Manual

Peptide Properties Calculations:

This utility allows you to calculate the following properties of a peptide based on the sequence:

1. Calculate the estimated molecular weight of a polypeptide
2. Estimate the partial specific volume of a protein and correct the v-bar value to the temperature of your experiment
3. Estimate the extinction coefficient at 280 nm of the denatured protein according to the method by Gill and von Hippel.
4. List the amino acid composition of a peptide

Note: This program will utilize the database backend of UltraScan. This functionality allows you to save certain files and data in a database, among them the peptide files analyzed with this module. If the database system is not supported or available on your system, you will get a warning message when opening this module.

The peptide properties calculation module can be loaded in one of three ways:

1. Click on "Calculate Protein MW and vbar" in the Utility Menu from the Main Menu of UltraScan.
2. Click on the vbar button from within any of the analysis windows in UltraScan
3. Through the database function "Commit Data to DB:Buffer"

When using the second option, the temperature of the experiment will automatically be used to correct the partial specific volume obtained from the sequence analysis.

Explanation of fields and buttons:

	Peptide Sequence Loading: You can load peptide sequences into this program by one of two methods: Load Sequence from HD Use this button to load an ASCII sequence file from your harddrive. A file dialog will open and allow you to search for *.pep files. The protein file should have the extension ".pep" and should be located in your UltraScan Root Directory. Query Peptide from DB If you are using a database backend for UltraScan you can query the database for peptide sequence files that have previously been committed to the database. Sequence files available in the database will be listed in the listbox once you click on this button. More information about various database functions is available elsewhere.
	Select Investigator: When searching for peptide sequences from the database, you can limit your search to only those sequences belonging to a specific investigator in the database. When you click on this button, the investigator selection dialog will open up and allow you to select the desired investigator. When the sequence is loaded from the database, this option is disabled, since an investigator is already associated with the selected sequence. Also, when committing a sequence to the database an investigator needs to be chosen to be associated with the sequence.
	Help: Show this help file Close: Close this function Save Peptide to DB: Use this function to save a peptide sequence to the database. Before storing a sequence in the database, you need to have a copy of the sequence stored locally, for example, on your harddrive. You can then select the sequence to be stored in the database from a filedialog and after selection, commit the sequence to the database. Delete Sequence from DB: Delete the selected sequence from the database. This function requires administrator privileges. Download Sequence: This button will open a Netscape window with a link to the Swiss Protein Database Search form. From there, you can search and download the sequence for your protein (provided it is already entered into the Swiss Protein Database). After finding the appropriate sequence, save it as "text" with the extension ".pep" in your UltraScan Root Directory More Information: Clicking on this button allows you to review the amino acid composition and the vbar calculation results. The results are always written to a file called <peptide file name>.res Peptide Reset: Clear the loaded peptide sequence from memory
	Selected File: The location of a Swiss Protein database sequence entry file that can be imported directly into UltraScan. See also the information on the "Download Sequence" button. This field is blank if the sequence was retrieved from the database, and only applies to sequences loaded from the harddrive. Protein Description: The name of the protein Temperature (in oC): The temperature of the experiment vbar (temperature): The partial specific volume of the protein corrected to the experimental temperature vbar (20 oC): The partial specific volume of the protein at 20 oC. All vbar calculations are based on the method by Cohn and Edsall (Proteins, Amino Acids and Peptides as Ions and Dipolar Ions. New York. Reinhold, 1943.) E280 (denatured): The extinction coefficient at 280 nm for the peptide in a denatured form based on the method by Gill and von Hippel in Anal. Biochem. 182:319-326 (1989). This number is based on the 280 absorbance of Tyrosine, Cysteine and Tryptophan in 6 M urea. Use at your own risk - measuring this value will be more accurate. UltraScan will require extinction coefficients for the calculation of equilibrium constants. # of Residues: The number of amino acids in the sequence Molecular weight: The theoretical molecular weight obtained by summing the partial amino acid molecular weights, and subtracting one less water molecules than amino acid residues present.

You can generate your own peptide sequence file, and import them into UltraScan, provided the sequence file follows the correct sequence file format:

Required Fields in Database file: When generating your own sequence file, you will have to follow the Swiss Protein Database file format convention. However, not all fields are required for correct sequence parsing. The fields that are required are:

• "DE" - Sequence Description tag. Prepend any description line with this tag to identify the description of the sequence. Note: Only the first line is parsed, all others are ignored. There can only be one sequence/file.


• "SQ" - Sequence Entry. Use this tag to identify the start of the sequence in a line preceeding the actual sequence, In the line following the sequence place the end-of-sequence marker "//"

DE   LYSOZYME C PRECURSOR (EC 3.2.1.17) (1,4-BETA-N-ACETYLMURAMIDASE C)
SQ   SEQUENCE   147 AA;  16238 MW;  81E85743FF579468 CRC64;
     MRSLLILVLC FLPLAALGKV FGRCELAAAM KRHGLDNYRG YSLGNWVCAA KFESNFNTQA
     TNRNTDGSTD YGILQINSRW WCNDGRTPGS RNLCNIPCSA LLSSDITASV NCAKKIVSDG
     NGMNAWVAWR NRCKGTDVQA WIRGCRL
//