Optimization of plasma sample pretreatment for quantitative analysis using iTRAQ labeling and LC-MALDI-TOF/TOF.
Shotgun proteomic methods involving iTRAQ (isobaric tags for relative and absolute quantitation) peptide labeling facilitate quantitative analyses of proteomes and searches for useful biomarkers. However, the plasma proteome's complexity and the highly dynamic plasma protein concentration range...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0101694&type=printable |
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| author | Magdalena Luczak Lukasz Marczak Maciej Stobiecki |
| author_facet | Magdalena Luczak Lukasz Marczak Maciej Stobiecki |
| author_sort | Magdalena Luczak |
| collection | DOAJ |
| description | Shotgun proteomic methods involving iTRAQ (isobaric tags for relative and absolute quantitation) peptide labeling facilitate quantitative analyses of proteomes and searches for useful biomarkers. However, the plasma proteome's complexity and the highly dynamic plasma protein concentration range limit the ability of conventional approaches to analyze and identify a large number of proteins, including useful biomarkers. The goal of this paper is to elucidate the best approach for plasma sample pretreatment for MS- and iTRAQ-based analyses. Here, we systematically compared four approaches, which include centrifugal ultrafiltration, SCX chromatography with fractionation, affinity depletion, and plasma without fractionation, to reduce plasma sample complexity. We generated an optimized protocol for quantitative protein analysis using iTRAQ reagents and an UltrafleXtreme (Bruker Daltonics) MALDI TOF/TOF mass spectrometer. Moreover, we used a simple, rapid, efficient, but inexpensive sample pretreatment technique that generated an optimal opportunity for biomarker discovery. We discuss the results from the four sample pretreatment approaches and conclude that SCX chromatography without affinity depletion is the best plasma sample preparation pretreatment method for proteome analysis. Using this technique, we identified 1,780 unique proteins, including 1,427 that were quantified by iTRAQ with high reproducibility and accuracy. |
| format | Article |
| id | doaj-art-86907b7beea74f658bd408ce85ec31d2 |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-86907b7beea74f658bd408ce85ec31d22025-08-20T03:01:19ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0197e10169410.1371/journal.pone.0101694Optimization of plasma sample pretreatment for quantitative analysis using iTRAQ labeling and LC-MALDI-TOF/TOF.Magdalena LuczakLukasz MarczakMaciej StobieckiShotgun proteomic methods involving iTRAQ (isobaric tags for relative and absolute quantitation) peptide labeling facilitate quantitative analyses of proteomes and searches for useful biomarkers. However, the plasma proteome's complexity and the highly dynamic plasma protein concentration range limit the ability of conventional approaches to analyze and identify a large number of proteins, including useful biomarkers. The goal of this paper is to elucidate the best approach for plasma sample pretreatment for MS- and iTRAQ-based analyses. Here, we systematically compared four approaches, which include centrifugal ultrafiltration, SCX chromatography with fractionation, affinity depletion, and plasma without fractionation, to reduce plasma sample complexity. We generated an optimized protocol for quantitative protein analysis using iTRAQ reagents and an UltrafleXtreme (Bruker Daltonics) MALDI TOF/TOF mass spectrometer. Moreover, we used a simple, rapid, efficient, but inexpensive sample pretreatment technique that generated an optimal opportunity for biomarker discovery. We discuss the results from the four sample pretreatment approaches and conclude that SCX chromatography without affinity depletion is the best plasma sample preparation pretreatment method for proteome analysis. Using this technique, we identified 1,780 unique proteins, including 1,427 that were quantified by iTRAQ with high reproducibility and accuracy.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0101694&type=printable |
| spellingShingle | Magdalena Luczak Lukasz Marczak Maciej Stobiecki Optimization of plasma sample pretreatment for quantitative analysis using iTRAQ labeling and LC-MALDI-TOF/TOF. PLoS ONE |
| title | Optimization of plasma sample pretreatment for quantitative analysis using iTRAQ labeling and LC-MALDI-TOF/TOF. |
| title_full | Optimization of plasma sample pretreatment for quantitative analysis using iTRAQ labeling and LC-MALDI-TOF/TOF. |
| title_fullStr | Optimization of plasma sample pretreatment for quantitative analysis using iTRAQ labeling and LC-MALDI-TOF/TOF. |
| title_full_unstemmed | Optimization of plasma sample pretreatment for quantitative analysis using iTRAQ labeling and LC-MALDI-TOF/TOF. |
| title_short | Optimization of plasma sample pretreatment for quantitative analysis using iTRAQ labeling and LC-MALDI-TOF/TOF. |
| title_sort | optimization of plasma sample pretreatment for quantitative analysis using itraq labeling and lc maldi tof tof |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0101694&type=printable |
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