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Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation.

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Title: Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation.
Authors: Riffault, Mathieu1, Moulin, David1, Grossin, Laurent1, Mainard, Didier1, Magdalou, Jacques1, Vincourt, Jean-Baptiste1 Jean-Baptiste.Vincourt@univ-lorraine.fr
Source: Journal of Proteomics. Jan2015, Vol. 114, p263-273. 11p.
Subjects: Proteomics, Cartilage cells, Liquid chromatography, Quantitative research, Matrix-assisted laser desorption-ionization, Secretion
Abstract: Proteomics users enjoy the rapid development of LC-MS-based label-free relative quantification methods but in practice these remain restricted to mass spectrometers using electrospray ionization. Here, tools dedicated to ion chromatogram extraction, time alignment, signal normalization and statistical analysis were used to interpret label-free relative difference between primary human chondrocyte secretomes and dilutions thereof, analyzed successively by LC-MALDI. The analysis of secretomes diluted into culture medium demonstrated that abundant proteins could be relatively quantified within 1.5–20-fold changes with satisfactory statistics. In addition, comparison of multiple samples requires analyzing most samples in TOF mode only, saving considerable machine-time usage. The method allowed identification and quantification of most secreted proteins relevant to the chondrocyte phenotype and evidenced their up- or down regulations by TGFβ1 and patient-to-patient differential expression. Novel targets of TGFβ1 were evidenced, such as pro-collagen C-proteinase enhancer protein 1, Metalloproteinase inhibitor 1, Fibulin-3, Tetranectin and Cartilage Intermediate Layer Protein 1, while others match previous findings. Several were verified by Western blot. This whole workflow is non-invasive, compatible with many cell culture protocols, technically straightforward and rapid, particularly regarding mass spectrometer time usage and could make label-free LC-MALDI analysis of low-complexity proteomes a major tool for routine cell culture characterization. Biological significance The present work presents the adaptation of label free relative protein quantification principles to LC-MALDI data to rapidly measure protein fold-changes between samples of relative complexity and its utility to characterize the secreted proteome of human primary chondrocytes. The method was employed to characterize the chondrocyte secretome regulation by TGFβ1 and is proposed as a routine tool to assess the quality of biomaterials designed for cartilage repair and to quantitatively investigate the influence of environmental factors upon it. [ABSTRACT FROM AUTHOR]
Copyright of Journal of Proteomics is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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DbLabel: Engineering Source
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  Data: <searchLink fieldCode="DE" term="%22Proteomics%22">Proteomics</searchLink><br /><searchLink fieldCode="DE" term="%22Cartilage+cells%22">Cartilage cells</searchLink><br /><searchLink fieldCode="DE" term="%22Liquid+chromatography%22">Liquid chromatography</searchLink><br /><searchLink fieldCode="DE" term="%22Quantitative+research%22">Quantitative research</searchLink><br /><searchLink fieldCode="DE" term="%22Matrix-assisted+laser+desorption-ionization%22">Matrix-assisted laser desorption-ionization</searchLink><br /><searchLink fieldCode="DE" term="%22Secretion%22">Secretion</searchLink>
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  Data: Proteomics users enjoy the rapid development of LC-MS-based label-free relative quantification methods but in practice these remain restricted to mass spectrometers using electrospray ionization. Here, tools dedicated to ion chromatogram extraction, time alignment, signal normalization and statistical analysis were used to interpret label-free relative difference between primary human chondrocyte secretomes and dilutions thereof, analyzed successively by LC-MALDI. The analysis of secretomes diluted into culture medium demonstrated that abundant proteins could be relatively quantified within 1.5–20-fold changes with satisfactory statistics. In addition, comparison of multiple samples requires analyzing most samples in TOF mode only, saving considerable machine-time usage. The method allowed identification and quantification of most secreted proteins relevant to the chondrocyte phenotype and evidenced their up- or down regulations by TGFβ1 and patient-to-patient differential expression. Novel targets of TGFβ1 were evidenced, such as pro-collagen C-proteinase enhancer protein 1, Metalloproteinase inhibitor 1, Fibulin-3, Tetranectin and Cartilage Intermediate Layer Protein 1, while others match previous findings. Several were verified by Western blot. This whole workflow is non-invasive, compatible with many cell culture protocols, technically straightforward and rapid, particularly regarding mass spectrometer time usage and could make label-free LC-MALDI analysis of low-complexity proteomes a major tool for routine cell culture characterization. Biological significance The present work presents the adaptation of label free relative protein quantification principles to LC-MALDI data to rapidly measure protein fold-changes between samples of relative complexity and its utility to characterize the secreted proteome of human primary chondrocytes. The method was employed to characterize the chondrocyte secretome regulation by TGFβ1 and is proposed as a routine tool to assess the quality of biomaterials designed for cartilage repair and to quantitatively investigate the influence of environmental factors upon it. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
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  Data: <i>Copyright of Journal of Proteomics is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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        Value: 10.1016/j.jprot.2014.10.026
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        Text: English
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      – SubjectFull: Cartilage cells
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      – SubjectFull: Liquid chromatography
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      – SubjectFull: Quantitative research
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      – SubjectFull: Matrix-assisted laser desorption-ionization
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      – SubjectFull: Secretion
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      – TitleFull: Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation.
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              Text: Jan2015
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