Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications

Application to glyceraldehyde-3-phosphate dehydrogenase

Jawon Seo, Jaeho Jeong, Young-Mee Kim, Narae Hwang, Eunok Paek, Kong Joo Lee

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.

Original languageEnglish (US)
Pages (from-to)587-602
Number of pages16
JournalJournal of Proteome Research
Volume7
Issue number2
DOIs
StatePublished - Feb 1 2008

Fingerprint

Glyceraldehyde-3-Phosphate Dehydrogenases
Post Translational Protein Processing
Peptides
Ions
Proteins
Electrospray ionization
Oxidative stress
Liquid chromatography
Proteomics
Mass spectrometry
Screening
Gels
Biological Phenomena
Mass Screening
Tandem Mass Spectrometry
Liquid Chromatography
Chemical analysis
Oxidative Stress

Keywords

  • GAPDH
  • Minor PTMs
  • Oxidative stress
  • Post-translational modifications
  • Proteomic analysis
  • SEMSA
  • Selectively excluded mass screening analysis

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Cite this

Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications : Application to glyceraldehyde-3-phosphate dehydrogenase. / Seo, Jawon; Jeong, Jaeho; Kim, Young-Mee; Hwang, Narae; Paek, Eunok; Lee, Kong Joo.

In: Journal of Proteome Research, Vol. 7, No. 2, 01.02.2008, p. 587-602.

Research output: Contribution to journalArticle

@article{4dcd81c7afff44b486f3b51cc8268e80,
title = "Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications: Application to glyceraldehyde-3-phosphate dehydrogenase",
abstract = "Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92{\%} peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.",
keywords = "GAPDH, Minor PTMs, Oxidative stress, Post-translational modifications, Proteomic analysis, SEMSA, Selectively excluded mass screening analysis",
author = "Jawon Seo and Jaeho Jeong and Young-Mee Kim and Narae Hwang and Eunok Paek and Lee, {Kong Joo}",
year = "2008",
month = "2",
day = "1",
doi = "10.1021/pr700657y",
language = "English (US)",
volume = "7",
pages = "587--602",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "2",

}

TY - JOUR

T1 - Strategy for comprehensive identification of post-translational modifications in cellular proteins, including low abundant modifications

T2 - Application to glyceraldehyde-3-phosphate dehydrogenase

AU - Seo, Jawon

AU - Jeong, Jaeho

AU - Kim, Young-Mee

AU - Hwang, Narae

AU - Paek, Eunok

AU - Lee, Kong Joo

PY - 2008/2/1

Y1 - 2008/2/1

N2 - Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.

AB - Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.

KW - GAPDH

KW - Minor PTMs

KW - Oxidative stress

KW - Post-translational modifications

KW - Proteomic analysis

KW - SEMSA

KW - Selectively excluded mass screening analysis

UR - http://www.scopus.com/inward/record.url?scp=39749099462&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=39749099462&partnerID=8YFLogxK

U2 - 10.1021/pr700657y

DO - 10.1021/pr700657y

M3 - Article

VL - 7

SP - 587

EP - 602

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 2

ER -