Bart's Cookbook--

Using Pepsort and Mobility

The programs Mobility and Pepsort can be used together to predict and plot the theoretical mobilities of peptides in two dimensional thin layer analysis. The plots show electrophoresis in the first dimension and ascending chromatography in the second dimension.

They are written with the GCG v8.1 procedure library and use GCG file formats and interface. You have to have the GCG suite of DNA analysis programs to run these programs. Additionally, to view the Mobility plot, and to print it, you need to use a terminal emulation program that will emulate a Tektronics 4105 terminal. Versaterm Pro is one such program. Regular Versaterm does not have this feature, nor do most Telnet tools.

The GCG programs can be confusing to run using Tektronics 4105 emulation. It is possible however, at least using Versaterm Pro, to run the program using VT220 emulation and switch to Tektronics 4105 emulation for the display and printing of the Mobility plot.

Pepsort is just a modification of the GCG program Peptidesort. Like Peptidesort, Pepsort reads a GCG peptide sequence file and prompts you for chromatography buffer type, the pH of the electrophoresis buffer, and the protease that you are using.

The output file contains all the same information as Peptidesort (peptide weight, HPLC retention, the chemical properties of the amino acids in the peptide, the composition of each peptide, and a summary of the composition of the whole protein).

Pepsort calculates two additional values, the electrophoretic mobility (Emobile) and the chromatographic mobility (Rf). These are the two values that are plotted by Mobility. The output file from Pepsort is used as the input to Mobility.

Mobility is simply a plotting program that plots the mobility of the peptides calculated by Pepsort. You therefore have to run Pepsort before Mobility.

To start Pepsort, type Pepsort at the $ prompt after starting GCG.

Pepsort has a few command line modifiers that most people will find useful. As is the case with all VMS command line switches, these must be typed into the command when you start the program. You cannot go back without quitting the program.

If you plan to oxidize your sample with performic acid, use the /CYSTEICACID command.

If you are interested in phosphorylated peptides, specify the residue number of the phosphorylated residues with the /PHOS= command.

If there are bonds that won't be cut, (for example, trypsin won't cut KP or RP bonds) specify the residue after which the virtual protease should not cut with the /DONTC= command.

Finally, you can have the program calculate data only for specific peptides. For this, use the /SELECT= command. The select command is of marginal value since you can easily instruct the Mobility program to plot only specific peptides. You can use all of these command line modifiers at one time. Just run them together with /'s and avoid inserting spaces.

 

Mobility


Mobility reads a Pepsort output file and prompts you for the number(s) of the peptide(s) you want to plot. Peptides that contain phosphorylated residues are marked by diamonds.

To start, type Mobility at the $ prompt.

Mobility customizes the axes that it uses to plot the peptides so that the set of peptides that are plotted uses the full width of the display. Therefore, plots of two different sets of peptides may have different axes and may therefore not be directly comparable visually. An alternative is to plot the Rfs and Electrophoretic Mobilities (Emobile) of the peptides calculated by Pepsort by hand on graph paper with a pencil. Those unfamiliar with this powerful but old-fashioned technique should consult senior colleagues. The Rf and Emobile values can be found in the Pepsort output file. They are the two values that precede the amino acid composition portion of the report. There is a table heading near the top of the pepsort file that indicates, somewhat crudely and confusingly, what each of the calculated values is.

Finally, be aware that the prediction of peptide mobility is a very inexact science. Protease digestion often does not go to completion. Additionally, proteases occasionally cleave non-canonical sites. Finally, peptides can undergo unwanted and unpredictable chemical modification during preparation and therefore exhibit unexpected electrophoretic or chromatographic properties. You should view the output of Pepsort and Mobility as helpful, but by no means definitive. On the other hand, it is extremely accurate in predicting the mobilities of the phosphopeptides containing tyrosines 394 and 505 of Lck.

 

The methods used in this program are described in Chapter 11 of Methods in Enzymology, v. 201, 1991, Academic Press.

 

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