Relative quantitation of protein nitration by liquid chromatography-mass spectrometry using isotope-coded dimethyl labeling and chemoprecipitation

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Abstract

Protein nitration has been recognized as an important biomarker for nitroxidative stress associated with various diseases. While identification of protein targets for nitration is important, its quantitative profiling also is necessary to understand the biological impact of this low-abundance posttranslational modification. We have previously reported an efficient and straightforward enrichment method for nitropeptides to reduce sample complexity and permit unambiguous site-specific identifications by LC-MS analyses. This approach relies on two chemical derivatization steps: specifically reductive methylation of aliphatic amines and, then, conversion of nitrotyrosines to the corresponding aminotyrosines before their selective capture by a solid-phase reagent we introduced previously. Hence, the method inherently offers the opportunity for relative quantitation of nitropeptides by using isotopic variants of formaldehyde for reductive methylation. This simple method was tested via LC-MS analyses of differently N-methylated nitropeptides and nitroubiquitin as a model nitroprotein enriched from human serum albumin digest and from human plasma, respectively.

Original languageEnglish
Pages (from-to)266-275
Number of pages10
JournalJournal of Chromatography A
Volume1232
DOIs
StatePublished - 6 Apr 2012

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Nitration
Methylation
Liquid chromatography
Liquid Chromatography
Isotopes
Labeling
Mass spectrometry
Mass Spectrometry
Plasma (human)
Biomarkers
Serum Albumin
Formaldehyde
Amines
Proteins
Post Translational Protein Processing
3-nitrotyrosine

Keywords

  • Enrichment
  • LC-MS
  • Protein nitration
  • Relative quantitation
  • Stable-isotope labeling
  • Tandem mass spectrometry

Cite this

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title = "Relative quantitation of protein nitration by liquid chromatography-mass spectrometry using isotope-coded dimethyl labeling and chemoprecipitation",
abstract = "Protein nitration has been recognized as an important biomarker for nitroxidative stress associated with various diseases. While identification of protein targets for nitration is important, its quantitative profiling also is necessary to understand the biological impact of this low-abundance posttranslational modification. We have previously reported an efficient and straightforward enrichment method for nitropeptides to reduce sample complexity and permit unambiguous site-specific identifications by LC-MS analyses. This approach relies on two chemical derivatization steps: specifically reductive methylation of aliphatic amines and, then, conversion of nitrotyrosines to the corresponding aminotyrosines before their selective capture by a solid-phase reagent we introduced previously. Hence, the method inherently offers the opportunity for relative quantitation of nitropeptides by using isotopic variants of formaldehyde for reductive methylation. This simple method was tested via LC-MS analyses of differently N-methylated nitropeptides and nitroubiquitin as a model nitroprotein enriched from human serum albumin digest and from human plasma, respectively.",
keywords = "Enrichment, LC-MS, Protein nitration, Relative quantitation, Stable-isotope labeling, Tandem mass spectrometry",
author = "Jia Guo and Katalin Prokai-Tatrai and Laszlo Prokai",
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doi = "10.1016/j.chroma.2011.12.100",
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T1 - Relative quantitation of protein nitration by liquid chromatography-mass spectrometry using isotope-coded dimethyl labeling and chemoprecipitation

AU - Guo, Jia

AU - Prokai-Tatrai, Katalin

AU - Prokai, Laszlo

PY - 2012/4/6

Y1 - 2012/4/6

N2 - Protein nitration has been recognized as an important biomarker for nitroxidative stress associated with various diseases. While identification of protein targets for nitration is important, its quantitative profiling also is necessary to understand the biological impact of this low-abundance posttranslational modification. We have previously reported an efficient and straightforward enrichment method for nitropeptides to reduce sample complexity and permit unambiguous site-specific identifications by LC-MS analyses. This approach relies on two chemical derivatization steps: specifically reductive methylation of aliphatic amines and, then, conversion of nitrotyrosines to the corresponding aminotyrosines before their selective capture by a solid-phase reagent we introduced previously. Hence, the method inherently offers the opportunity for relative quantitation of nitropeptides by using isotopic variants of formaldehyde for reductive methylation. This simple method was tested via LC-MS analyses of differently N-methylated nitropeptides and nitroubiquitin as a model nitroprotein enriched from human serum albumin digest and from human plasma, respectively.

AB - Protein nitration has been recognized as an important biomarker for nitroxidative stress associated with various diseases. While identification of protein targets for nitration is important, its quantitative profiling also is necessary to understand the biological impact of this low-abundance posttranslational modification. We have previously reported an efficient and straightforward enrichment method for nitropeptides to reduce sample complexity and permit unambiguous site-specific identifications by LC-MS analyses. This approach relies on two chemical derivatization steps: specifically reductive methylation of aliphatic amines and, then, conversion of nitrotyrosines to the corresponding aminotyrosines before their selective capture by a solid-phase reagent we introduced previously. Hence, the method inherently offers the opportunity for relative quantitation of nitropeptides by using isotopic variants of formaldehyde for reductive methylation. This simple method was tested via LC-MS analyses of differently N-methylated nitropeptides and nitroubiquitin as a model nitroprotein enriched from human serum albumin digest and from human plasma, respectively.

KW - Enrichment

KW - LC-MS

KW - Protein nitration

KW - Relative quantitation

KW - Stable-isotope labeling

KW - Tandem mass spectrometry

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DO - 10.1016/j.chroma.2011.12.100

M3 - Article

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VL - 1232

SP - 266

EP - 275

JO - Journal of Chromatography A

JF - Journal of Chromatography A

SN - 0021-9673

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