Isobaric protein-level labeling strategy for serum glycoprotein quantification analysis by liquid chromatography-tandem mass spectrometry

Song Nie, Andy Lo, Jianhui Zhu, Jing Wu, Mack Ruffin, David M. Lubman

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

While peptide-level labeling using isobaric tag reagents has been widely applied for quantitative proteomics experiments, there are comparatively few reports of protein-level labeling. Intact protein labeling could be broadly applied to quantification experiments utilizing protein-level separations or enrichment schemes. Here, protein-level isobaric labeling was explored as an alternative strategy to peptide-level labeling for serum glycoprotein quantification. Labeling and digestion conditions were optimized by comparing different organic solvents and enzymes. Digestions with Asp-N and trypsin were found highly complementary; combining the results enabled quantification of 30% more proteins than either enzyme alone. Three commercial reagents were compared for protein-level labeling. Protein identification rates were highest with iTRAQ 4-plex when compared to TMT 6-plex and iTRAQ 8-plex using higher-energy collisional dissociation on an Orbitrap Elite mass spectrometer. The compatibility of isobaric protein-level labeling with lectin-based glycoprotein enrichment was also investigated. More than 74% of lectin-bound labeled proteins were known glycoproteins, which was similar to results from unlabeled and peptide-level labeled serum samples. Finally, protein-level and peptide-level labeling strategies were compared for serum glycoprotein quantification. Isobaric protein-level labeling gave comparable identification levels and quantitative precision to peptide-level labeling.

Original languageEnglish (US)
Pages (from-to)5353-5357
Number of pages5
JournalAnalytical Chemistry
Volume85
Issue number11
DOIs
StatePublished - Jun 4 2013

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Liquid chromatography
Labeling
Mass spectrometry
Glycoproteins
Proteins
Peptides
Lectins
Mass spectrometers
Enzymes
Organic solvents
Trypsin
Experiments

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

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abstract = "While peptide-level labeling using isobaric tag reagents has been widely applied for quantitative proteomics experiments, there are comparatively few reports of protein-level labeling. Intact protein labeling could be broadly applied to quantification experiments utilizing protein-level separations or enrichment schemes. Here, protein-level isobaric labeling was explored as an alternative strategy to peptide-level labeling for serum glycoprotein quantification. Labeling and digestion conditions were optimized by comparing different organic solvents and enzymes. Digestions with Asp-N and trypsin were found highly complementary; combining the results enabled quantification of 30{\%} more proteins than either enzyme alone. Three commercial reagents were compared for protein-level labeling. Protein identification rates were highest with iTRAQ 4-plex when compared to TMT 6-plex and iTRAQ 8-plex using higher-energy collisional dissociation on an Orbitrap Elite mass spectrometer. The compatibility of isobaric protein-level labeling with lectin-based glycoprotein enrichment was also investigated. More than 74{\%} of lectin-bound labeled proteins were known glycoproteins, which was similar to results from unlabeled and peptide-level labeled serum samples. Finally, protein-level and peptide-level labeling strategies were compared for serum glycoprotein quantification. Isobaric protein-level labeling gave comparable identification levels and quantitative precision to peptide-level labeling.",
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Isobaric protein-level labeling strategy for serum glycoprotein quantification analysis by liquid chromatography-tandem mass spectrometry. / Nie, Song; Lo, Andy; Zhu, Jianhui; Wu, Jing; Ruffin, Mack; Lubman, David M.

In: Analytical Chemistry, Vol. 85, No. 11, 04.06.2013, p. 5353-5357.

Research output: Contribution to journalArticle

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