The Use of MALDITOF Mass Spectrometry and Amino Acid Sequence Analysis in Characterising Small Amounts of N-Blocked Protein
One hundred picomoles of a high mobility group (HMG) protein were isolated by reverse phase hplc from an extract of pea nuclei. Amino acid sequence and composition analysis of half the sample showed the protein to be blocked at the N-terminus. Of the remaining material, 40pmol was subjected to digestion with proteinases and the peptides from the tryptic digest were separated on reverse phase hplc. Each eluted peak was examined by matrix-assisted laser desorption time-of-flight (MALDITOF) mass spectrometry and amino acid sequence analysis. From the resulting information, it was clear that the target protein sequence correlated with the inferred sequence of a previously isolated pea leaf cDNA encoding an HMG-I-like protein. The expressed protein was smaller than the DNA sequence suggested. From a series of further digests on 1–2pmol of protein, the likely identity of the N-terminal block was established as well as several sites of C-terminal processing. This works illustrates how extensive amounts of data can be derived from a small amount of protein by the combined use of sequence analysis and MALDITOF mass spectrometry.
KeywordsReverse Phase Hplc High Mobility Group Amino Acid Sequence Analysis Cyanogen Bromide High Mobility Group Protein
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- Bartlett-Jones, W.J., Hansen, H.F. & Pappin, D.J.C., 1995, The use of volatile N-terminal degradation reagents for rapid, high-sensitivity sequence analysis of peptides by generation of sequence ladders, this volume Google Scholar
- Johns, E.W., 1982, The HMG Chromosomal Proteins, Academic Press, LondonGoogle Scholar
- Pwee, K-H, Webster, C.I. & Gray, J.C., 1994, HMG protein binding to an A/T-rich positive regulatory region of pea plastocyanin gene promoter, Plant Mol. Biol., in pressGoogle Scholar
- Talbo, G & Maim, M.,1994, Distinction between phosphorylated and sulfated peptides by matrix assisted laser desorption ionization reflector mass spectrometry at the sub-picomole level, in Techniques in Protein Chemistry V, J Crabb (ed), Academic Press, New York. pp 105–113Google Scholar