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Metal ion-mobilizing additives for comprehensive detection of femtomole amounts of phosphopeptides by reversed phase LC-MS

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Abstract

It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase μLC-ICP-MS or nanoLC-ESI-MS. Using μLC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of >10 pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga3+-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.

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Abbreviations

MS:

Mass spectrometry

EDTA:

Ethylendiaminetetraacetic acid

RP-LC:

Reversed phase liquid chromatography

capLC:

Capillary liquid chromatography

DFO:

Deferoxamine

IMAC:

Immobilized metal ion affinity chromatography

FA:

Formic acid

Ni-NTA:

Nickel nitrilotriacetic acid

ESI:

Electrospray ionization

ICP:

Inductively coupled plasma

mimLC:

Metal ion-mobilizing LC

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Acknowledgments

We thank R. Pipkorn for expert peptide synthesis.

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Authors and Affiliations

  1. Molecular Structure Analysis, German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany

    Joerg Seidler, Nico Zinn, Martin E. Boehm & Wolf D. Lehmann

  2. Core Facility Proteomics, Center for Systems Biology ZBSA, Habsburger Str. 49, 79104, Freiburg, Germany

    Erik Haaf & Andreas Schlosser

  3. Department of Pathology, Harvard Medical School and Children’s Hospital, Boston, MA, 02115, USA

    Dominic Winter

Authors
  1. Joerg Seidler
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  2. Nico Zinn
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  3. Erik Haaf
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  4. Martin E. Boehm
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  5. Dominic Winter
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  6. Andreas Schlosser
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  7. Wolf D. Lehmann
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Corresponding author

Correspondence to Wolf D. Lehmann.

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Seidler, J., Zinn, N., Haaf, E. et al. Metal ion-mobilizing additives for comprehensive detection of femtomole amounts of phosphopeptides by reversed phase LC-MS. Amino Acids 41, 311–320 (2011). https://doi.org/10.1007/s00726-010-0647-7

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  • DOI: https://doi.org/10.1007/s00726-010-0647-7

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