User's manual for AASeq∗ - Bruno Zanuttini
Contents
1. You may also specify a relative path to the file for instance myacids txt However the absolute path will be evaluated relatively to the working directory i e the directory from which you run AASeq and not relatively to the directory which contains the command file Molecular mass The option name is molecular mass and its value is a range for the mass of the sequences to generate modified by the mass of wa ter see below This range can be specified in two manners either in the form massi mass2 meaning that AASeq must generate sequences weighing between massi and mass2 included or in the form mass precision mean ing that AASeq must generate sequences weighing mass up to precision or equivalently between mass precision and mass precision Importantly you must not give AASeq an m z value you must give the molecular mass of the sequence Here are some example lines in this context we assume that the mass spec ified for water is 0 see below gt 123 456 Da up to 5 456 i e between 118 0 and 128 912 Da molecular mass 123 456 5 456 gt Between 123 456 and 234 0 Da molecular mass 123 456 234 Mass of water The mass of a peptide is usually measured including a water molecule That is why AASeq automatically deduces the mass of this molecule from the masses you specify for sequences Option water mass lets you give this mass Anyway if you want to deduce it yourself or if your measures take o2. The subsequence is followed by exactly pos 1 acids in the sequences and in the order in which it is given e sub u N pos or sub N pos u The subsequence occurs after exactly pos 1 acids in the sequences but maybe in a different order e sub u C pos or sub C pos u The subsequence is followed by ex actly pos 1 acids in the sequences but maybe in a different order Once again subsequences may overlap and also overlap with N terminal and C terminal required subsequences For instance sequence GNLFRF is considered to begin with GNL to end with LFRF to contain FL u and to contain NLF N2 at the same time Here is finally a whole example of subsequences specification gt The following lines all together impose that each generated gt sequence satisfies the three following constraints gt i it contains either KLF or RF or FR gt ii it begins with xNL where x is any acid gt iii either it ends with RKxxxx or it ends with KRxxxx or it gt contains ALI or it contains WS subsequences KLF RF u subsequences NL N2 subsequences RK u C5 ALI WS 4 4 Databases As previously evoked most rules that apply to the syntax of command files also apply to the syntax of database files Acid names must be capital letters masses can range from 0 000001 to 3999 999999 Da lines beginning with character gt and blank lines are ignored and there must be one acid specified per line The s
3. 5 5 gt When C terminal amino acid F gains 5 5 Da cter modification F 5 5 When C terminal amino acid F looses 5 5 Da cter modification F 5 5 Two special differential modifications are available amidation and pyroglu tamate Amidation induces the same mass loss for every acid in the database when C terminal you must specify both the fact that amidation occurs in your peptide with option amide without any value and the mass loss with option amide mass loss and value mass loss without sign As for pyroglutamate it concerns the often encountered mass loss of glu tamine when N terminal You must specify that pyroglutamate occurs with option pyroglutamate without any option the mass loss occurring with op tion pyroglutamate mass loss and value mass loss without sign and the name of glutamine with option glutamine name and value name Here are some example lines gt Every acid looses 0 984 Da when C terminal amide amide mass loss 0 984 Acid named Q looses 17 0265 Da when C terminal pyroglutamate pyroglutamate mass loss 17 0265 glutamine name Q What makes AASeq take amidation into account is option amide If your command file contains option amide mass loss but not option amide then the sequences will not be considered to be amidated Similarly if your command file contains option pyroglutamate mass loss option glutamine name or both but not option pyroglutamate glutamine wil
4. User s manual for AASeq Bruno Zanuttini Joel Henry GREYC Universit de Caen LBBM Universit de Caen Boulevard du Mar chal Juin Esplanade de la Paix 14 032 Caen Cedex France 14 032 Caen Cedex France zanutti info unicaen fr joel henry unicaen fr May 2005 Contents 1 Presentation of AASeq 2 2 Installation 3 3 Usage 3 daly Commands 1o oru PRA A wer eus RM 3 3 2 vA dn example xis tod ui hash DR MER AM dox Rus EGER RR 4 3 9 Important note sss ra oe pa dedeiC8cy dew uexXowod 49 6 0A Oe 4 3 4 Errors and returned values llle 4 3 5 Command prompts d ae pie gan aeaa e a ER Ei 5 4 Command files and databases 5 41 Syntaxe e eae BE T AR3 WE n E r aE 5 4 2 Mandatory constraints and options 6 4 3 Other available constraints and options 7 ZAA Databases 4s ce yale Bee mex pm Gohl ee Bux eae te 11 5 Technical support 12 The version of AASeq at the time we write this manual is version 5 0 There fore this is the version concerned here Please visit regularly the website of AASeq at http www info unicaen fr zanutti aaseq for upgrades f Groupe de Recherches en Informatique Image Automatique et Instrumentation de Caen tLaboratoire de Biologie et Biotechnologies Marines 1 Presentation of AASeq AASeq is a software for helping de novo sequencing of peptides Used together with a mass spectrometer it allows to circumvent the difficulty of de novo sequencing due to the absence of database
5. amino acids must be capital letters A Z and must be the same in the database and in the command file A sequence of amino acids is written as the sequence of all their names without any space Finally note that spaces and case are not important except when explicitly mentionned below However once again do not split a constraint or option option name option value over two or more lines and do not write several constraints or options on the same line The file named command txt and distributed together with AASeq recalls the syntax of all options When you want to create your own command files you may also copy the file named template txt and use it as a basis 4 2 Mandatory constraints and options There are three mandatory constraints and options the database of amino acids the mass of the sequence and the mass of water Database of amino acids The option name is database and its value is the name or path of the file containing the database of amino acids The amino acids together with their masses specified in this file will be those composing the generated sequences The syntax of database files is explained at the end of this section Here are some example lines specifying the database in a command file gt Under Linux database aaseq aasacids txt gt Under Windows database c Program Files AASeq aasacids txt database c Program Files AASeq myacids txt database c Documents and Settings myacids txt
6. ample database of acids with syntax explanation for use if you want to create your own database e template txt A template command file meant to be used as a basis for creating your own command files There is no particular requirement for the directories where AASeq must be installed However the simpler is certainly to create a new directory and to store the five files into it Installation indeed consists in copying the executable file and if desired the text files to this directory However if you do not want to install all the files in the same directory this will not alter AASeq s functioning You can get the files from AASeq s website at http www info unicaen fr zanutti aaseq where precise directives are given for both Linux and Windows or you can ask them to the authors Once the files on your computer you are ready to use AASeq 3 Usage AASeq generates sequences from the options and constraints given in a com mand file which is simply a text file with a special syntax In this section we assume that you have written this file and we explain how to do that in next section 3 1 Commands The method for asking AASeq to generate the sequences that match the con straints and options specified in your command file is 1 getting a command prompt and going to the right directory see the end of this section if you do not know about command prompts 2 typing aaseq name of command file and pressing enter If yo
7. can all be stored on your disk but also so that your other softwares can handle all of them 3 4 Errors and returned values If an error occurs during reading of the command file or of the database of acids or during the generation of sequences a message will be displayed and 3Of course those who are used to these mechanisms may add the path to AASeq to their path environment variable then run AASeq from any directory etc However note that the name of the database of acids when relative is evaluated with respect to the working directory not to the directory that contains the command file where the database is specified This is explained in more details in next section generation will be aborted Messages are as explicit as possible and should guide you for correcting the error if it is your responsibility If generation went well AASeq returns 0 and in any other case it returns 1 3 5 Command prompts Because AASeq does not have any graphical interface yet running it requires that you get a so called command prompt Linux users should be used to that they need to launch a terminal for instance an xterm from their applications menu As for Windows users they need to launch a Command Prompt from their Start menu Both under Linux and under Windows you will then have to go to the direc tory where you installed AASeq with a cd command Type cd directory in the command prompt e g cd aaseq Linux cd c Progra
8. ecular mass 123 456 5 456 123 456 234 mass of seq 123 456Da up to 5 456 mass of seq gt 123 456Da and lt 234Da water mass 18 01 gt masses of acids molecular mass 18 01Da size 5 7 nb acids in seq gt 5 and lt 7 7 nb acids in seq lt 7 5 nb acids in seq gt 5 nter modification G 5 5 when N terminal G gains 5 5Da G 5 5 when N terminal G looses 5 5Da cter modification F 5 5 when C terminal F gains 5 5 Da F 5 5 when C terminal F looses 5 5Da amide no value all acids loose the same mass when C ter amide mass loss 0 984 if amidation occurs mass loss is 0 984 pyroglutamate no value glutamine looses some mass when N ter pyroglutamate mass loss 17 0265 if pyroglutamate occurs mass loss is 17 0265 glutamine name Q pyroglutamate concerns acid symbol Q acid L 2 4 nb of L in seq is gt 2 and lt 4 L 4 nb of L in seq is lt 4 L 2 nb of L in seq is gt 2 nterminal GNL GNI seq begins with GN L or GNT cterminal RF FR seq ends with RF or FR subsequences KLF RF u seq contains KLF or RF or FR NL N2 seq contains NL in 2nd position RK u C5 ALI WS seq RK xxx K Rreze ALI or WS Table 1 List of all constraints and options available in command files
9. eptide 3 Use this information as constraints for AASeq 4 Test the MS MS spectrum against the sequences generated by AASeq The use of AASeq is thus completely independent from the software you use for analyzing your MS MS spectra you can use any one that can read sequences in fasta format Authors and conditions of use AASeq is developed at the University of Caen France by Jo l Henry laboratory LBBM IBFA and Bruno Zanuttini laboratory GREYC You are free to download and use it without any fee You may also redistribute it for free or modify it under the terms of the GNU General Public Licence 1Companion software of AASeq See http www info unicaen fr zanutti aaseq 2 Available at http www gnu org copyleft gpl html 2 Installation Installing and using AASeq does not require any special hardware configuration As for operating systems AASeq is available both for Linux and for Windows 95 or later There is no other software requirement The standard installation of AASeq is composed of an executable file named aaseq Linux or aaseq exe Windows and of four text files Only the exe cutable one is necessary The files have the following roles e aaseq exe Executable file i e the software itself e aasacids txt Database of the twenty common amino acids with their average masses e command txt An explanation of every constraint and option available for generation of sequences e database txt An ex
10. h it is given then nothing has to be specified Otherwise simply specify u for un ordered inside the parentheses this means that the subsequence must occur in the sequences to generate but maybe in a different order Finally if you know the position of the subsequence from the N terminal extremity specify N pos inside the parentheses meaning there are pos 1 acids in the sequence before the beginning of the subsequence if you know the position of the subsequence from the C terminal extremity specify C pos pos 1 acids after the end of the subsequence finally if you do not know the position do not specify anything You cannot specify a position from both the N terminal and the C terminal extremities Also note that a subsequence specified with option subsequence may occur at the N terminal or C terminal extremity Finally if two precisions are given separate them by a comma All together here are all the possible precisions for a subsequence e sub or sub O The subsequence occurs at any position in the sequences in the order in which it is given e sub u The subsequence occurs at any position but maybe in a different order than that in which it is given for instance IRF u means that either IRF or IFR or FIR or FRI or RIF or RFI must occur somewhere in the sequences e sub N pos The subsequence occurs after exactly pos 1 acids in the sequences and in the order in which it is given 10 e sub C pos
11. l be considered to weigh its normal mass even when N terminal Numbers of occurrences of amino acids This constraint allows you to specify that a given amino acid occurs a given number of times in each sequence You can specify a maximum number of occurrences a minimum one or both and you can do that for as many acids as you desire For a given acid this is specified by option acid name of acid minimum number maximum number omit ting one of the bounds if desired bounds are included Note that in particular this allows you to specify that a given acid cannot occur in the sequence thanks to line acid name of acid 0 this can also be achieved by removing the acid from the database Here are some example lines gt Acid L occurs at least two and at most four times acid L 2 4 gt Acid L occurs at most four times acid L 4 gt Acid L occurs at least two times acid L 2 gt Acid L does not occur at all acid L 0 Subsequences This last group of options allows you to impose some subse quences to the sequences to be generated First of all you can specify the pos sible N terminal and or C terminal subsequences with options nterminal and cterminal At most one value can be specified for each of these options because the sequence of a peptide cannot begin or end with two different subsequences but the value is a list of possible subsequences More precisely N terminal possi ble subsequences are given in the f
12. m Files MAASeq cd d MyPrograms AASeq Windows etc and press enter You will then be ready to use AASeq as explained above 4 Command files and databases AASeq uses text files as command files and databases of amino acids You may create such text files by using any text editor for instance NotePad Emacs Vi etc Nevertheless if you use editors such as Word remember to save the file in plain text txt format Also name your command files something txt Constraints and options available in command files are summarized in Ta ble 1 on Page 13 4 1 Syntax A command file must contain one option or constraint per line in the form option name option value However lines beginning with character are not taken into account which allows you to write comments in your file for remembering whatever you want when you read it again Similarly blank lines are not taken into account The order of lines is not meaningful Masses are expressed in Daltons Da may it be for the mass of the sequences to generate or for differential modifications The maximum precision is six decimal digits and the maximum mass allowed is 4000Da you may specify some more Daltons but the result is not guaranteed moreover remind the note in previous section about the size of the output file Thus you can specify masses between 0 000001 Da and 3999 999999 Da The same restrictions apply to the mass of each amino acid in the database The names of
13. nly the masses of amino acids into account and thus you wish to specify the exact mass of the sequences to generate sum of the masses of their acids then simply set the mass of water to 0 Here are some example lines gt The two following lines together mean 105 446 123 456 18 01 gt Sum of masses of acids in a sequence is 105 446 Da up to 5 456 nolecular mass 123 456 5 456 water mass 18 01 gt The two following between 105 446 and 215 99 Da nolecular mass 123 456 234 water mass 18 01 gt The two following between 123 456 and 234 Da nolecular mass 123 456 234 water mass O 4 3 Other available constraints and options While the previous constraints and options are mandatory the following are optional Thus you may specify some of them or none at all Importantly the more constraints you specify the less sequences you will obtain thus speeding up future search The available constraints and options concern The size of the sequences N and C terminal differential modifications numbers of occurrences of amino acids and imposed subsequences Size of sequences This option allows you to constrain the generated se quences to those within a given size range It is useless to specify this option if the values can be deduced from the mass of the sequences and the masses of the amino acids Otherwise you may specify a lower bound an upper bound or both in the form size lower bound upper bound omi
14. orm nterminal sub1 sub2 sub3 and similarly for C terminal subsequences Here are some example lines gt The sequence of the peptide begins either with GNL or with GNI nterminal GNL GNI gt The sequence of the peptide begins with GNL nterminal GNL gt The sequence of the peptide ends with RF or FR cterminal RF FR gt The sequence of the peptide ends with RF cterminal RF Importantly subsequences may overlap for instance if you require GNL as an N terminal subsequence and LFRF as a C terminal subsequence then sequence GNLFRF is considered to match both constraints together You can also specify subsequences that are not necessarily N terminal or C terminal This can be done combining the following precisions Whether you know the order of the subsequence and whether you know its position from the N terminal or C terminal extremity Similarly to the case of N terminal and C terminal subsequences you may specify various possibilities e g subsequences subi sub2 means that your peptide contains either sub1 or sub2 or both But you may also specify several such constraints e g specifying both subsequences subi sub2 and subsequences sub3 sub4 sub5 means that your peptide contains either sub1 or sub2 but also contains either sub3 or sub4 or subb5 As for precisions they are given inside parentheses after the concerned sub sequence If the subsequence must appear in the exact order in whic
15. s of known sequences to which new spectra can be compared in traditional sequencing AASeq does not analyze MS spectra neither does it give the sequence of a peptide What it is able to do is to create databases of sequences that can be used to feed other softwares More precisely it is able to create a virtual database of all the sequences of amino acids that match given constraints The constraints concern the mass of the sequences and if desired their size their subsequences N terminal C terminal or internal the number of certain amino acids etc Consequently if the peptide to be sequenced indeed satisfies these constraints then its sequence will be in the database generated by AASeq and consequently if the MS MS spectrum is good the sequence will be found out All the databases generated by AASeq are in fasta format Thus AASeq can be used helpfully as soon as enough information has been collected about the peptide for the number of candidate sequences to be rea sonable This information can be obtained thanks to a mass spectrometer as concerns the approximate mass of the peptide through testing of its MS MS spectrum against random sequences for instance generated by AARand as con cerns size subsequences and numbers of given amino acids by intuition from knowledge about the family of the peptide etc The use of AASeq can be summarized as follows 1 Get a MS MS spectrum of your peptide 2 Collect information about your p
16. tting one of the bounds if desired bounds are included Here are some example lines gt Only generate sequences containing between 5 and 7 amino acids size 5 7 gt Only generate sequences containing at most 7 amino acids size 7 gt Only generate sequences containing at least 5 amino acids size 5 N and C Terminal modifications This option allows you to specify differ ential modifications on the mass of some amino acids when positionned at the N or C terminal extremity The option value is name of acid modification where modification is of the form mass if the amino acid looses mass Daltons and of the form mass if the amino acid gains mass Daltons You can specify as many modifications as you desire however the behaviour of AASeq is not defined if the same amino acid is subject to several N terminal modifications or to several C terminal modifications including the case when the peptide is amidated and an amino acid is subject to another C terminal modification Similary the behaviour of AASeq is not defined if an amino acid is subject to both an N terminal and a C terminal modification and the sequence contain ing only this amino acid matches all other constraints indeed in this case the amino acid is both N terminal and C terminal Here are some example lines gt When N terminal amino acid G gains 5 5 Da nter modification G 5 5 gt When N terminal amino acid G looses 5 5 Da nter modification G
17. ur command file is named something txt then you will get the sequences in file something fasta in the same directory This file will contain all the desired sequences in fasta format and will be ready to be tested against your MS MS spectrum using your favourite software for that purpose There are also two useful options for AASeq e aaseq version displays version and license information e aaseq help displays a quick help about basic usage 3 2 An example The most basic use consists in asking AASeq to generate all sequences with a given mass To generate all sequences with a total molecular mass of 500 Da plus or minus 1Da type the following into a file named for instance seqs500 txt in the directory where you installed AASeq see below for the meaning of option water mass database aasacids txt molecular mass 500 1 water mass 0 Save the file then get a command prompt and go to the right directory see below and type aaseq seqs500 txt Press enter the desired sequences are now stored in file seqs500 fasta in the directory where you installed AASeq 3 3 Important note Do not use AASeq for e g generating all sequences weighing between 500 and 1000Da The only thing you will get is a full disk AASeq can generate sequences very quickly but it is designed to generate every sequence it is asked to It is your own responsibility to know in advance whether there will be a reasonable number of them so that they
18. yntax for one acid is simply acid name mass for instance gt The database contains an acid named Y and weighing 128 01 Da Y 128 01 gt The database contains an acid named Z and weighing 82 00 Da Y 82 The file named aasacids txt and distributed together with AASeq contains a standard database of the twenty common amino acids together with their standard names and their average masses with a precision of four decimal digits If you want to create your own database you can proceed by copying the file named database txt and using it as a basis 11 5 Technical support Technical support can be obtained from Bruno Zanuttini current e mail ad dress zanutti info unicaen fr In case of error or observed bug please communicate the corresponding command and database files as well as every thing displayed by AASeq As already evoked there is a website dedicated to AASeq and AARand this site is currently located at http www info unicaen fr zanutti aaseq New versions will be published there as well as known bugs if any You can also register there as an AASeq user so that we can support you efficiently Finally according to the GNU General Public Licence you are allowed to modify and redistribute AASeq The source files can be obtained from the web site and explanations can be asked to Bruno Zanuttini 12 I Option Example values Meaning database c AASeq myacids txt database file mol
Download Pdf Manuals
Related Search