SYSTEM REQUIREMENTS FOR WINDOWS (tested on XP/Vista/7)
******************************************************

* Perl 5.10 and above (you can download and install free Activeperl community edition for Windows from 

http://www.activestate.com/activeperl/downloads or http://www.perl.org/get.html - the program is tested with Activeperl community edition 5.12.4.1205) ----> Click on the *.exe files and the installation is self explanatory
	
* (OPTIONAL) MinGW (Minimalist GNU for Windows for C code compilation) installation (see http://www.mingw.org/wiki/Getting_Started for detailed installation guidelines)
An executable (.exe) is already packed in the Source_code directory for the c++ codes (works with XP/Vista/7) and normally this step is not required. However, in case of any incompatibility with the .exe, install MinGW and re-compile the c++ codes from command line (cmd) in Windows as follows:
OPTIONAL - see above.
# cd to the Path where you have extracted the file \DisConnect\DisConnect_Windows\Source_Code

# g++ new_comb_gen.cpp -o comb_win 
# g++ new_comb_gen-msn.cpp -o comb_win_MSn 

******************************************************************

INSTALLATION AND USAGE ON WINDOWS 
*********************************

	* Extract DisConnect.zip or DisConnect.tar.gz
	
	* Go to the folder DisConnect_Windows

	* Update peak_mass_ms/2/n.txt with m/z values of MS/MS2/MSn experiments respectively

	* Update prot_seq.txt with the protein sequence of interest, when assigning proteolytic fragment ions (MS ions).

	* Double click on run.bat and choose the option to assign either proteolytic or MS2/n fragment ions 

	* Key in the inputs as prompted at the window opened

	* The calculation window closes automatically once the job finishes

#Probale warning messages to be ignored:

*********************************************************************

INPUT/OUTPUT OPTIONS FOR WINDOWS
********************************

Enter 1 / 2 if you are assigning proteolytic or MS2/n fragment ions respectively

********************************************************************
>> If 1 is chosen at this stage (i.e., assignment of MS data for proteolytic fragments) >>
Before Starting: 
	# Paste the protein sequence, ending with a * symbol, in the file "prot_seq"
	# Put the m/z values, in the format 'm/z value'space'charge state' (e.g. 1020.2 3, where the m/z value 
        is 1020.2 and charge state 3)in the file 'peak_mass_ms'


	1. Enter the name/symbol for the protein of interest

	2. Enter 1 / 2 for monoisotopic / average mass

	3. Enter 1 / 2 if the protein C-terminus is Amidated / Non-amidated

	4. Enter your choice of proteases

	Enter 1 for Trypsin
	Enter 2 for Trypsin-P
	Enter 3 for V8(DE)
	Enter 4 for V8(D)
	Enter 5 for Asp-N
	Enter 6 for Lys-C
	Enter 7 for Arg-C
	Enter 8 for CNBr
	Enter 10 for user defined protease

	5. Enter the number of additional proteases to be used

	6. Enter the number of allowed proteolytic miscleavages (1-3; upto three miscleavages allowed)

	7. Enter window range for match with MS data depending on the resolution of the experimental setup.

	8. Results will be stored in a folder named Results_MS in the format "match_MS_protein name/symbol"  
	   within the DisConnect_Windows folder. Also a file called "inp_MSn_match_MS_protein_name.out" will 
         be provided for input to subsequent MSn experiments for each MS fragment ion.


********************************************************************
>> If 2 is chosen at this stage (i.e., assignment of MS2/n data) >>
#Before Starting: 
	# Put the m/z values of the fragment ions, in the format 'm/z value'space'charge state' (e.g. 1020.2 3, where the 
	  m/z value is 1020.2 and charge state 3)in the file 'peak_mass_ms2' (for MS2) or 'peak_mass_msn' (for MSn)

	1. Enter 1 / 2 for monoisotopic / average mass (Depnding upon the querried m/z value average or monoisotopic 
                                                      mass needs to be chosen)

	2. Enter 1 / 2 if you are calculating on whole sequence (MS2) / daughter fragments (MSn; n > 2)

		If Step 2 = (1) ---> Enter the complete polypeptide sequence: (Polypeptide sequence can be entered using 
												   the standard one letter codes. For peptide 
												   containing multiple chains, like insulin, 
												   a letter X should be entered between the two 
												   chains(e.g if a peptide conatins two 
												   polypeptide chains with sequence GVCSF and 
												   RLTCY then the input is GVCSFXRLTCY). For 
												   user benefit, if these peptides are results 
												   of proteolytic digestion then an input file 
												   in the Result_MS folder is created, named 
												   "inp_MSn_match_MS_protein name.out", that 
												   contains the peptide sequence in the required 
												   format for the MS2 analysis. The user can copy 
												   the respective sequence from there and paste it) 	  																																				   

		If Step 2 = (2) ---> Enter the complete polypeptide sequence: (As above)
                     Enter the fragment polypeptide sequence of interest:(Format for this input sequence is given 
									  inside the Resut_MSn folder, termed as
 									  inp_Result_MSn_rigorous/smart_Entered complete/
									  fragment sequence. This is obtained during the
									  the structure determination of the parent ion from
									  the preceeding stage of MS/MS ions. 
									  Copy the corresponding structure of the ion 
									  undergoing MSn fragmentation from this file).


	3. Enter 1 / 2 for smart / rigorous mode of calculation (Smart mode considers only the Cys residue mass values 
										   of 135 & 69 Da, and rigorous considers all the four 
										   possible values of 101, 103, 135, 69. Users are advised 
										   to use Smart mode for posstiive ion CID fragmentation, 
										   as it is the prevalent mode of fragmentation under this 
										   condition)
										   

	4. Enter the mass error tolerance based on the resolution of the experimantal setup

	5. Enter 1/2/3/4/5 for normal peptide fragments/fragments with CO loss/fragments with 
	   H2O loss/fragments with NH3 loss/fragments with NH3+CO loss
	
	6. Depending on the number of C-termini in the sequence (say x) the code will ask for 
	   the amidated(enter 16) / non-amidated(enter 17) status for each C-term
	   
	   Enter mass to be added for C-term 1 status
	   .
	   .
	   .
	   Enter mass to be added for C-term x status
 	

	7. Enter 1/2 for ion trap/ no ion trap filtering 
	
	8. Results will be stored in the path ./Results_MSn in the format Result_MS2/n_smart/rigorous_Entered 
	   complete/fragment sequence/Result_*" within the DisConnect_Windows folder.

	9. For MSn experiments, the input parent fragment ion structure needs to be inserted twice. 
	   
	   (a)		At first, at Step 2(2) stage. Here the input ion has to retain a numbering scheme (which has been removed from the 
	   final output for the clarity of presentation) for technical purposes. An output file named "input_for_MSn_rigorous/smart_Entered 
	   complete/fragment sequence" is also given within the Results_MSn folder, retaining this numbering scheme for the fragment ions. 
	   For any MSn experiments, the input of the corresponding parent ion structure should be taken from this output file. 
	   It is to be noted that this file contains all the ion structures, without ion trap filtering. The user need
	   to select the corresponding numbered structure of the ion shown in the result file "Result_*".

	   (b) 		Next, it needs to be inserted again after Step 7. Here, the Input should be in the output format and thus can directly 
	    be copied from the MS2 output file, termed as "Result_MS2/n_smart/rigorous_Entered complete/fragment sequence/Result_*". 
	
	 
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All the benchmark data used in the text are given in the folder ./benchmark-data

For more details refer to the Methods in the Documentation folder and the reference given below. The program package 
,DisConnect, is distributed under the BSD License (see License.txt).

If you are using this package, please also refer to (Rapid Mass Spectrometric Determination of Disulfide Connectivity in Peptides and Proteins. Bhattacharyya M, Gupta K, Gowd KH, Balaram P , Mol. BioSyst.,2013,XX,XX-XX)

Please contact the following in case of any difficulties:

moitrayeebhattacharyya@gmail.com
talkingtothekg@gmail.com

Thank you for using DisConnect !!!




