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Secretome analysis to elucidate metalloprotease-dependent ectodomain shedding of glycoproteins during neuronal cells differentiation

ABSTRACT: N1E-115 cells were seeded two days before sample preparation, and cultured in fresh medium containing 2% fetal bovine serum for 12 h before sample preparation. Cells were washed with serum-free medium for three times, and cultured in fresh serum-free DMEM with or without 50 μM GM6001 for 9 h. The conditioned media were collected and centrifuged to remove cell debris, and concentrated up to ~45-fold. Concentrated media were acidified by acetate acidic buffer, and oxidized by NaIO4. Proteins were precipitated with methanol and chloroform, reduced, alkylated, and digested by trypsin. Digested peptides were purified using C18 column, concentrated, diluted with coupling buffer (Biorad), and incubated with Aff-Gel Hydrazide Gel (Biorad). N-glycosylated peptides were eluted and labeled with 18O by treatment with PNGase F (NEB). Eluted peptides were labeled with CH2O (light, DMSO-treated samples) or 13CD2O (heavy, GM6001-treated samples). Each sample was divided into two parts and subjected to LC-MS/MS analysis respectively.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Atsuko Sehara-Fujisawa

PROVIDER: PXD004699 | JPOST Repository | Sun Jan 15 00:00:00 GMT 2017

REPOSITORIES: jPOST

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Secretome analysis to elucidate metalloprotease-dependent ectodomain shedding of glycoproteins during neuronal differentiation.

Tsumagari Kazuya K Shirakabe Kyoko K Ogura Mayu M Sato Fuminori F Ishihama Yasushi Y Sehara-Fujisawa Atsuko A

Genes to cells : devoted to molecular & cellular mechanisms 20170113 2

Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, processes referred to as ectodomain shedding. Shedding ectodomains of membrane-bound ligands results in activation of downstream signaling pathways, whereas shedding those of cell adhesion molecules causes loss of cell-cell contacts. Secreted proteomics (secretomics) using high-resolution mass spectrometry would be strong tools for both comprehensive identification and quantitative measurement of membrane ...[more]

PMID: 28084684

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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.

			Action	DRS
	150406_kt_CSC1.maxq.mgf	Mgf
	150406_kt_CSC1.raw	Raw
	150406_kt_CSC1.wizd.mgf	Mgf
	150406_kt_CSC2.maxq.mgf	Mgf
	150406_kt_CSC2.raw	Raw