Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome

Shin, Jihye; Kwon, Yumi; Lee, Seonjeong; Na, Seungjin; Hong, Eun Young; Ju, Shinyeong; Jung, Hyun Gyo; Kaushal, Prashant; Shin, Seung Sook; Back, Ji Hyun; Choi, Seon Young; Kim, Eun Hee; Lee, Su Jin; Park, Yae Eun; Ahn, Hee‐Sung; Ahn, Younghee; Kabir, Mohammad Humayun; Park, Seong Jun; Yang, Won-Mo; Yeom, Jeonghun; Bang, Oh Young; Ha, Chul‐Won; Lee, Jinwon; Kang, Un Beom; Kim, Hye Jung; Park, Kang Sik; Lee, J. Eugene; Lee, Ji Eun; Kim, Jin Young; Kim, Kwang Pyo; Kim, Youngsoo; Hirano, Hisashi; Yi, Eugene C.; Cho, Je Yoel; Paek, Eunok; Lee, Cheolju

doi:10.1021/acs.jproteome.9b00475

Cited by 23 publications

(19 citation statements)

References 17 publications

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“…LC–MS/MS was performed, as previously described 66 . Peptides were reconstituted in 0.4% acetic acid and analyzed on a reversed-phase C18 column (20 cm × 75 μm i.d., 3 μm, 300 Å, packed in-house; Dr. Maisch GmbH) using an Eksigent MDLC system at a flow rate of 300 nL/min.…”

Section: Methodsmentioning

confidence: 99%

Multi-layered proteogenomic analysis unravels cancer metastasis directed by MMP-2 and focal adhesion kinase signaling

Kwon

Park

Nguyen

et al. 2021

Sci Rep

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The role of matrix metalloproteinase-2 (MMP-2) in tumor cell migration has been widely studied, however, the characteristics and effects of MMP-2 in clinical sample of metastatic colorectal cancer (CRC) remain poorly understood. Here, in order to unveil the perturbed proteomic signal during MMP-2 induced cancer progression, we analyzed plasma proteome of CRC patients according to disease progression, HCT116 cancer secretome upon MMP-2 knockdown, and publicly available CRC tissue proteome data. Collectively, the integrative analysis of multi-layered proteomes revealed that a protein cluster containing EMT (Epithelial-to-Mesenchymal Transition)-associated proteins such as CD9-integrin as well as MMP-2. The proteins of the cluster were regulated by MMP-2 perturbation and exhibited significantly increased expressions in tissue and plasma as disease progressed from TNM (Tumor, Node, and Metastasis) stage I to II. Furthermore, we also identified a plausible association between MMP-2 up-regulation and activation of focal adhesion kinase signaling in the proteogenomic analysis of CRC patient tissues. Based on these comparative and integrative analyses, we suggest that the high invasiveness in the metastatic CRC resulted from increased secretion of MMP-2 and CD9-integrin complex mediated by FAK signaling activation.

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Section: Methodsmentioning

confidence: 99%

Multi-layered proteogenomic analysis unravels cancer metastasis directed by MMP-2 and focal adhesion kinase signaling

Kwon

Park

Nguyen

et al. 2021

Sci Rep

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“…In addition to supporting efforts by the ISEV, we suggest two additional steps for reporting in vitro‐derived EV data. With the recent publication of the common repository of FBS proteins, we advocate for investigators to confirm that the putative proteins identified from isolated in vitro‐derived EVs be cross‐referenced with this database, and whenever possible, to provide additional quantitative measures of relative abundance (Shin et al., 2019). Widespread participation will ensure a more accurate interrogation of the cell‐derived EV proteome.…”

Section: Potential Solutions To Refining Culture Protocolsmentioning

confidence: 99%

“…In addition to supporting efforts by the ISEV, we suggest two additional steps for reporting in vitro-derived EV data. With the recent publication of the common repository of FBS proteins, we advocate for investigators to confirm that the putative proteins identified from isolated in vitro-derived EVs be cross-referenced with this database, and whenever possible, to provide (Wei et al, 2016;Turchinovich et al, 2011) No for basal medium, but Yes for chemically-supplemented medium (Auber et al, 2019) Cell physiology Cell lines Affected (Eitan et al, 2015) Affected, but may be adapted (Lee et al, 2019;Li et al, 2015) Stem/primary cells Affected (Liao et al, 2017;Driedonks et al, 2019;Tosar et al, 2017) Affected, may need addition of growth factors (Vallabhaneni et al, 2015;Zhu et al, 2006) EV release May be affected (Wei et al, 2016;Driedonks et al, 2019) Affected (Gudbergsson et al, 2016;Li et al, 2015;Sun et al, 2014), yet may be cell-type dependent (De Jong et al, 2012) Cell-derived EV Profile Need to determine Affected (Gudbergsson et al, 2016;Sun et al, 2014), yet may be adapted with consistency and without undermining therapeutics (Gimona et al, 2017) Cost Cost usually high when preparing or purchasing EV-depleted FBS Less when using basal medium, and may increase when using chemically-supplemented medium additional quantitative measures of relative abundance (Shin et al, 2019). Widespread participation will ensure a more accurate interrogation of the cell-derived EV proteome.…”

Section:  Potential Solutions To Refining Culture Protocolsmentioning

confidence: 99%

Foetal bovine serum influence on in vitro extracellular vesicle analyses

Lehrich

Liang

Fiandaca

2021

J of Extracellular Vesicle

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Foetal bovine serum influence on in vitro extracellular vesicle analyses  INTRODUCTION Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles most notably from either endosomal (i.e., exosomes) or plasma membrane origins (i.e., microvesicles/ectosomes) and released from nearly all mammalian cells (Colombo et al., 2014). An interest in EV research has increased over the past decade, in part due to their participation in complex intercellular communication (Roy et al., 2018). Though EVs are abundant in blood and other biofluids, the investigation of in vitro-derived EVs provides a critical tool for understanding various mechanisms associated with their biogenesis, molecular composition, packaging of specific payloads, and cellular trafficking. Once released, EVs traffic to target cells where they may be taken up to release their payloads via specific mechanisms, and thereby exert their physiological influence (Colombo et al., 2014; Kowal et al., 2014). Although engineered micelles and liposomes have previously been utilized as lipid nanocarriers (Fiandaca & S., 2013; Fiandaca et al., 2011) for many therapeutic applications, EVs have garnered recent interest as drug delivery vehicles (Elsharkasy et al., 2020). Currently, there exist vastly heterogeneous cell culture conditions for EV production and isolation (Consortium, 2017). Therefore, there is a current need to define more standard cell culture conditions for investigating EVs that may accelerate the translation of therapeutic clinical-grade EVs (Lener et al., 2015; Lötvall et al., 2014; Théry et al., 2018). Herein, we present a mini-review on recent investigations reporting the influence of foetal bovine serum (FBS)-supplemented media formulations on cultured cell physiology, EV production/release, and its contaminating presence of vesicular and non-vesicular particles. Additionally, we describe potential solutions and provide recommendations to aid in vitro EV investigators.  CELL CULTURE CONDITIONS FOR EV INVESTIGATIONS: SERUM USAGE AND CONCERNS An international survey observed 83% of International Society for Extracellular Vesicles (ISEV) respondents utilize conditioned cell culture media as their starting material (Gardiner et al., 2016). FBS is a common additive in cell culture and 52% of ISEV respondents utilize serum-containing media for downstream EV analyses, with 59% and 57% of those respondents performing in vitro and in vivo functional studies, respectively (Gardiner et al., 2016). Serum usage, in part due to its ill-defined composition, provides a variety of contaminating particles (e.g., EVs, lipoproteins, and protein complexes, which differ in their physical properties, yet also have similar size, density, and/or RNA components) that confound these investigative results.  FBS SUPPLEMENTATION AND GENERAL CONCERNS The growth factors and other constituents within FBS appear to provide a nourishing ecosystem for many cultured cells (Bettger & Mckeehan, 1986). Despite this nourishing milieu, the presence of FBS in culture has raised spec...

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“…We note that 22% (17/75) of the enriched proteins in SG2 can be contaminants from the FBS which would be expected because in late‐eluting fractions the proportion of soluble proteins should increase. Some of these proteins include lactotransferrin (LTF), inter‐alpha‐trypsin inhibitor heavy chain H2 (ITIH2), antithrombin‐III (SERPINC1), complement C4‐A (C4‐A), and fibulin‐1 (FBLN1) (Shin et al., 2019). Despite these putative impurities, our results suggest that EVs in SG2 constitute a mixture of small vesicles that maintain their functional capabilities as corroborated in the uptake experiments.…”

Section: Discussionmentioning

confidence: 99%

Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer‐based precipitation and size exclusion chromatography

Martínez‐Greene

Hernández‐Ortega²,

Quiroz-Báez

et al. 2021

J of Extracellular Vesicle

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The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer‐based precipitation and size exclusion chromatography (Pre‐SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early‐ and late‐eluting EV fractions, we performed a quantitative proteomic analysis of MDA‐MB‐468‐derived EVs. We identified 286 exclusive proteins in early‐eluting fractions and 148 proteins with a differential concentration between early‐ and late‐eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies.

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Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome

Cited by 23 publications

References 17 publications

Multi-layered proteogenomic analysis unravels cancer metastasis directed by MMP-2 and focal adhesion kinase signaling

Multi-layered proteogenomic analysis unravels cancer metastasis directed by MMP-2 and focal adhesion kinase signaling

Foetal bovine serum influence on in vitro extracellular vesicle analyses

Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer‐based precipitation and size exclusion chromatography

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