Optimizing the identification of citrullinated peptides by mass spectrometry: utilizing the inability of trypsin to cleave after citrullinated amino acids

Tue Bennike, Kasper B. Lauridsen, Michael Kruse Olesen, Vibeke Andersen, Svend Birkelund, Allan Stensballe

Research output: Contribution to journalJournal articleResearchpeer-review

23 Citations (Scopus)

Abstract

Citrullinated proteins have been associated with several diseases and citrullination can most likely function as a target for novel diagnostic agents and unravel disease etiologies. The correct identification of citrullinated proteins is therefore of most importance. Mass spectrometry (MS) driven proteomics can with bottom up strategies analyze protein profiles and PTMs in complex samples. However, the site-specific characterization of citrullination using MS remains problematic, especially in complex samples where no sensitive chemical modification technique exists. A tryptic missed cleavage after citrulline is therefore often used as a marker for citrullination post processing. However, C-terminal tryptic citrullinated peptides have also been reported. In this study, we therefore aimed at optimizing the identification of citrullinated peptides in complex samples.

To assess the cleavage properties of trypsin, digestion was performed on synthetic peptide sets containing either arginine or citrulline. The peptide sequences originated from disease-associated in vivo citrullinated proteins; some reported as being C-terminal tryptic citrullinated peptides. Furthermore, the proteolytic activity was verified using digested synovial fluid samples from a rheumatoid arthritis patient. The samples were analyzed using liquid chromatography/tandem MS with electrospray ionization.

Our in vivo and in vitro studies clearly demonstrate the inability of trypsin to cleave after citrulline residues. Based on our findings, we present a strategy for verifying citrullinated sites in complex samples post processing, in proteomics shotgun experiments. By requiring a missed cleavage for the identification of citrullinated peptides, we demonstrate that 64% of false-positively annotated citrullination sites could be removed. We furthermore demonstrated likely pitfalls of applying the strategy.

In conclusion, manual annotation of citrullinated peptide spectra remains essential to ensure correct annotation. Implementing a missed cleavage requirement significantly reduces the number of spectra needing manual verification with minimal loss. This method may help future proteomics studies identify citrullinated proteins in complex samples.
Original languageEnglish
JournalJournal of Proteomics & Bioinformatics
Volume6
Issue number12
Pages (from-to)288-295
Number of pages8
ISSN0974-276X
DOIs
Publication statusPublished - 15 Dec 2013

Fingerprint

Trypsin
Peptides
Mass spectrometry
Amino acids
Mass Spectrometry
Amino Acids
Citrulline
Proteins
Proteomics
Pulse time modulation
Electrospray ionization
Arginine
Synovial Fluid
Liquid chromatography
Chemical modification
Firearms
Processing
Tandem Mass Spectrometry
Liquid Chromatography
Digestion

Keywords

  • Citrulline
  • Citrullination
  • missed cleavage
  • trypsin
  • digestion
  • synovial fluid

Cite this

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title = "Optimizing the identification of citrullinated peptides by mass spectrometry: utilizing the inability of trypsin to cleave after citrullinated amino acids",
abstract = "Citrullinated proteins have been associated with several diseases and citrullination can most likely function as a target for novel diagnostic agents and unravel disease etiologies. The correct identification of citrullinated proteins is therefore of most importance. Mass spectrometry (MS) driven proteomics can with bottom up strategies analyze protein profiles and PTMs in complex samples. However, the site-specific characterization of citrullination using MS remains problematic, especially in complex samples where no sensitive chemical modification technique exists. A tryptic missed cleavage after citrulline is therefore often used as a marker for citrullination post processing. However, C-terminal tryptic citrullinated peptides have also been reported. In this study, we therefore aimed at optimizing the identification of citrullinated peptides in complex samples.To assess the cleavage properties of trypsin, digestion was performed on synthetic peptide sets containing either arginine or citrulline. The peptide sequences originated from disease-associated in vivo citrullinated proteins; some reported as being C-terminal tryptic citrullinated peptides. Furthermore, the proteolytic activity was verified using digested synovial fluid samples from a rheumatoid arthritis patient. The samples were analyzed using liquid chromatography/tandem MS with electrospray ionization.Our in vivo and in vitro studies clearly demonstrate the inability of trypsin to cleave after citrulline residues. Based on our findings, we present a strategy for verifying citrullinated sites in complex samples post processing, in proteomics shotgun experiments. By requiring a missed cleavage for the identification of citrullinated peptides, we demonstrate that 64{\%} of false-positively annotated citrullination sites could be removed. We furthermore demonstrated likely pitfalls of applying the strategy.In conclusion, manual annotation of citrullinated peptide spectra remains essential to ensure correct annotation. Implementing a missed cleavage requirement significantly reduces the number of spectra needing manual verification with minimal loss. This method may help future proteomics studies identify citrullinated proteins in complex samples.",
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Optimizing the identification of citrullinated peptides by mass spectrometry : utilizing the inability of trypsin to cleave after citrullinated amino acids. / Bennike, Tue; Lauridsen, Kasper B.; Olesen, Michael Kruse; Andersen, Vibeke; Birkelund, Svend; Stensballe, Allan.

In: Journal of Proteomics & Bioinformatics, Vol. 6, No. 12, 15.12.2013, p. 288-295.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Optimizing the identification of citrullinated peptides by mass spectrometry

T2 - utilizing the inability of trypsin to cleave after citrullinated amino acids

AU - Bennike, Tue

AU - Lauridsen, Kasper B.

AU - Olesen, Michael Kruse

AU - Andersen, Vibeke

AU - Birkelund, Svend

AU - Stensballe, Allan

PY - 2013/12/15

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N2 - Citrullinated proteins have been associated with several diseases and citrullination can most likely function as a target for novel diagnostic agents and unravel disease etiologies. The correct identification of citrullinated proteins is therefore of most importance. Mass spectrometry (MS) driven proteomics can with bottom up strategies analyze protein profiles and PTMs in complex samples. However, the site-specific characterization of citrullination using MS remains problematic, especially in complex samples where no sensitive chemical modification technique exists. A tryptic missed cleavage after citrulline is therefore often used as a marker for citrullination post processing. However, C-terminal tryptic citrullinated peptides have also been reported. In this study, we therefore aimed at optimizing the identification of citrullinated peptides in complex samples.To assess the cleavage properties of trypsin, digestion was performed on synthetic peptide sets containing either arginine or citrulline. The peptide sequences originated from disease-associated in vivo citrullinated proteins; some reported as being C-terminal tryptic citrullinated peptides. Furthermore, the proteolytic activity was verified using digested synovial fluid samples from a rheumatoid arthritis patient. The samples were analyzed using liquid chromatography/tandem MS with electrospray ionization.Our in vivo and in vitro studies clearly demonstrate the inability of trypsin to cleave after citrulline residues. Based on our findings, we present a strategy for verifying citrullinated sites in complex samples post processing, in proteomics shotgun experiments. By requiring a missed cleavage for the identification of citrullinated peptides, we demonstrate that 64% of false-positively annotated citrullination sites could be removed. We furthermore demonstrated likely pitfalls of applying the strategy.In conclusion, manual annotation of citrullinated peptide spectra remains essential to ensure correct annotation. Implementing a missed cleavage requirement significantly reduces the number of spectra needing manual verification with minimal loss. This method may help future proteomics studies identify citrullinated proteins in complex samples.

AB - Citrullinated proteins have been associated with several diseases and citrullination can most likely function as a target for novel diagnostic agents and unravel disease etiologies. The correct identification of citrullinated proteins is therefore of most importance. Mass spectrometry (MS) driven proteomics can with bottom up strategies analyze protein profiles and PTMs in complex samples. However, the site-specific characterization of citrullination using MS remains problematic, especially in complex samples where no sensitive chemical modification technique exists. A tryptic missed cleavage after citrulline is therefore often used as a marker for citrullination post processing. However, C-terminal tryptic citrullinated peptides have also been reported. In this study, we therefore aimed at optimizing the identification of citrullinated peptides in complex samples.To assess the cleavage properties of trypsin, digestion was performed on synthetic peptide sets containing either arginine or citrulline. The peptide sequences originated from disease-associated in vivo citrullinated proteins; some reported as being C-terminal tryptic citrullinated peptides. Furthermore, the proteolytic activity was verified using digested synovial fluid samples from a rheumatoid arthritis patient. The samples were analyzed using liquid chromatography/tandem MS with electrospray ionization.Our in vivo and in vitro studies clearly demonstrate the inability of trypsin to cleave after citrulline residues. Based on our findings, we present a strategy for verifying citrullinated sites in complex samples post processing, in proteomics shotgun experiments. By requiring a missed cleavage for the identification of citrullinated peptides, we demonstrate that 64% of false-positively annotated citrullination sites could be removed. We furthermore demonstrated likely pitfalls of applying the strategy.In conclusion, manual annotation of citrullinated peptide spectra remains essential to ensure correct annotation. Implementing a missed cleavage requirement significantly reduces the number of spectra needing manual verification with minimal loss. This method may help future proteomics studies identify citrullinated proteins in complex samples.

KW - Citrulline

KW - Citrullination

KW - missed cleavage

KW - trypsin

KW - digestion

KW - synovial fluid

U2 - 10.4172/jpb.1000293

DO - 10.4172/jpb.1000293

M3 - Journal article

VL - 6

SP - 288

EP - 295

JO - Journal of Proteomics & Bioinformatics

JF - Journal of Proteomics & Bioinformatics

SN - 0974-276X

IS - 12

ER -