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Published online before print November 5, 2004

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(Journal of Leukocyte Biology. 2005;77:190-198.)
© 2005 by Society for Leukocyte Biology

In silico evaluation of two mass spectrometry-based approaches for the identification of novel human leukocyte cell-surface proteins

I. C. Nicholson^*,,1, M. Ayhan^*,, N. J. Hoogenraad^*, and H. Zola^*,

^* Co-operative Research Centre for Diagnostics and
Child Health Research Institute, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; and
Department of Biochemistry, Latrobe University, Bundoora, Victoria, Australia

¹ Correspondence: Child Health Research Institute, 72 King William Road, North Adelaide 5006, South Australia, Australia. E-mail: ian.nicholson{at}adelaide.edu.au

The identification and quantitation of cell-surface proteins expressed by leukocytes currently use the wide availability of monoclonal antibodies (mAb) in immunohistochemical and flow cytometric assays. Presently, 400 such proteins have been characterized; however, analysis of the completed human genome sequence indicates that it may contain several thousand as-yet unidentified molecules, which may be expressed on the leukocyte cell surface. Recent advances in protein isolation and analysis using mass spectrometry illustrate that it is now feasible to identify the protein composition of a complex sample such as a plasma membrane extract. Such an approach may be useful for the identification of the cell-surface proteins that have not been identified using mAb techniques. Here, we detail the results of an in silico evaluation of the peptides isolated using two methods used to label plasma membrane proteins to determine whether these methods are suitable for the identification of known leukocyte cell-surface proteins by mass spectrometry. The labeling of cell-surface proteins before isolation and characterization is a valuable means of differentiating between plasma membrane and internal membrane proteins The results indicate that although the majority of cell-surface proteins can be identified using either of the approaches, others known to be important diagnostically and/or therapeutically would not be identified using either approach. The implication of this for the use of these techniques in the discovery of new leukocyte cell-surface proteins is discussed.

Key Words: proteomics • protein identification • MASCOT search • cell-surface biotinylation • glycoprotein identification

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