The Homepage of Sequence V1.5

Sequence V1.5 is a piece of software for collision induced dissociation mass spectrometric sequencing of peptides prepared by combinatorial split-and-pool methodology. Sequence V1.5 works for both cyclic and linear peptides, and combines library enumeration and sequencing into one package. Any number of mass spectra may be supplied, with arbitrary MSn steps. Common peptide fragmentations are understood, and the user may specify unnatural residues and end groups.

Supplied with a simple to use Excel spreadsheet user interface, the software can be mastered without detailed knowledge of mass spectrometry or computer programming. All results are conveniently gathered in regular worksheets for viewing, printing and archiving. The expert user can tailor the software using VBA, or another language of choice, to create a customized interface.

Kombyonyx.com are pleased to be able to offer other free software for download. Our barcoding software creates standard barcodes in Excel and the MOtoPOV utility helps to generate POV-Ray raytraced pictures of molecular orbitals calculated using MOPAC6. The isotopes calculator is a simple program for calculating isotope distributions from a molecular formula.

Introduction to Sequence V1.5

The split-and-pool combinatorial synthesis method enables the efficient solid-phase preparation of libraries consisting of a single library member per resin bead. Miniaturized assays are used to screen the library and determine the activity of the library members. A means of determining the structure of the library members is required and a variety of methods have been proposed for this purpose. For libraries of peptides and peptide-like compounds single bead Edman degredation is a possibility, although due to the requirement for an N-terminus it does not work for all classes of compounds (e.g. capped peptides, cyclic peptides). Sequencing by mass spectrometry is an attractive option due to the requirement for only a small sample size and the high speed of analysis. Various strategies for chemical encoding and ladder preparation to aid mass spectrometric structure determination have been described, although many of these require additional complications to the library synthesis, and may result in contamination of the library products with the encoding molecules.

Direct sequencing of peptides by collision induced dissociation (CID) or post source decay (PSD) has commonly been applied in proteomics applications. We wished to employ this methodology for sequence determination of library peptides as it does not require any encoding/capping steps during the library synthesis. Using automated spectral acquistion and data analysis this technique has the potential for great speed, permitting sequencing of both library "hits" and inactive compounds for construction of preliminary QSAR.

The program Sequence V1.5 was developed specifically for the sequencing of peptides from split-and-pool combinatorial libraries using CID mass spectrometry.

The Sequence V1.5 software was first publicly released at the Fall Meeting of the American Chemical Society, Boston, 2002. It was originally developed and tested by James E. Redman, Keith M. Wilcoxen and M. Reza Ghadiri at The Scripps Research Institute. JER thanks the Wellcome Trust for a postdoctoral fellowship (061454).

How does it work?

Sequence V1.5 permits the user to specify the residues used at each stage of a split-and-pool library synthesis of linear or cyclic peptides. The program determines the compounds that the library should contain, taking into account the cyclic permutation symmetry of cyclic peptides.

The user supplies the program with mass spectra of the library member of interest. Normally these would be a parent ion spectrum and a CID spectrum, although any combination of MS and MSn spectra are acceptable. The potential library members are scored according to how well a predicted mass spectrum agrees with that experimentally observed. This allows elimination of unlikely candidates. Spectra are considered in order, progressively eliminating more sequences, to finally leave a ranked short-list of sequences. Ordinarily a parent ion spectrum would be considered first, followed by the CID spectrum. The software understands commonly observed fragmentations of the peptide backbone and losses from side chains.

In an optional final step, pairs of sequences on the short list are compared to each other. This calculation which we call "critical analysis" (CA) highlights any ambiguities and can sometimes identify the correct sequence even when it is not at the top of the shortlist. Other features include the ability to generate graphical overlays of predicted and experimental spectra.

Sequence V1.5 is provided as a dynamic link library (DLL) for use on Windows based PCs. It can be scripted using VBA so can be incorporated into many other commercial applications such as databases and spreadsheets. We supply an Excel user interface that provides easy menu and worksheet based access to most features of the software, without any need for a knowledge of programming.

How do I get it?

Sequence V1.5 is available free of charge. The full package includes the Sequence program itself, the Excel spreadsheet user interface, the full documentation in PDF format, and some example files which are necessary to work through the tutorial. To obtain a copy please email request~kombyonyx.com where you will need to substitute ~ with @. The subject line of your email should be the word "sequence".

Once your request for the software has been received, you will be emailed a URL and password to permit download. The software is supplied either as a self-installing executable (4 Mb) or as a zip archive (400 kB). The installer will copy all of the necessary files to your hard disk and add a shortcut to the start menu. It is recommended unless you have a slow network connection or need to fit the program on a floppy disk.

Use the program entirely at your own risk. No guarantees are made regarding its performance and there is no support. Please refer to the full disclaimer in the documentation.

Tutorial

This tutorial gives an overview of the steps involved in sequencing a small cyclic peptide using Sequence V1.5 and Excel 2000 running under Windows XP. It assumes that the software and example files have already been installed correctly.

1. Simply double click the SequenceV1_5.xls icon. There is likely to be a warning about macros. Macros must be enabled – you may need to alter your security settings. Also some antivirus software may give a warning about ActiveX controls. If this happens then the control should be allowed to run.
   The SequenceV1.5 control worksheet will then appear. The worksheet will contain many blank cells which will be filled with processing parameters in the next stage.
2. Select Open Job from the Sequence menu, then choose the example parameter file "in.txt" from the file open dialog box. This file will be located wherever you unzipped the Sequence V1.5 package.
   The blank cells in the worksheet will then be filled with a typical set of parameters. Most of these can be left alone, but the spectrum filenames will need to be changed.
3. Change the filenames in the "Parent Spectrum" and "Fragment Spectrum" cells to the mass spectrum files that are to be processed.
   To save typing, right click in the cell and choose "Insert Filename" from the pop-up menu. A file open dialog box will then appear from which a file can be chosen.
   The example files are KWLWKEparent.stx and KWLWKEfrag.stx and are for the cyclic peptide c[KWLWKE] (italic residues in the D configuration).
4. Select Open Masses from the Sequence menu. Choose the file "monomstab.txt" which contains the standard amino acid residue masses.
5. Choose Process All from the Sequence menu.
   In a few seconds a new worksheet should appear which will contain a summary of the processing parameters and a table showing the 30 top scoring sequences and scores calculated by pairwise comparisons.
   The experimental spectrum overlaid with the calculated peaks of the "best" sequence is shown at the bottom of the worksheet. Using the example parameters provided, the program assumes that the peptide originated from a library with the first two residues fixed to KW and the remaining four residues can be any of EFHKLNRSW.

That's all there is to it! A convenient batch process command will automate steps 3 and 5 if you have many files that need to be processed using identical parameters. The results will be summarized in a single worksheet, along with the more detailed analyses in separate sheets.