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Using HPTLC/DESI-MS for peptide identification in 1D separations of tryptic protein digests

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Abstract

Desorption electrospray ionization mass spectrometry (DESI-MS) was investigated as a method to detect and identify peptides from tryptic digests of cytochrome c and myoglobin separated on ProteoChrom® HPTLC Silica gel 60 F254s plates and ProteoChrom® HPTLC Cellulose sheets. Full-scan mass spectra and data-dependent tandem mass spectra were acquired in separate plate scans and used to identify peptide ions. Peptide distributions along the development lane were mapped for each separated protein digest. Signal levels ranged over several orders of magnitude. In general, highest signal levels were obtained for the peptides with the highest R f values on a plate, while peptides with very low R f values were often not detected. Sequence coverages for cytochrome c were 58% for the digest separated on the silica gel plate and 72% for the separation on the cellulose sheet; myoglobin sequence coverages were 62% and 68% on silica gel and cellulose, respectively. Weak correlations between peptide hydrophilicity and R f values on the silica gel and cellulose plates were found, with the more hydrophilic peptides having lower R f values.

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Acknowledgements

S.P.P. acknowledges an Oak Ridge National Laboratory (ORNL) appointment through the ORNL Postdoctoral Research Associates Program. Dr. Julian Philips (Thermo Fisher Scientific) is thanked for the loan of the LCQ DECA mass spectrometer. The MicroIonSpray head used to fabricate the DESI emitter was provided through a cooperative research and development agreement (CRADA) with MDS Sciex (ORNL02-0662). This research was supported by the Battelle Memorial Institute Technology Maturation Fund. The surface scanning platform and associated control software used in this study was developed with support from ORNL Technology Transfer and Economic Development (TTED) Royalty Funds. ORNL is managed and operated by UT-Battelle, LLC, for the United States Department of Energy under contract DE-AC05-00OR22725.

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Authors and Affiliations

  1. Organic and Biological Mass Spectrometry Group, Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831-6131, USA

    Sofie P. Pasilis, Vilmos Kertesz & Gary J. Van Berkel

  2. Thin-Layer Chromatography Laboratory, Performance and Life Science Chemicals, Merck KGaA, 64293, Darmstadt, Germany

    Michael Schulz & Susanne Schorcht

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  1. Sofie P. Pasilis
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  2. Vilmos Kertesz
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  3. Gary J. Van Berkel
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  4. Michael Schulz
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  5. Susanne Schorcht
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Correspondence to Gary J. Van Berkel.

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This manuscript has been authored by a contractor of the US Government under contract DE-AC05-00OR22725. Accordingly, the US Government retains a paid-up, nonexclusive, irrevocable, worldwide license to publish or reproduce the published form of this contribution, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, or allow others to do so, for US Government purposes.

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Pasilis, S.P., Kertesz, V., Van Berkel, G.J. et al. Using HPTLC/DESI-MS for peptide identification in 1D separations of tryptic protein digests. Anal Bioanal Chem 391, 317–324 (2008). https://doi.org/10.1007/s00216-008-1874-6

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  • DOI: https://doi.org/10.1007/s00216-008-1874-6

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