Evaluation of Chemical Derivatisation Methods for Protein Identification using MALDI MS/MS *,** Janice L. Joss, 1 Mark P. Molloy, 2 Lyn A. Hinds 3 and Elizabeth M. Deane 1,4 (Accepted March 17, 2006; online publication April 21, 2006) In proteomic studies, assigning protein identity from organisms whose genomes are yet to be completely sequenced remains a challenging task. For these organisms, protein identification is typically based on cross species matching of amino acid sequence obtained from collision induced dissociation (CID) of peptides using mass spectrometry. The most direct approach of de novo sequencing is slow and often difficult, due to the complexity of the resultant CID spectra. For MALDI-MS, this problem has been addressed by using chemical derivatisation to direct peptide fragmentation, thereby simplifying CID spectra and facilitating de novo interpretation. In this study, milk whey proteins from the tammar wallaby (Macropus eugenii) were used to evaluate three chemical derivatisation methods compatible with MALDI MS/MS. These methods included (i) guanidination and sulfonation using chemically-assisted fragmentation (CAF), (ii) guanidination and sulfonation using 4-sulfophenyl isothiocyanate (SPITC) and (iii) derivatising the epsilon-amino group of lysine residues with Lys Tag 4H. Derivatisation with CAF and SPITC resulted in more protein identification than Lys Tag 4H. Sulfonation using SPITC was the preferred method due to the low cost per experiment, the reactivity with both lysine and arginine terminated peptides and the resultant simplified MS/MS spectra. KEY WORDS: cross species matching; de novo sequencing; MALDI MS/MS; guanidination; sulfonation. INTRODUCTION Proteomics research encompasses an increasing array of techniques to separate, identify and cha- racterise proteins from a wide range of organisms. A typical scenario includes protein separation by either two dimensional electrophoresis (2D PAGE) or HPLC, proteolytic cleavage with trypsin, followed by mass spectrometry (MS) for protein identification. For organisms with a sequenced genome, the peptide mass spectra is compared to theoretical masses in protein databases for identity. Greater confidence in correct protein assignment can be achieved by con- ducting collision induced dissociation (CID) of pep- tides to create candidature MS/MS sequence for database matching. When studying species that do not have a well defined genome, protein identification can often be obtained by using cross species matching. This tech- nique relies on the presence of highly conserved regions of amino acid sequence between similar pro- teins from different species, and the existence of these sequences in protein databases. However, where amino acid substitutions have occurred between * Australian Peptide Conference Issue. **This project was funded by an ARC Linkage grant to Deane supported by TGR Biosciences and facilitated by access to the Australian Proteome Analysis Facility established under the Australian GovernmentÕs Major National Research Facilities program. 1 Division of Environmental and Life Sciences, Department of Bio- logical Science, Macquarie University, Sydney, NSW, 2109, Australia. 2 Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW, 2109, Australia. 3 CSIRO, Division of Entomology, GPO Box 700 Canberra ACT, Australia. 4 Correspondence should be addressed to: Elizabeth M. Deane, Division of Environmental and Life Sciences, Department of Biological Science, Macquarie University, Sydney, NSW, 2109, Australia. Tel.: +61-98508418; Fax: +61-98509671; e-mail: ede- ane@els.mq.edu.au. International Journal of Peptide Research and Therapeutics, Vol. 12, No. 3, September 2006 (Ó 2006), pp. 225–235 DOI: 10.1007/s10989-006-9026-3 225 1573-3149/06/0900–0225/0 Ó 2006 Springer Science+Business Media, Inc.