A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics

Kristin L M Boylan, Somaieh Afiuni-Zadeh, Melissa A. Geller, Kayla Hickey, Timothy J. Griffin, Stefan E. Pambuccian, Amy P N Skubitz

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Abstract

Background: The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically "normal" results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance. Results: The average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40% of the proteins identified were extracellular or localized to the plasma membrane. Almost 20% of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a "Normal Pap test Core Proteome" consisting of 153 proteins. Conclusions: Residual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases.

Original languageEnglish (US)
Article number30
JournalClinical Proteomics
Volume11
Issue number1
DOIs
StatePublished - Jul 14 2014

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Cytology
Papanicolaou Test
Feasibility Studies
Proteomics
Mass spectrometry
Cell Biology
Mass Spectrometry
Fluids
Proteins
Fixatives
Biomarkers
Proteome
Tandem Mass Spectrometry
Western Blotting
Silver Staining
Residual Volume
Body fluids
Body Fluids
Cell membranes

Keywords

  • Biomarker discovery
  • Mass spectrometry
  • Pap test
  • Proteomics

Cite this

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title = "A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics",
abstract = "Background: The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically {"}normal{"} results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance. Results: The average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40{\%} of the proteins identified were extracellular or localized to the plasma membrane. Almost 20{\%} of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a {"}Normal Pap test Core Proteome{"} consisting of 153 proteins. Conclusions: Residual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases.",
keywords = "Biomarker discovery, Mass spectrometry, Pap test, Proteomics",
author = "Boylan, {Kristin L M} and Somaieh Afiuni-Zadeh and Geller, {Melissa A.} and Kayla Hickey and Griffin, {Timothy J.} and Pambuccian, {Stefan E.} and Skubitz, {Amy P N}",
year = "2014",
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language = "English (US)",
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journal = "Clinical Proteomics",
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T1 - A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics

AU - Boylan, Kristin L M

AU - Afiuni-Zadeh, Somaieh

AU - Geller, Melissa A.

AU - Hickey, Kayla

AU - Griffin, Timothy J.

AU - Pambuccian, Stefan E.

AU - Skubitz, Amy P N

PY - 2014/7/14

Y1 - 2014/7/14

N2 - Background: The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically "normal" results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance. Results: The average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40% of the proteins identified were extracellular or localized to the plasma membrane. Almost 20% of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a "Normal Pap test Core Proteome" consisting of 153 proteins. Conclusions: Residual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases.

AB - Background: The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically "normal" results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance. Results: The average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40% of the proteins identified were extracellular or localized to the plasma membrane. Almost 20% of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a "Normal Pap test Core Proteome" consisting of 153 proteins. Conclusions: Residual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases.

KW - Biomarker discovery

KW - Mass spectrometry

KW - Pap test

KW - Proteomics

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