Quantitative pattern analysis of the N-terminally processed isoforms of platelet factor-4 in serum

Jin Young Kim, Jae Ryoung Lee, Sunkyu Choi, Eun Min Kim, Nak Kyun Jung, Young Hwan Kim, Jong Shin Yoo, Seung Won Lee

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Rationale: Platelet factor 4 (PF4) is a small cytokine belonging to the CXC chemokine family which has been shown to play a role in inflammation and in the regulation of angiogenesis. In general, chemokines are susceptible to proteolytic cleavage in amino and carboxy terminal regions, which usually Results in dramatic changes to the chemokine bioactivity. The purpose of this study was to identify various platelet factor-4 (PF4) isoforms caused by proteolytic processing and to quantify their levels in normal serum. Methods: First, we identified the N-terminally truncated PF4 isoforms from a standard purified PF4 protein sample by using mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. Then, we used high-performance liquid chromatography (HPLC) to semi-purify PF4 from serum samples, and the levels of the four most abundant PF4 isoforms were quantitatively determined using selected reaction monitoring (SRM) assays on a nano-LC/triple-quadrupole mass spectrometer. Results:We have identified seven N-terminally processed PF4 isoforms and compared the levels of major PF4 isoforms from nine serum samples. Pro-p1 (EAEEDGDLQCLCVK-; average MW, 7765.2) is the major PF4 isoform in serum whereas the PF4 isoforms, designated Prot-p4 (FASAEAEEDGDLQCLCVK- ;average MW, 8141.5), Prot-p3 (SAEAEEDGDLQCLCVK- ; average MW, 7923.3), and Prot-p2 (AEEDGDLQCLCVK- ; average MW, 7836.3), are at about 16%, 3%, and 2% levels of the major one, respectively. Conclusions: This study is the first report on the levels of N-terminally processed PF4 isoforms in serum. Also, this study shows the usefulness of SRM in determining concentrations of protein isoform variants, which can be often overlooked in immunoassay analysis.

Original languageEnglish
Pages (from-to)521-530
Number of pages10
JournalRapid Communications in Mass Spectrometry
Volume27
Issue number4
DOIs
Publication statusPublished - 28 Feb 2013
Externally publishedYes

Fingerprint

Platelet Factor 4
Protein Isoforms
Chemokines
Mass spectrometry
CXC Chemokines
Monitoring
High performance liquid chromatography
Mass spectrometers
Bioactivity
Assays

ASJC Scopus subject areas

  • Spectroscopy
  • Analytical Chemistry
  • Organic Chemistry

Cite this

Quantitative pattern analysis of the N-terminally processed isoforms of platelet factor-4 in serum. / Kim, Jin Young; Lee, Jae Ryoung; Choi, Sunkyu; Kim, Eun Min; Jung, Nak Kyun; Kim, Young Hwan; Yoo, Jong Shin; Lee, Seung Won.

In: Rapid Communications in Mass Spectrometry, Vol. 27, No. 4, 28.02.2013, p. 521-530.

Research output: Contribution to journalArticle

Kim, Jin Young ; Lee, Jae Ryoung ; Choi, Sunkyu ; Kim, Eun Min ; Jung, Nak Kyun ; Kim, Young Hwan ; Yoo, Jong Shin ; Lee, Seung Won. / Quantitative pattern analysis of the N-terminally processed isoforms of platelet factor-4 in serum. In: Rapid Communications in Mass Spectrometry. 2013 ; Vol. 27, No. 4. pp. 521-530.
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abstract = "Rationale: Platelet factor 4 (PF4) is a small cytokine belonging to the CXC chemokine family which has been shown to play a role in inflammation and in the regulation of angiogenesis. In general, chemokines are susceptible to proteolytic cleavage in amino and carboxy terminal regions, which usually Results in dramatic changes to the chemokine bioactivity. The purpose of this study was to identify various platelet factor-4 (PF4) isoforms caused by proteolytic processing and to quantify their levels in normal serum. Methods: First, we identified the N-terminally truncated PF4 isoforms from a standard purified PF4 protein sample by using mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. Then, we used high-performance liquid chromatography (HPLC) to semi-purify PF4 from serum samples, and the levels of the four most abundant PF4 isoforms were quantitatively determined using selected reaction monitoring (SRM) assays on a nano-LC/triple-quadrupole mass spectrometer. Results:We have identified seven N-terminally processed PF4 isoforms and compared the levels of major PF4 isoforms from nine serum samples. Pro-p1 (EAEEDGDLQCLCVK-; average MW, 7765.2) is the major PF4 isoform in serum whereas the PF4 isoforms, designated Prot-p4 (FASAEAEEDGDLQCLCVK- ;average MW, 8141.5), Prot-p3 (SAEAEEDGDLQCLCVK- ; average MW, 7923.3), and Prot-p2 (AEEDGDLQCLCVK- ; average MW, 7836.3), are at about 16{\%}, 3{\%}, and 2{\%} levels of the major one, respectively. Conclusions: This study is the first report on the levels of N-terminally processed PF4 isoforms in serum. Also, this study shows the usefulness of SRM in determining concentrations of protein isoform variants, which can be often overlooked in immunoassay analysis.",
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AU - Kim, Jin Young

AU - Lee, Jae Ryoung

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AU - Kim, Eun Min

AU - Jung, Nak Kyun

AU - Kim, Young Hwan

AU - Yoo, Jong Shin

AU - Lee, Seung Won

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AB - Rationale: Platelet factor 4 (PF4) is a small cytokine belonging to the CXC chemokine family which has been shown to play a role in inflammation and in the regulation of angiogenesis. In general, chemokines are susceptible to proteolytic cleavage in amino and carboxy terminal regions, which usually Results in dramatic changes to the chemokine bioactivity. The purpose of this study was to identify various platelet factor-4 (PF4) isoforms caused by proteolytic processing and to quantify their levels in normal serum. Methods: First, we identified the N-terminally truncated PF4 isoforms from a standard purified PF4 protein sample by using mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. Then, we used high-performance liquid chromatography (HPLC) to semi-purify PF4 from serum samples, and the levels of the four most abundant PF4 isoforms were quantitatively determined using selected reaction monitoring (SRM) assays on a nano-LC/triple-quadrupole mass spectrometer. Results:We have identified seven N-terminally processed PF4 isoforms and compared the levels of major PF4 isoforms from nine serum samples. Pro-p1 (EAEEDGDLQCLCVK-; average MW, 7765.2) is the major PF4 isoform in serum whereas the PF4 isoforms, designated Prot-p4 (FASAEAEEDGDLQCLCVK- ;average MW, 8141.5), Prot-p3 (SAEAEEDGDLQCLCVK- ; average MW, 7923.3), and Prot-p2 (AEEDGDLQCLCVK- ; average MW, 7836.3), are at about 16%, 3%, and 2% levels of the major one, respectively. Conclusions: This study is the first report on the levels of N-terminally processed PF4 isoforms in serum. Also, this study shows the usefulness of SRM in determining concentrations of protein isoform variants, which can be often overlooked in immunoassay analysis.

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