RelEx: A Computer Program for the Calculation of Ion-Current Ratios from Liquid Chromatography - Mass Spectrometry Data

The Name: RelEx is short for Relative Expression.

Background: Most quantitative mass spectrometry methods are based on the measurement of relative ion-intensity abundances in the mass spectrometer between a compound with natural abundance stable isotopes (unlabeled isotopomer) and a 'spiked' internal standard that is structurally identical with the with the exception of atom(s) that are enriched with a 'heavy' stable isotope (labeled isotopomer) -- usually 2H, 13C, 15N, or 18O. Approaches have been developed for incorporating a stable isotope label into a protein using either metabolic labeling, enymatic approaches, or chemical derivatization. After mixing the unlabeled sample with the labeled sample, the mixture is prepared and analyzed by mass spectrometry. After mixing, a peptide containing a 'heavy' stable isotope should undergo the same sample preparation and measurement biases as the respective unlabeled peptide.

The most error-prone step in determining the unlabeled and labeled isotopomer ratios is the assessment of the respective mass spectrometer ion-intensity ratios. This ion-intensity ratio is normally calculated using the areas under the ion-chromatograms of the unlabeled and enriched isotopomer m/z (or m/z range). To determine the area under any peak, an integration routine must i) decide where the peak starts and ends and ii) decide the contribution from background on which the peak is superimposed. Automated assessment of these parameters is dependent on the chromatographic peak shape -- making the process for an individual ion-chromatogram highly subjective.

Our Approach: We have adapted a least-squares correlation for to calculate the background-subtracted intensity ratio between two extracted ion-chromatograms from peptide isotopomers.

Publication: M.J. MacCoss, C.C. Wu, H. Liu, R. Sadygov, J.R. Yates III, A Correlation Algorithm for the Automated Quantitative Analysis of Shotgun Proteomics Data, Analytical Chemistry (2003)

Availability: RelEx is available through a Software Transfer Agreement. Academic and nonprofit use is free of charge. Commercial use is available for a licensing fee.

Platform: RelEx was designed to be used under Microsoft Windows. We have successfully run RelEx under Windows 2000 and Windows XP Professional. In addition, RelEx can be configured to run under Linux without modification using Wine.

Required Software: RelEx itself requires no additional software to calculate ratios from ion chromatograms. However to produce chromatograms in the correct format, RelEx contains an extraction program (Extract-Chro) that will extract the appropriate ion-chromatograms from ThermoFinnigan Xcaliber datafiles. Extract-Chro requires that a version of Xcaliber with a valid license is installed on the same computer as RelEx. Additionally, Extract-Chro needs information within DTASelect output files to correctly determine the m/z range and scan range to produce extracted ion chromatograms.

Chromatogram File Format: Extract-Chro reads the DTASelect output and produces the following file with a .chro extension from data in both the DTASelect file and the Xcaliber raw file. Unfortunately, Extract-Chro is only capable of reading Xcaliber raw files. However, because RelEx reads an intermediate data file instead of the raw file directly, anyone willing to write the code to produce the .chro files from their instrument datafiles can use RelEx to calculate the background subtracted ratios from their data.

Program:	Extract-Chro for Windows
Version:	 0.5 
M/Z Window:	auto
M/Z Shift:	
Label APE:	98.0
Before:	50 scans
After:	50 scans
M/Z Range Sample:	 1186.7 - 1189.4 
M/Z Range Reference:	 1199.2 - 1202.4 
Peptide Corr Factor:	 1.023 

Locus:	ORFP:YKL152C
Description:	GPM1 SGDID:S0001635, Chr XI from 163643-164386, reverse complement
Raw File:	112401-CNBr-mem-40NaCl-07.RAW
MS/MS Scan Number:	1735

XCorr:	0.3088
DeltaCN:	0.2534
Charge:	2
Unique:	Yes

1537	32.8275	4727570	4509290
1541	32.8978	11206681	4377465
1545	32.9718	4298401	4713328
1549	33.0477	2975233	9286918
1553	33.126	1617696	6768966
1557	33.1998	6139429	7091311
1561	33.2725	4531165	5408441

Versions and Updates:

Version 0.2 -- Added peak shift function in integration parameters to accomodate  
                   deuterated references.                                          
               Added an intensity filter to remove chromatograms of low intensity. 
               Added a regression coefficient filter to remove chromatograms that  
                   integrate with a poor regression coefficient.                   
Version 0.3 -- Improved the peak integration routine to select the peak closest    
                   to the scan that the MS/MS file was acquired.                   
               Allowed RelEx to find locus information from most recent version of 
Version 0.4 -- Added a 15N correction factor using the isotope distribution        
                   from IDCalc                                                     
               RelEx can save and retrieve integration settings for each chr       
               Converted the intensity filter to a pseudo S/N filter               
               Added a 5-point and 7-point quadratic Savitzky-Golay filter         
               Added a tool to merge output files into a single file               
               Added protein Std Dev to the output                                 
               Peptides are removed from the protein calculation if they have a    
                   ratio > 2 SD's from the mean                                    
Version 0.5 -- Changed the text file output format to make it more compatable with 
                   reopening into RelEx                                            
               Added function to reopen RelEx-Output.txt file                      
Version 0.6 -- Made RelEx compatible with both DTASelect 1.8 and 1.9               
               Added progress bar to lower right hand corner of GUI                
               Made RelEx capable of handling DTASelect -t 0 output and remove     
                   respective duplicate peptide identifications                    
               Added filters to IntSettings() so that they wouldn't have to be run 
                   separately each time.                                           
Version 0.7 -- Fixed issue with duplicate peptide identifications.  Duplicates     
                   are identified before assessing outliers not after.             
               Added Q-test for proteins with <= 10 chromatograms.                 
               Added scaling correlation filter.                                   
               Modified Savitsky-Golay filter so that it could handle smoothing    
                   of any number of points                                         
               Modified peak-shift to be the non-detected peak instead of always   
                   the reference peak                                              
               Added an intensity filter using log10(Ref*Sam) as a cutoff          
Version 0.8 -- Added an extraction program to read Xcaliber RAW files              
               A new file format is made with a .chro extension that prevents the  
                   need to reread the DTASelect-filter.txt file.                   
               Full support for the new format has been added.                     
               The correct X-axis scaling in PlotChro() has been added.            
               Fixed a bug in MinMax where the program hung when it reached a      
                   chromatogram where there were no peaks with s/n > 2.            
               Modified the Chro() array to include scan numbers too.              
Version 0.9 -- Implemented the peptide and protein filters correctly in the        
                    Integration Options form.                                      

Reporting Bugs: Although we can't promise to fix every bug. We will definately consider all requests for fixes and additions. Please direct questions and problems to Michael MacCoss

Screen Shot of RelEx's GUI in Windows: