren R Nakajima Daisuke D Sato Hironori H Endo Yusuke Y Ohara Osamu O Kawashima Yusuke Y

International journal of molecular sciences 20210304 5

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 m ...[more]

PMID: 33806404

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Project description:Study question: Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? Summary answer: A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable. What is known already: There are no data in the literature on what non-declared proteins are present in un-conditioned (fresh media in which no embryos have been cultured) commercial embryo media. Study design, size, duration: The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analysed for each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analysed. Participants/materials, setting, methods: For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulphate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis. Main results and the role of chance: Using advanced mass spectrometry and high confidence criteria for accepting proteins (p< 0.01), a total of 110 proteins other than HSA were identified. The average serum albumin content was found to be 94% (92% - 97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins. Limitations, reasons for caution: For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions. Wider implications of the findings: The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analysing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in un-conditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring.

Dataset Information

In-depth proteome analysis of Hela cell culture conditioned media containing fetal bovine serum.

Publications

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

Similar Datasets

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