Mass Spectrometry-based Quantitative Proteomic Profiling of Human Pancreatic and Hepatic Stellate Cell Lines

Joao A. Paulo; Vivek Kadiyala; Peter A. Banks; Darwin L. Conwell; Hanno Steen

doi:10.1016/j.gpb.2013.01.009

Mass Spectrometry-based Quantitative Proteomic Profiling of Human Pancreatic and Hepatic Stellate Cell Lines

Joao A. Paulo, Vivek Kadiyala, Peter A. Banks, Darwin L. Conwell, Hanno Steen

Research output: Contribution to journal › Article › peer-review

23 Scopus citations

Abstract

The functions of the liver and the pancreas differ; however, chronic inflammation in both organs is associated with fibrosis. Evidence suggests that fibrosis in both organs is partially regulated by organ-specific stellate cells. We explore the proteome of human hepatic stellate cells (hHSC) and human pancreatic stellate cells (hPaSC) using mass spectrometry (MS)-based quantitative proteomics to investigate pathophysiologic mechanisms. Proteins were isolated from whole cell lysates of immortalized hHSC and hPaSC. These proteins were tryptically digested, labeled with tandem mass tags (TMT), fractionated by OFFGEL, and subjected to MS. Proteins significantly different in abundance (P<0.05) were classified via gene ontology (GO) analysis. We identified 1223 proteins and among them, 1222 proteins were quantifiable. Statistical analysis determined that 177 proteins were of higher abundance in hHSC, while 157 were of higher abundance in hPaSC. GO classification revealed that proteins of relatively higher abundance in hHSC were associated with protein production, while those of relatively higher abundance in hPaSC were involved in cell structure. Future studies using the methodologies established herein, but with further upstream fractionation and/or use of enhanced MS instrumentation will allow greater proteome coverage, achieving a comprehensive proteomic analysis of hHSC and hPaSC.

Original language	English
Pages (from-to)	105-113
Number of pages	9
Journal	Genomics, Proteomics and Bioinformatics
Volume	11
Issue number	2
DOIs	https://doi.org/10.1016/j.gpb.2013.01.009
State	Published - Apr 2013

Bibliographical note

Funding Information:
Funds were provided by the following NIH grants: 1 F32 DK085835-01A1 (JP), 1 R21 DK081703-01A2 (DC), 5 P30 DK034854-24 (Harvard Digestive Diseases Center; DC), as well as a grant from the American College of Gastroenterology: ACG – 042103580 (JP). We would like to thank the Burrill family for their generous support through the Burrill Research Grant. We would also like to thank members of the Steen Laboratory at Boston Children’s Hospital, in particular John FK Sauld and Ali Ghoulidi for their technical assistance and critical reading of the manuscript. In addition, we thank members of the Center for Pancreatic Disease at Brigham and Women’s Hospital, particularly Shadeah Suleiman for her technical assistance.

Keywords

Fibrosis
Pancreas
Proteomics
Tandem mass tag

ASJC Scopus subject areas

Biochemistry
Molecular Biology
Genetics
Computational Mathematics

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10.1016/j.gpb.2013.01.009

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@article{31583da4f0ad4842ad8062a2182aeac5,

title = "Mass Spectrometry-based Quantitative Proteomic Profiling of Human Pancreatic and Hepatic Stellate Cell Lines",

abstract = "The functions of the liver and the pancreas differ; however, chronic inflammation in both organs is associated with fibrosis. Evidence suggests that fibrosis in both organs is partially regulated by organ-specific stellate cells. We explore the proteome of human hepatic stellate cells (hHSC) and human pancreatic stellate cells (hPaSC) using mass spectrometry (MS)-based quantitative proteomics to investigate pathophysiologic mechanisms. Proteins were isolated from whole cell lysates of immortalized hHSC and hPaSC. These proteins were tryptically digested, labeled with tandem mass tags (TMT), fractionated by OFFGEL, and subjected to MS. Proteins significantly different in abundance (P<0.05) were classified via gene ontology (GO) analysis. We identified 1223 proteins and among them, 1222 proteins were quantifiable. Statistical analysis determined that 177 proteins were of higher abundance in hHSC, while 157 were of higher abundance in hPaSC. GO classification revealed that proteins of relatively higher abundance in hHSC were associated with protein production, while those of relatively higher abundance in hPaSC were involved in cell structure. Future studies using the methodologies established herein, but with further upstream fractionation and/or use of enhanced MS instrumentation will allow greater proteome coverage, achieving a comprehensive proteomic analysis of hHSC and hPaSC.",

keywords = "Fibrosis, Pancreas, Proteomics, Tandem mass tag",

author = "Paulo, {Joao A.} and Vivek Kadiyala and Banks, {Peter A.} and Conwell, {Darwin L.} and Hanno Steen",

note = "Funding Information: Funds were provided by the following NIH grants: 1 F32 DK085835-01A1 (JP), 1 R21 DK081703-01A2 (DC), 5 P30 DK034854-24 (Harvard Digestive Diseases Center; DC), as well as a grant from the American College of Gastroenterology: ACG – 042103580 (JP). We would like to thank the Burrill family for their generous support through the Burrill Research Grant. We would also like to thank members of the Steen Laboratory at Boston Children{\textquoteright}s Hospital, in particular John FK Sauld and Ali Ghoulidi for their technical assistance and critical reading of the manuscript. In addition, we thank members of the Center for Pancreatic Disease at Brigham and Women{\textquoteright}s Hospital, particularly Shadeah Suleiman for her technical assistance. ",

year = "2013",

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doi = "10.1016/j.gpb.2013.01.009",

language = "English",

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AU - Conwell, Darwin L.

AU - Steen, Hanno

N1 - Funding Information: Funds were provided by the following NIH grants: 1 F32 DK085835-01A1 (JP), 1 R21 DK081703-01A2 (DC), 5 P30 DK034854-24 (Harvard Digestive Diseases Center; DC), as well as a grant from the American College of Gastroenterology: ACG – 042103580 (JP). We would like to thank the Burrill family for their generous support through the Burrill Research Grant. We would also like to thank members of the Steen Laboratory at Boston Children’s Hospital, in particular John FK Sauld and Ali Ghoulidi for their technical assistance and critical reading of the manuscript. In addition, we thank members of the Center for Pancreatic Disease at Brigham and Women’s Hospital, particularly Shadeah Suleiman for her technical assistance.

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N2 - The functions of the liver and the pancreas differ; however, chronic inflammation in both organs is associated with fibrosis. Evidence suggests that fibrosis in both organs is partially regulated by organ-specific stellate cells. We explore the proteome of human hepatic stellate cells (hHSC) and human pancreatic stellate cells (hPaSC) using mass spectrometry (MS)-based quantitative proteomics to investigate pathophysiologic mechanisms. Proteins were isolated from whole cell lysates of immortalized hHSC and hPaSC. These proteins were tryptically digested, labeled with tandem mass tags (TMT), fractionated by OFFGEL, and subjected to MS. Proteins significantly different in abundance (P<0.05) were classified via gene ontology (GO) analysis. We identified 1223 proteins and among them, 1222 proteins were quantifiable. Statistical analysis determined that 177 proteins were of higher abundance in hHSC, while 157 were of higher abundance in hPaSC. GO classification revealed that proteins of relatively higher abundance in hHSC were associated with protein production, while those of relatively higher abundance in hPaSC were involved in cell structure. Future studies using the methodologies established herein, but with further upstream fractionation and/or use of enhanced MS instrumentation will allow greater proteome coverage, achieving a comprehensive proteomic analysis of hHSC and hPaSC.

AB - The functions of the liver and the pancreas differ; however, chronic inflammation in both organs is associated with fibrosis. Evidence suggests that fibrosis in both organs is partially regulated by organ-specific stellate cells. We explore the proteome of human hepatic stellate cells (hHSC) and human pancreatic stellate cells (hPaSC) using mass spectrometry (MS)-based quantitative proteomics to investigate pathophysiologic mechanisms. Proteins were isolated from whole cell lysates of immortalized hHSC and hPaSC. These proteins were tryptically digested, labeled with tandem mass tags (TMT), fractionated by OFFGEL, and subjected to MS. Proteins significantly different in abundance (P<0.05) were classified via gene ontology (GO) analysis. We identified 1223 proteins and among them, 1222 proteins were quantifiable. Statistical analysis determined that 177 proteins were of higher abundance in hHSC, while 157 were of higher abundance in hPaSC. GO classification revealed that proteins of relatively higher abundance in hHSC were associated with protein production, while those of relatively higher abundance in hPaSC were involved in cell structure. Future studies using the methodologies established herein, but with further upstream fractionation and/or use of enhanced MS instrumentation will allow greater proteome coverage, achieving a comprehensive proteomic analysis of hHSC and hPaSC.

KW - Fibrosis

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KW - Proteomics

KW - Tandem mass tag

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Mass Spectrometry-based Quantitative Proteomic Profiling of Human Pancreatic and Hepatic Stellate Cell Lines

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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