Chapter

1

Introduction

Getting started with GPMAW (General Protein/Mass Analysis for Windows).

What is GPMAW 1.1

As the name implies, GPMAW is a program for the analysis of protein primary structures, particularly using mass spectrometry.

The initial purpose of the program was to answer the simple question: given a certain peptide mass how many peptides can be constructed that fit with a mass within the given precision (see Chapter 6). The answer to this trivial question is easily solved with a computer while it is rather tedious and error-prone using a calculator.

Since then the program has expanded in a number of directions, but most of these are characterized by the fact that they are based upon previous knowledge of a known sequence. This has necessitated the interfacing to protein databases, with all the problems of exponentially growing data-input (from less than 10,000 proteins in 1988 to more than 700,000 proteins today, EMBL non-redundant database).

Having loaded or entered your sequence (GPMAW interfaces to a number of other file formats and hardware), the sequence can be post-translationally modified and cross-links established (Chapter 3). The protein can be cleaved based on a number of parameters (using enzymes or chemicals) and the resulting peptides can be sorted, viewed, and further characterized based on a number of parameters (Chapter 9).

One of the most recent advances in mass spectrometric protein identification, digest mass search, is shown in Chapter 8, but also features like ms/ms fragmentation are covered (Chapter 10).

When performing mass spectrometry or proteins and peptides you are very likely to perform additional protein chemical procedures. For this purpose a number of features has been included: Searching a protein for amino acid composition, displaying the hydrophobicity index, reversed phase retention data of peptides, predicting secondary structure, comparing sequences with dot-plots, charge vs. pH graphs of peptides and proteins, digest HPLC chromatogram and mass spectra of peptide mixtures, etc.

Installation 1.2

Pre-installation notes

i Note: As the installation system is likely to change over time, particularly the actual content on the distribution media, you should always check the installation package for additional information.

GPMAW is presently (April 2002) distributed on a single CD-ROM containing the program and all auxiliary files including two protein databases (see Appendix B for more information on databases). Alternatively, the program may be shipped on three floppy disks without protein databases.

The installation takes place by running the setup program from Windows. In the case of CD-ROM based installation:

If you have auto-notification turned on, the installation program will start as soon as you insert the CD-ROM in your computer. If you do not have auto-notification turned on, you will have to either open the file explorer and navigate to your CD-ROM drive and start the ‘Setup’ program. Alternatively you can select the ‘Start’ button. From the menu select ‘Run…’. In the ‘Run’ dialog box you enter ‘D:SETUP’ and select ‘OK’ (assuming that ‘D:’ is your CD-ROM drive). Follow the instructions.

The file on the CD-ROM called Setup.exe is the installation program. This file contains all the components for installation except for Gpmaw.chk, which is the separate license file, also present in the root directory. WizardUI.exe is a wizard that runs just after the installation and prompts you for the basic settings of GPMAW.

The installation program also contains two protein databases, the Swiss-Prot database and the EMBL non-redundant protein database. The databases cannot be accessed on the CD-ROM, but have to be installed. They can be installed across a network on a shared drive for access by several persons.

i NOTE: The Swiss-Prot database is copyrighted. If you are a commercial company you need a license in order to use the database. For non-commercial users (e.g. universities) there is no license requirement.

Installation

The default directory for the installation GPMAW is C:\GPMAW. It is recommended that you choose this directory for installation of GPMAW, as future upgrades and faultfinding will be simplified.

The program starts with checking for previously installed components, specifically it checks for the location of the gpmaw3.exe program file. If the program is found, its location will be taken as the default for an upgrade, otherwise c:\gpmaw will be taken as the default.

The installation starts with a splash screen (a lighthouse) followed by a welcome dialog. Note: the actual installation of files will not take place before all dialog boxes are handled (the last box ends with a ‘Finish’ button).

You are then asked to acknowledge the End User License Agreement before proceeding wit the installation.

i Hint: If you want to perform an additional installation, you can rename the \gpmaw\bin\ directory to something else (e.g. \gpmaw\bin1\) before running the installation program. In this case you have to specify a different destination directory.

The next dialog box gives you the following options:

GPMAW: Installs GPMAW. This option is selected by default – you may deselect it if you only want to install a database.

DBindex: Installs the FastA database indexing utility. For more information, please see Chapter 12.3).

Local BLAST Installs a local copy of NCBI BLAST program. The program works on a local FastA formatted database. Details are presented in Chapter 7.2.

Swiss-Prot: Copies the Swiss-Prot database to a directory on your harddisk (default C:\gpmaw\database\).

EMBL-nr: Copies the EMBL non-redundant protein database to a directory on your harddisk (default C:\gpmaw\database\). As an alternative the NCBI non-redundant may be included. They are based on the same basic databases and should be (almost) identical.

Note that when you an item in the list box is selected the combined size necessary for installation is displayed below the check-box.

If a previous version of GPMAW was detected in the initial part of the installation, the following screen will give you the option of saving all files replaced during the installation in a directory called \gpmaw\backup\. The main files are gpmaw3.exe and gpmaw.hlp. Restoring these to the \gpmaw\bin\ directory will in most cases restore previous installation.

If the installation program did not detect a previous installation of GPMAW it will jump directly to the following options:

Place GPMAW shortcut icon on the desktop. A shortcut will be created on the desktop. Double-clicking on this will open GPMAW.

Associate the .seq extension with GPMAW. When you associate the .SEQ extension it means that when you double-click on a file in Explorer that has the extension .SEQ GPMAW will automatically open and read the file.

You are then asked for a startup folder for the program. As default a name of ‘GPMAW’ is suggested, but you can also choose another name, or select an existing startup folder from the list below the edit line.

The next (last) dialog box informs you that installation will proceed when you press the ‘Finish’ button.

The GPMAW setup wizard

When the program has been installed, the setup wizard takes over. This program quickly walks you through the basic settings of GPMAW. You can at any point select ‘Cancel’ whereupon the default settings of GPMAW will take effect. Only when you run through the complete wizard, the settings chosen on the various pages will take effect.

i Notice: All the settings in the wizard can be set at any point in the program by selecting Setup|Setup system (see Chapter 5 for details).

The individual pages in the wizard work with ‘Back’ and ‘Next’ buttons that enable you to go both forward and backwards in order to customize GPMAW.

The left hand side of each page gives a short description of the functions, further details can be found in the respective chapters (sequence – Ch. 3; peptide – Ch. 9; general setup – Ch. 5).

Errors during installation

If something goes wrong during installation, you may still have the right programs copied to the hard disk, but in the wrong places. Please see Appendix A for directory structure and file information.

If you are unable to reconstruct the program, try to delete all parts of the program and perform a re-installation. If you still do not have a functional installation, call Lighthouse data (Chapter 1.9) either by fax or e-mail.

Un-installation

The best way to remove GPMAW from your system is to open the Control Panel and select the ‘Add/Remove Programs’ applet. This shows you a list of installed programs. From the list you select GPMAW and click on the ‘Remove’ button. All files and registry entries created during installation will be removed, only files created after installation (e.g. sequences etc.) will remain to be removed manually.

Alternatively you can uninstall manually by activating the uninstall program called unwise.exe located in the \gpmaw\ folder.

i As all files are installed under the same directory (C:\GPMAW) you can remove everything by deleting this directory and everything beneath it (remember to move valuable sequence files first). The shortcuts on the desktop and/or the Start menu may have to be removed manually.

Upgrading

Starting with version 3.0 the program has been made ‘upgradeable’ compared to previous versions. This means that if you can connect to the Internet you will be able to download minor upgrades free of charge. In this way, we hope to make bug fixes and improvements immediately available to users without the usual hassle and time delays. Please check the GPMAW web site http://welcome.to/gpmaw (or http://www.protein.sdu.dk/gpmaw/) for more details. If this site is unavailable contact the author on php@bmb.sdu.dk. Free upgrades are available for at least one year after purchase, but may be available for a longer time.

When you buy an upgrade, you will get a full installation while downloaded upgrades contains only the gpmaw3.exe and gpmaw3.hlp files. In the case of the full installation your existing mass and modification files may be overwritten if the names coincide with files from the installation. In this case the installation program should warn you, but the safest course is to make a backup of your \gpmaw\system directory before upgrading. In the case of a ‘raw’ upgrade, only the .exe and the .hlp files are replaced.

When upgrading you will experience an installation program like for a normal installation except that the ‘Desktop icon’ option will be checked, and you will have an extra option ‘Upgrade only’. If you un-check this option you will have the option of specifying a different installation directory for the upgrade (gpmaw3.exe and gpmaw3.hlp). You should only do this if you know you have an installation that is not found, or if you want the new files separately.

When you buy an upgrade, it will always be accompanied by a manual, while upgrades downloaded from the Internet will have to do with the on-line help.

The frequent upgrades have the downside that the printed documentation will often be slightly out of date. In this case you are referred to the on-line documentation, which will be kept up to date and available along with the upgrades. Pressing the <F1> key or selecting ‘Help’ from the main menu can access the online help. <F1> will usually give context sensitive help that is help information pertaining to the dialog box currently open.

Problems during upgrading

When GPMAW is upgraded, the first time it is started, it will sometime complain about a missing file. As the program will in most cases auto-generate a default copy of this file, you should close the program and restart it. If the problem persists, you may contact Lighthouse data (Chapter 1.9).

If, after performing an upgrade, you are informed that your license is no longer valid you will have to ‘downgrade’ to your previous version of GPMAW. You may then contact Lighthouse data to obtain a renewed license (an upgrade). The upgrade price of GPMAW is 50% of the current price for a full version. The upgrade includes a copy of the most recent manual.

Conventions

Angle brackets '<' and '>' are used to denote function and special keys. <F1> to <F12> are the function keys, <Esc> the escape key, <Tab> the tabulator key, <up>, <down>, <left>, <right> the arrow keys and <Ins> (insert), <Del> (delete), <Home>, <End>, <PgUp> (page up), <PgDn> (page down) the page control keys. <Enter> is the ‘enter’ or ‘return’ key.

When the main or sequence/daughter window has the focus you can always access the menu by pressing <F10> or you can access individual menu items by pressing <Alt> + the underlined letter in the menu. When a dialog box has the focus, you can move between controls using <Alt> + underlined letter of the text describing the control or by use the <Tab> key to cycle through all controls. Once a control has the focus, indicated by highlighting or a stippled line around the associated text, you can activate it using the space bar if the control is a toggle function (button, check box etc).

Menu selections like selecting the menu option 'Edit sequence' from the main menu 'Edit' are shown as: Edit|Edit sequence.

A large number of notes, hints and tips are partly framed and marked with the i symbol in the margin.

Mouse functions: Controls are activated in the usual manner using the left mouse button after positioning the mouse cursor on the control. Most windows also have an associated local menu, which is activated by positioning the mouse cursor inside the dialog box or control and press the right mouse button. Commands can then be activated by using the left mouse button as usual, or by pressing the underlined character.

References are either shown in brackets [ ] or listed at the end of each chapter.

Program layout 1.3

Main content

The GPMAW program is built around a few central themes:

Protein sequence management: Reading and importing protein sequences from a variety of sources. Modify the sequences (cross-links, chemical modifications etc.) and saving the sequences to local files for easy retrieval. The sequence can be viewed and colored in various ways for easy navigation.

Protein cleavage: Cleave the protein into specific peptides by enzymatic or chemical means. From the resulting peptide list a large number of parameters can be calculated and the list can be viewed, sorted and displayed in various ways.

Mass search: Given a peptide mass, where in a given protein can it be found. This question can be expanded to search for modifications, ion types etc.

Database mass search: Given a list of peptide masses that originate from a single or a few proteins, what protein(s) does it originate from? A large number of options can be specified, both when searching and when analyzing the results. You may also search a database for a protein of a given mass.

Various: A number of functions which are helpful in protein analysis, but not directly related to the above groups. This comprises functions like composition search, charge vs. pH graph, dot-plot analysis, BLAST searches, ClustalW multiple sequence alignment etc.

Protein sequence management

The simplest way of getting a protein sequence into GPMAW is to enter it directly in the sequence editor (Ch. 4.1). Although perfectly feasible, it is a tiresome and error prone undertaking. The easiest alternative is to import directly from the web (Ch. 2.7), but you may also download a database in FastA or Swiss-Prot format for even speedier access (Ch. 2.6). If you are already in possession of a protein sequence, you can transfer it directly through the clipboard or a text file (Ch. 2.5). The main criterion for importing a sequence into GPMAW is that it has to be in standard 1-letter code.

Once a sequence is entered into GPMAW you should save it to a file on disk. You have the choice of either saving a single sequence to a file or saving several sequences to the same file, resulting in a sequence library. The sequence is saved in a format proprietary to GPMAW (the format is explained in Appendix A) and contains in addition to the name and the sequence, information on cross-linked residues (Ch. 3.5), modified residues (Ch. 3.6) and a free text annotation page (Ch. 3.9).

In order to navigate the sequence without effort, you can display in either 1- or 3-letter code, you may color specific residues or sequences (Ch. 3.3) and you may underline residues. Furthermore, you may highlight parts of the sequence to locate peptides and to calculate coverage.

The sequence window is the basis for most other functions in GPMAW as indicated in the figure above and in Chapter 3.

Protein cleavage

The protein cleavage is based upon a sequence window and a cleavage specification in a particular GPMAW notation (Ch. 9). A large number of proteolytic enzymes and chemical cleavages are pre-defined, and additional ones can easily be added. The cleavage can be modified in a number of ways like limiting the mass range, modify the new terminals, combine cleavage specifications etc.

The results of a cleavage is a peptide list which is displayed in a separate window. The peptide list is shown with a number of paramters and can be sorted by mass, charge, number etc., displayed in 1- or 3- letter code, printed and exported to other programs. You can even select to have a peptide from the list as a the basis for a new sequence window.

In order to easily navigate the peptide list you can color residues or sequence parts of the parent sequence window which will be ‘carried on’ to the peptide window. In the other direction you may underline a sequence in the parent sequence based on a selected peptide in the peptide window.

Based on the peptide window (Ch. 9.4) a number of other windows can be generated. This comprises simulated reversed phase HPLC chromatograms, N-glycosylated mass values, MS/MS of selected peptides, simulate a mass spectrum and compare it to a recorded experimental spectrum, cross-linking mass spectrometric experiments etc.

Mass search

The mass search function tries to answer the question: ‘ Where in this protein can I find this mass’. Like the protein cleavage above, this function is based on a sequence window. You then supply a mass list, either with manual input, read from a disk file or transferred through the clipboard. GPMAW is able to read the peak files from a number of mass spectrometric analysis programs.

The search parameters can be modified in a number of ways to cater for different ms instruments (mass type, precision, ion type) and sample parameters (enzyme, modifications).

The results are displayed in a ‘hit’ list window. The results are shows with a number of parameters, and can be displayed in various ways. A potential hit can be linked to the parent window, and coverage can be calculated.

The ‘hit’ list can be exported to a mass spectrum (GRAMS only) and saved to disk or to clipboard.

Digest mass search

The digest mass search has received its name because it is based on the masses of the peptides obtained from a proteolytic or chemical digest of a protein. The protein is typically obtained from a SDS gel. Similar to mass search above, the digest mass search is based on a mass list that can be entered and read in an identical way.

The mass list is then compared to a derivatized database based on a FastA formatted database. These databases are available on the web, and can also be obtained on CD-ROM from NCBI or EMBL. The derivatization of the database is performed in GPMAW prior to first use (Ch. 8.2).

The result of a digest mass search is a list of proteins ranked in by high scores (Ch. 5.5). The ‘hit’ list can be saved to disk for later analysis or comparison with other digest parameters. Individual entries in the list can be viewed as a detailed report (Ch. 8.5) and can also be retrieved into GPMAW for more detailed analysis.

Starting GPMAW 1.4

Main GPMAW window

You may start GPMAW either by double clicking on the icon on the desktop or by selecting Start|Programs|Gpmaw|GPMAW (Windows 95/98). You are then greeted with an empty window, unless. If you have enabled ‘Autoload last sequence’ (Chapter 5.1) in the most recently loaded sequence will be loaded.

While the program loads a splash screen displaying a lighthouse will be displayed for five seconds. You can remove the picture sooner by clicking inside the picture. The reason for the splash screen is to give you something to look at while auxiliary files are loaded, and also to identify the actual version of GPMAW that is running.

The title bar of the program shows the name (GPMAW), the version (4.04), the program type (32-bit) and the currently loaded user (Default – see Chapter 5.7).

GPMAW is an MDI application (Multiple Document Interface) in a manner similar to a word processor. This means that you can load any number of sequences (documents) simultaneously, each in its own window and all bounded by the main GPMAW window. From each sequence window you can derive a number of daughter-windows, each with a specific function (e.g. peptide window, mass search results, HPLC graph etc.).

You normally control the program in by a combination of the following:

Ø Use the mouse or the keyboard to activate the menu options.

Ø Use the mouse to activate the speed buttons in the toolbar.

Ø Right-click the mouse in the relevant window (this opens a context-sensitive pop-up menu).

Ø Use the keyboard shortcuts (see page ii after ‘Contents’).

i Note: The pop-up menu that is accessed by right-clicking in the given window is both context sensitive (that is the content depends on the window) and may vary for different parts of a given window. This will usually be the quickest way of accessing commands.

Functions

Most functions in GPMAW work directly on protein sequences, which means that you have to get a sequence into the program. This is accomplished either by reading a sequence in GPMAW- or text format (Chapter 2.1), reading from a database (Chapter 2.6), pasting from the clipboard (Chapter 2.5), or entering a sequence directly into the sequence editor (Chapter 4.1).

Without a sequence loaded into the program, the options available can be summarized as loading a sequence, performing a digest mass search (Chapter 8), a few peptide mass comparison functions (Chapter 6.2) and the utilities described in Chapter 12.

As soon as a sequence is loaded into a sequence window (Chapter 3), a number of menu options are enabled and the menu line is expanded

Whenever you switch between sequence windows and other daughter windows (peptide windows, graphs etc.), the content of the menu will change to reflect the options available.

The Toolbar

The main window toolbar consist of several smaller toolbars that can be moved around and rearranged. You move a toolbar by grabbing the vertical bars at the left end of the toolbar with the mouse and drag the toolbar to the position wanted. The other toolbars may rearrange to accommodate the new position.

i NB. The presence and position of each individual toolbar is saved in the ‘ini’ file when the program closes. This means that they will appear in the same positions when the program re-opens.

Each small toolbar can be turned on and off, either by clicking on the down-arrow next to the ‘Help’ question mark or by right-clicking in an unused part of the main toolbar window (thus calling on the pop-up menu). Each visible toolbar is indicated by a check-mark.

All toolbar buttons perform commands that are duplicated in the menu. Furthermore, some of the commands can be accessed through the pop-up menu (right-click the mouse) local to each individual window.

Eight toolbars are available. Some of the commands will only be available when a sequence window is has the focus (e.g. ‘Save’, graph commands etc.).

Below the commands of the individual toolbars are mentioned and in which chapter to look for more information.

FILE

Open sequence (file, chapter 2).

Close sequence window.

Save sequence to file (chapter 2.3).

Print

Printer setup

EDIT

Edit current sequence (chapter 4.1).

Edit new sequence (chapter 4.1).

Edit cross-links of current sequence (chapter 3.5).

IMPORT / EXPORT

Import from file (chapter 2.5).

Import from clipboard (chapter 2.5).

Search FastA formatted database (chapter 2.6).

Search Web Entrez (chapter 2.7).

Copy to clipboard.

BASIC

System setup (chapter 5).

On-line help.

SS-button – enable/disable disulfide bridges (oxidized/reduced Cys). See below.

Mass file selection list (drop-down list). The list is gray when the standard mass file (AA_MASS) is selected, white for all other mass files. See also chapter 4.2.

Exit – close GPMAW.

GRAPH

Hydrophobicity (chapter 11.3).

Secondary structure prediction (GOR - chapter 11.2).

Charge vs. pI (titration - chapter 9.4).

Dot-plot graph (chapter 11.6).

SEQUENCE

Highlight residues (motifs – chapter 3.3).

Search for mass (chapter 6.1).

Automatic cleavage (digest - chapter 9.1).

Ms/ms fragmentation (chapter 10.1).

WEB

Internet based sequence retrieval based on accession number (chapter 2.7).

VARIOUS

Digest (peptide) mass search (PMS – chapter 8).

Simulated 2D gel (chapter 12.4).

Composition calculator (chapter 12.2).

When using the mouse you can get additional information on a number of controls (particularly the toolbar buttons) by letting the mouse cursor rest on top of the control for a second or two. A tool tip will appear explaining the function of the control.

The 'Print' button is shared for all MDI windows (sequence and daughter windows). Most local pop-up menus (right-click with the mouse) have a copy of the print command as have the ‘File’ option of the main menu. This button cannot be accessed when displaying terminal dialog boxes, but these will have their own print button (if applicable). Most printing from GPMAW will be black and white, even if you have a color printer connected to your computer. When you copy a graph to the clipboard (Chapter 11.1) it will be pasted in color enabling you to print graphs in color through other programs.

The 'SS' button enables and disables cross-links. In the 'SS' state, Cys residues are calculated in the oxidized state (mass 102 Da), while in the 'SH' state Cys residues are calculated in the reduced state (mass 103 Da). If the mass defined in the current mass file (Edit mass file, Chapter 4) does not correspond to 102/103 Da., this button will be disabled (this will typically be the case when cysteine residues are carboxymethylated). The default state of the 'SS' button is defined in 'Setup system parameters' (Chapter 5.1).

The 'Mass file drop-down list' enables you to change the masses of any (or all) amino acid residues on all opened sequences by selecting a new mass file. In addition to sequence windows, peptide windows are modified. All other daughter windows will not be changed. You have to recreate the derived windows (like mass search) in order for the modified masses to be displayed. The most typical use of this function is when you modify an amino acid residue, for example when you carboxymethylate cysteine residues (for more details see Chapter 4.2, Edit mass files).

The background of the drop-down list will be gray when the standard mass file (AA_MASS) is selected and white when another mass file is selected.

The 'Exit' button closes all MDI windows and exits the program. In the 'System setup' (Chapter 5.1) you can enable a dialog box that asks you before closing down. If you have made changes to a sequence you will also be asked to save the sequence before closing down.

Non-sequence dependent functions

A few functions are not dependent on a pre-defined sequence. This comprises 'Database mass search' (Chapter 8), the 'Mass difference' functions (Chapter 6.2, Mass difference) and the functions of the Utilities menu (Chapter 12).

Essential tables 1.5

A number of tables in GPMAW are essential for the function of the program. Premier among these tables is the mass table that defines the composition, name and abbreviation of each amino acid residue. The mass table works in concert with the atom mass table that defines the mass of each atom used for calculating the mass of compositions.

Modification tables specify the mass of chemical modifications that are also user definable.

In addition a number of tables are hard-coded into the program (they cannot be changed by the user). These include tables for calculating HPLC retention times, pI values, etc.

More information and the editing of the various mass and modification tables are explained in detail in Chapter 3.

Atom mass table.

The atom mass table is edited through the Edit|Edit mass file command (Ch. 4.2). The file is global to all mass calculations carried out in the program. For this reason there is only one copy of the table that is saved in the ‘ini’ file (initiation file). The atom mass table can at present contain 10 atoms.

i Note: Any change made to this file will affect all mass calculations carried out by GPMAW!

Mass file.

The mass files consist of a table of amino acid residues that are active in the current session of GPMAW. The table consists of 31 entries, each of which contains 1- and 3- letter abbreviation, name and the atomic composition of an amino acid residue.

i Note: The mass of each residue is not saved as part of the table, but is calculated based on the values in the ‘Atom mass table’ (i.e. changes to the atom mass table will change the values of the current mass file).

The first entry denotes an unknown residue ‘X’ that has a mass close to 110 Da (the mass of an average amino acid residue). The following 20 entries contain the 20 ‘standard’ residues, while the last 10 entries can contain any kind of modified residue (see also ‘Modification table’ below).

The mass file is also global to all mass calculation tables in GPMAW, but can be easily changed through the drop-down box in the main window toolbar.

i Note: Although the mass file is global, already calculated values may be fixed. However, most windows like the peptide window will change on-the-fly.

Different mass files are usually created in order to accommodate modifications to amino acid residues that are global to a given sequence. A typical example is the carboxylation of cysteine residues. The standard installation of GPMAW is supplied with a number of the most common cysteine modifications (pyridyl ethylation, carbamido methylation, cysteic acid etc.). These files can also be downloaded from the web site.

i Note: If you use some of the ‘extra’ amino acid residues (after residue 20) you will have to include them in all the modification files you use (e.g. both in aa_mass.mss and pe_cys.mss (pyridylethylated cys) if you use both files).

Modification file

Modification files are tables of amino acid compositions that represent potential modifications to amino acid residues. An example could be methylation of carboxylic acid residues (i.e. a change of +C1H2 valid for Glu, Asp and the C-terminus). As modifications can result in both the addition and removal of atoms, the compositions can be both positive and negative (see Chapter 4.4 for composition formulas).

Only a single modification file can be loaded at any time. The scope of a modification file is global (e.g. a single file covers all sequences loaded at a given time). This means that if you have different sequences loaded, you should construct your modification files to contain common modifications. However, in many cases (e.g. sequence windows) information is extracted from a sequence file and the information stored along with the sequence. This means that the information does not disappear or change when you load a different modification file.

The modification files are used in a number of cases: Modification of residues in sequences (Chapter 3.6), when comparing peptide masses (Chapter 12), and searching for mass matches in a sequence (Chapter 6.1).

Modification files can contain 30 entries each and are saved in the ‘System’ directory under \gpmaw\.

N- and C-terminal modification

The N- and C-terminal modifications are similar to the entries in the modification file (above), but are restricted to modifications of the respective terminals. The state of the terminals can be set when editing the sequence (Chapter 4.1), but also when you digest a protein, can you set the state of the newly generated peptide terminals.

The composition and modifications of the terminals are edited through the menu Edit|Edit modifications (Chapter 4.3) and are saved in a file called ‘TERMINALS.MSS’. You are limited to 10 modifications (+ 1 normal state of the terminals, ‘H’ and ‘OH’ for the N- and C-terminal respectively).

Window menu 1.6

The 'Window' entry in the main menu is only concerned with the overall control of MDI windows. The menu contains five commands:

Cascade (Ctrl+F5)

All open windows on the desktop will be resized to the same format, and each window will be positioned slightly below and to the right of the previous one in order to make the title bar of all daughter-windows visible.

Tile (Shift+F5)

All open windows on the desktop will be tiled, that is resized, and rearranged so they cover the complete desktop

Fit sequence window

The height of the sequence window will be changed (increased or decreased) to fit the sequence displayed.

Tile sequence window

When you have multiple daughter windows open (e.g. hydrophobicity, peptide, mass search, secondary prediction etc.) the display can quickly become confusing and difficult to manage. By selecting this option, the currently selected sequence window will move to the bottom of the display (the height will be made to fit) and the daughter windows will be tiled above. Non-related windows will be buried under these windows.

Arrange icons

All iconized daughter windows will be arranged at the bottom left of the GPMAW main window.

Minimize all

All daughter windows will be iconized and arranged from the bottom left corner of the GPMAW main window.

MDI windows on the desktop

Below the window arranging commands all open MDI (sequence and daughter) windows are listed. An underlined number precedes each window (up to 9). This means that you can change the focus to a particular window by pressing <Alt>+W followed by the underlined number.

Recent program changes (from version 3.0) 1.7

Version 3.0 to 3.1

Only the most important changes are presented.

Peptide mass search: 1) Results are now presented in a non-modal window. This means that if you have the database on-line you can retrieve sequences from the database and work with the sequences without closing the search results. The result windows stay on top to remind you to close it before carrying out a new search (you can move it out of the way in order to work in GPMAW). 2) Hard copies now include sequences used in addition to masses. 3) Masses are now shown in long printouts. 4) The derived mass file is now a combination of average and monoisotopic masses. 5) You can select a cross-over point for monoisotopic to average masses. 6) A detailed report can be viewed, copied and printed. 7) Sorting by optimized score now also shows the optimized peptides and more.

The mass search results now support a local menu (right mouse click) with the option of saving the results in a tab-delimited format for spreadsheet import.

Import ASCII is now divided into 'from file' and 'from clipboard'. Numbers are ignored even if they are part of the letters in the current mass file.

Mass column input tables now has cut and paste of entire columns (mass search and digest mass search). Import of Bruker peak data files in mass column tables. You can paste mass values from GRAMS (peak data) directly into mass column tables.

Sequence info dialog box shows inverted and underlined sequence regions. Multiply charged ions now have a separate page.

Peptide list printout has been changed - it is now possible to print both monoisotopic and average masses. It is possible to select part of a peptide list . The maximum number of peptides reported after cleavage has been increased to 1000 (16-bit) and 4000 (32-bit). There is now a ‘disulphide linked peptide predictor’ accessible from peptide list window.

The protein cleavage dialog box has changed as new features have been added (mass limit, no cleavage if modified residues, manual cleavage w. pre-cleavage, exchange of all exchangeable hydrogen).

pI is now calculated both as SH (pK 8.3) and SS in sequence and peptide info. In other places (peptide mass search list) it is calculated based on the status of the SS button. You can now show the charge vs. pI curve for intact proteins (Graph | Charge vs. pI). The graph display has been improved.

When right-clicking on the total mass in a sequence window (upper left corner) a pop-up menu will show the one to six times charged mass.

The open sequence dialog box now also shows the length of the protein and the size of the sequence database. The maximum size of the sequence database has been increased to 62000 bytes. When a sequence is saved to an existing file, the old file is saved with the extension '.BAK'.

Both sequence and peptide windows are recalculated when changing mass files.

Double clicking on the residue and selecting color at the bottom of the ‘Change modification’ dialog box can color individual residues. Highlighting of sequence motifs are now reflected in peptide windows. You can clear underlines and convert highlighted areas to underlines.

You can now select ‘autoload’ to automatically retrieve the most recently accessed sequence.

‘Ladder sequence’ can now use N- and C-terminal modifications from parent sequence. You can show more than 20 sequences in ladder sequences.

Sequence windows will now print in line modula 5 (3-letter) or modula 10 (1-letter). Limited color support in sequence printing (when selected from the print dialog box): Underline, highlight and modified residues (non- highlighted areas), cross-links.

Version 3.1 to 5.0

Digest mass search (Chapter 8): The results dialog has been redesigned, with a window for comparing input and database masses, a graph for judging the precision of mass hits and a slightly redesigned result table. The speed of the search has been increased. More information is included in the extended report.

It is now possible to do multiple searched in semi-automatic mode with post-analysis of the data based on peak files on disk.

The generation of the peptide mass database is now a four page ‘Wizard’ that leads you through the creation process.

Automatic cleavage: You can modify terminals in the digest. Multiple digests can be combined. Overlapping residues can be removed.

A Cleavage analysis window (Ch. 9.2) enables you to get a quick view of the effect of choosing various cleavage agents, making the decision on the type of cleavage agent faster.

Ms/ms and sequence ladder windows accept modified residues. Multiply charged peptide masses can be listed.

A formula editor enables secure input of formulas. Also present as an independent window in the Utilities section.

‘Search for cross-linker’ enables the mass search for cross-linked peptides based on defined linker masses and linked residues. Linkers can be input by the user and saved in a database on disk. The resulting list of peptides can be searched with a peak list. Linking can be performed either between two different proteins or within the same protein.

A mass peak difference window enables the identification of modification and sequence ladders. Can be called from mass search and as an independent window.

pI can be calculated based on different tables.

Export of peptide lists to spreadsheet has been enhanced.

A peptide in the peptide list can be opened as an independent sequence window.

Web access to the Entrez search engine for retrieval of protein sequences and MedLine search. Retrieval of sequences based on accession numbers directly from the main toolbar (Chapter 2.7).

Several of the toolbars are now detachable and can be torn off (using the mouse), rearranged and placed as free-floating windows on the desktop.

A few functions are now independent and are placed in a new menu item called ‘Utilities’ (Chapter 12).

The charge vs pH graph now has a panel showing the charge at various pH values.

The Dot-plot window (Ch. 11.6) has a separate control toolbar that additionally shows the sequence alignment. The PAM250 matrix has been implemented for homology comparison.

Simulated 2-D gel (Ch. 12.4) tries to give an impression of the looks of a 2-D gel separation (mass vs pI) of a database and/or proteins loaded on the desktop. Proteins loaded from the desktop can be trimmed from either terminal. Phosphorylations can be simulated.

The layout and content of the sequence editor (Ch. 4.1) has been improved.

Several more user options have been added to the setup (Ch. 5).

Settings can be saved for different users or projects (Ch. 5.7).

Resizable frames can be opened with additional information in sequence windows, ms/ms windows and mass search results.

Sequence homology searching can be done based on FastA formatted protein databases and a local copy of BLAST.

The peptide information window displays a bar graph and a list of the isotopic distribution of the peptide.

When reading a complete entry from the Swiss-Prot or Entrez database, the complete entry is placed on the annotation page. The feature table of the Swiss-Prot entry is parsed and placed in a separate page for easy import into GPMAW.

The main window toolbar is now a collection of smaller toolbars that can be turned on and off and rearranged.

Help 1.8

Help index

The menu command Help|Help index opens the on-line help on the index page. From this page you can either navigate to the help wanted or you can use the search function of the help program.

An alternative way of using the help system is to press the <F1> button to open the help system in a context sensitive way that is the help will open on the page containing help relevant to the window/dialog that currently has the focus (active window). Most dialog boxes have a help button that likewise opens the context sensitive help.

The on-line help system will always be updated with the most recent features, unlike the manual which is more infrequently updated.

About

Help|About displays the version and contact information for GPMAW. The top line displays the version number of the current installation. The version number, e.g. 4.23, means main version 4, minor version 2, update version 3. Both main and minor versions have an accompanying manual while update version only has an updated help file. If you want additional copies of the current manual, please contact Lighthouse data for price and availability.

Then follows the license number and supplier of the program. Please use the license number whenever you contact Lighthouse data concerning GPMAW.

Below the name of the supplier, you will find contact information for Lighthouse data or the actual supplier. (e.g. fax number, e-mail address, and web address). If you double-click on the e-mail, your default e-mail system will load, and the address of Lighthouse data/supplier in the ‘Send to’ field of empty e-mail. If you double-click on the web address, your default web browser will load and open the index page of the Lighthouse data web site.

At the bottom of the section, the license date (month and year) and the version number of the originally installed copy of GPMAW is displayed. The license date is useful information when you upgrade, as you can only upgrade for free for a certain time (minimum one year, actual time will be stated at the web site). If you use the ‘Info’ button (see below), the program will calculate whether your license is eligible for the current upgrade.

Each version of GPMAW (both major and minor versions) has its own picture of a lighthouse. This picture is shown in the start-up ‘splash’ screen and is also presented in the left side of the About box. Below the contact information is a short description of where in the world the lighthouse is located.

Below the description of the lighthouse is the ‘OK’ button that will close the dialog along with the compilation date of the current version of GPMAW.

If you have an Internet connection, you can press the ‘Info’ button at the bottom of the dialog box. This will make the program contact the GPMAW web site and retrieve the latest information about upgrades, bugs and other relevant information. It will also calculate whether your license is valid for the latest upgrade. The actual upgrade you will have to download from the web site using your favorite browser.

When making an inquiry to Lighthouse data concerning errors, problems in handling etc. it is very important to include the license number and the current version (e.g. version 5.02) of your GPMAW installation.

Lighthouse data 1.9

GPMAW is developed by

Lighthouse data

Engvej 35

DK-5230 Odense M

Denmark

Fax: (+45) 66 19 33 96

E-mail: php@bmb.sdu.dk

www: http://welcome.to/gpmaw or http://www.protein.sdu.dk/gpmaw

If you cannot access the web site please contact Peter Højrup, Lighthouse data by e-mail at the above address.

Updates to the program will be available for download every 3-4 month. You are entitled to free upgrades for one year from purchase date (actual update period may be slightly longer, please see above). A mailing list is maintained for information on updates, critical errors and similar information. If you want to be informed, please send e-mail to php@bmb.sdu.dk with ‘GPMAW mailing list’ in the body.

In order to continue development of the program, feedback on the current versions as well as input of new ideas are always appreciated.

Technical support: If you have any problems with the program, please send a fax or e-mail, and we will try to answer in a day or two. Please remember to include license number and version number of you copy of GPMAW when you contact Lighthouse data. Both items can be found in the ‘About box’ (see 1.8).

The web site contains additional information pertaining to the program, links to other web resources and downloads of updated versions of GPMAW.