Hydrophilic Strong Anion Exchange (hSAX) Chromatography for Highly Orthogonal Peptide Separation of Complex Proteomes

Stella Ritorto, Ken Cook, Kshitiz Tyagi, Patrick G. A. Pedrioli, Matthias Trost

    Research output: Contribution to journalArticle

    42 Citations (Scopus)

    Abstract

    Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis.
    Original languageEnglish
    Pages (from-to)2449-2457
    Number of pages9
    JournalJournal of Proteome Research
    Volume12
    Issue number6
    Early online date7 Jan 2013
    DOIs
    Publication statusPublished - 2013

    Fingerprint

    Proteome
    Chromatography
    Anions
    Ion exchange
    Peptides
    Hydrophobic and Hydrophilic Interactions
    Macrophages
    Liquid chromatography
    Hydrophobicity
    Proteins
    Ion Exchange Chromatography
    Hydrophilicity
    Reverse-Phase Chromatography
    Fractionation
    Liquid Chromatography
    Phase separation
    Proteomics
    Mass spectrometry
    Cations
    Mass Spectrometry

    Cite this

    Ritorto, Stella ; Cook, Ken ; Tyagi, Kshitiz ; Pedrioli, Patrick G. A. ; Trost, Matthias. / Hydrophilic Strong Anion Exchange (hSAX) Chromatography for Highly Orthogonal Peptide Separation of Complex Proteomes. In: Journal of Proteome Research. 2013 ; Vol. 12, No. 6. pp. 2449-2457.
    @article{15e4b93028c54e9dbcbc1c4e3f57b7df,
    title = "Hydrophilic Strong Anion Exchange (hSAX) Chromatography for Highly Orthogonal Peptide Separation of Complex Proteomes",
    abstract = "Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28{\%}, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis.",
    author = "Stella Ritorto and Ken Cook and Kshitiz Tyagi and Pedrioli, {Patrick G. A.} and Matthias Trost",
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    Hydrophilic Strong Anion Exchange (hSAX) Chromatography for Highly Orthogonal Peptide Separation of Complex Proteomes. / Ritorto, Stella; Cook, Ken; Tyagi, Kshitiz; Pedrioli, Patrick G. A.; Trost, Matthias.

    In: Journal of Proteome Research, Vol. 12, No. 6, 2013, p. 2449-2457.

    Research output: Contribution to journalArticle

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    AU - Ritorto, Stella

    AU - Cook, Ken

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    AU - Pedrioli, Patrick G. A.

    AU - Trost, Matthias

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    N2 - Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis.

    AB - Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis.

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