Introduction to Proteomics – Aug 6,2010– Database Exercise

A. C. Harms and M. M. Vestling

Goal:  To learn how to use several public calculators and small molecule databases

1.a)  Find the largest unmodified tryptic peptide (no missed cleavages) in the protein human histone H2B.  (Hint, you can change the format to  FASTA instead of the default GenPept in the display option.  Be careful not to choose a fragment or a histone-like protein.)

http://ncbi.nlm.nih.gov

http://prospector.ucsf.edu [MS-Digest]

1.b)  Find the known post-translational modifications for human histone H2B.  The following database is at your fingertips:

http://www.expasy.uniprot.org

2.)  Phosphokinase A is itself phosphorylated.  One bit of phosphorylated sequence found in its tryptic digest is KGSEQESVK.  Use BLAST to see if other proteins contain this sequence.

http://www.ncbi.nlm.nih.gov/BLAST

[M. J. Chalmers, K. Hakansson, R. Johnson, R. Smith, J. Shen, M. R. Emmett, A. G. Marshall, Protein kinase A phosphorylation characterized by tandem Fourier transform ion cyclotron resonance mass spectrometry, Proteomics 4: 970-981 (2004).]

3.) Your peptide with the sequence SLHTLFGDELCK has the unexpected mass of 1419.694.  How is it modified?  Hint: first calculate the mass of the unmodified peptide.  Then go to the ABRF web site for Delta Mass.  Be alert to what values each program is giving you:  M+, [M+H]+, [M-H]-, monoisotopic, or average.

http://prospector.ucsf.edu [MS-Product]

http://www.ionsource.com/programs/pepcalc.htm

http://rna.rega.kuleuven.ac.be/masspec/pepcalc.htm

http://www.abrf.org/index.cfm/dm.home

4.)  You think your peptide, KGSEQESVK, may be phosphorylated.  Use the mass of the peptide plus Delta Mass to calculate what negative ion you should look for.