README

PhosCalc 1.2 (Standalone Version) 
Requirements: Perl, Math::Combinatorics Module 
(http://search.cpan.org/~allenday/Math-Combinatorics-0.08/lib/Math/Combinatorics.pm#AUTHOR) 

About PhosCalc 
PhosCalc is a system for estimating the site of phosphorylation in a 
phosphorylated peptide with ambiguous sites. 
It does this by generating ions from peptide sequences and calculating their 
masses, generating different ions and masses for water-loss, different cysteine 
modifications and different charge states. These are then compared with peaks 
in the spectrum from which the peptide was identified to asess the most likely 
site of p The process is carried out as described in MacLean et al 

Feature additions over version 1: 
-will now accept mgf file 
-will add water-loss ions to predicted set 
-will deal with different charge states 
-allows selection of masses for different cysteine modifications 
-asesses calculated probabilities of combinations of phosphorylation sites to 
produce a best guess formatted string 

Running PhosCalc: 
Quick Start 

Input 
As a minimum PhosCalc requires a peptide spectrum list file and spectrum files to run. 
The spectra may be either .dta or .mgf format. Default is .dta. 
The peptide spectrum list must contain each peptide to be assessed and the 
name of the corresponding spectrum. The info should be in two columns, the 
peptide on the left and it's spectrum on the right, seperated by a tab character 

eg THISISAPEPTIDE aspectrumfile.dta 

If you like you can build the peptide list file in excel and save it as a 'tab delimited' file. 
If you are using .dta files, the spectrum column should contain the name of the 
.dta file, if you are using .mgf then the spectrum column should contain the 
name of the specturm as given in the TITLE= line. 

To run PhosCalc

Using .dta files 
Put the peptide spectrum list file and the .dta files in the same folder as the script. 
From the command line simply type perl phoscalc1.2.pl -p <peptide spectrum list> 

PhosCalc will run with 
experiment type MS2 
error margin of 0.4 
filtering the four strongest peaks from each 100 m/z window of the spectrum 
using a best guess range of 3 orders of magnitude (1000) 
output will go into a file with the name of your peptide spectrum list appended with '_phoscalc_output.tab' 

Using .mgf files 
Put the spectrum list file and the .mgf file in the same folder as the script. 
From the command line type phoscalc1.2.pl -p <peptide spectrum list> -t mgf -m  <mgf file name> 

PhosCalc will run with 
experiment type MS2 
error margin of 0.4 
filtering the four strongest peaks from each 100 m/z window of the spectrum 
using a best guess range of 3 orders of magnitude (1000) 
output will go into a file with the name of your peptide spectrum list appended with '_phoscalc_output.tab' 

Other settings 
You can customise the behaviour of PhosCalc using the command line flags. To see a summary of these type phoscalc1.2.pl -h at the command line. 
-p <some file> defines the peptide spectrum list file 
-t <'dta' or 'mgf'> defines the spectrum file type (defaults to dta if ommitted) 
-m <some file> defines the .mgf file (required if -t is set) 
-x defines the experiment type (defaults to MS2 if ommitted) 
-e defines the width of window for peak matching. (defaults to 0.4 if 
ommitted)
-D calculate additional ions with loss of water 
-M use carboxmethylated cysteine value (mutually exclusive with -A) 
-A use carboxamidomethylated cysteine (mutually exclusive with -M) 
unmodified cysteine mass = 103.00919 
alkylated cysteine (carboxmethylation) = 103.00919 + 58.00548 
alkylated cysteine (carboxamidomethylation) = 103.00919 + 57.021464 
-o <some file> defines the output file (defaults to 
input_file_phoscalc_output.tab if ommitted) 
-b <a number> defines the range of p values over which to consider a phosphorylation site equivalent for best guess formatting. eg 10 = 1 order of magnitude, 1000 = 3 orders of magnitude, 1000000 = 6 orders of magnitude. (defaults to 1000 if ommitted). 
-h print the help and quit 

References 
MacLean D, Burrell MA, Studholme DJ and Jones AMEJ (2008) PhosCalc: A tool for 
determining the likelihood of peptide phosphorylation from Mass Spectrometer 
data.BMC Research Notes .1:30 (PubMed)