TWINPEAKS is a software program developed in the Emili Laboratory at the University of Toronto to facilitate high throughput sequencing of peptide MS/MS spectra using the MCAT (Mass Coded Abundance Tagging) approach. TWINPEAKS is used to process large LC-MS datafiles to isolate sister peptide pairs for de novo sequence prediction by MCATSCAN. TWINPEAKS is essentially a tool for high-throughput proteomics experiments when many hundreds or thousands of tandem mass spectra must be analyzed.

User requirements

The program is written in Perl and will execute, as packaged, on Unix- or Linux-based platforms or on Mac computers operating on OS10. For Windows platforms, the standard Perl module can be downloaded from www.cpan.org (Perl5.6.1.629-MSWin32x86multi-thread) and installed prior to launching MCATSCAN from the command line. Implementation of the current version of MCATSCAN Express requires preinstallation of EXTRACTMS, a program written by Jimmy Eng (Institute of Systems Biology, Seattle, WA; www.systemsbiology.org).

Input

TWINPEAKS uses the output of EXTRACTMS, a program written by Jimmy Eng (Institute of Systems Biology, Seattle, USA; www.systemsbiology.org). EXTRACTMS summarizes the salient features of .dat files (that may contain dozens to thousands of .dta files) typically generated during LC-MS/MS experiments and creates a document (extractms.txt) listing all the MS/MS spectra. For each candidate peptide spectrum, it creates a file (your_spectrum_here.dta) that lists the parent ion (precursor) mass and predicted charge on the first line, followed by mass and intensity values for each peak in the spectrum.

An example of extractms.txt:

EXTRACTMS

An example of a .dta file:

TWINPEAKS scans and parses the extractms.txt file to identify parent ion scans that are likely to be MCAT pairs (ie. offset by 42, 21 or 14 mass units for plus1, plus2, or plus3 ions respectively, within a predetermined number of scan and relative elution order, of each other).

Two text files are generated as output. MCATpairs.txt is a verbose file suitable for manual analysis. An example is shown here:

TWINPEAKS ver.1.0
Proteomics and Bioinformatics
Univeristy of Toronto

Parent ions offset by 42 m/z units:
-----------------------------------

Scan:117 Parental Ion: 403.1
AE250401_BigZipyWCE_PlusMinusIsothio_RatioQuan_1unmod_to_19mo.0117.0117.1.dta
Scan:118 Parental Ion: 452.2
AE250401_BigZipyWCE_PlusMinusIsothio_RatioQuan_1unmod_to_19mo.0118.0118.1.dta

Scan:285 Parental Ion: 416.7
AE250401_BigZipyWCE_PlusMinusIsothio_RatioQuan_1unmod_to_19mo.0285.0285.1.dta
Scan:290 291 Parental Ion: 462.0
AE250401_BigZipyWCE_PlusMinusIsothio_RatioQuan_1unmod_to_19mo.0290.0291.1.dta

offset_dtas.txt is a list of .dta files where paired MCAT spectra are listed in succession. This file is used by the high-throughput version of MCATSCAN (MCATSCANexpress).

Operation

The program is executed by placing the following in a single directory:

1. The extractms.txt file corresponding to a particular mass spectrometry run.
2. The TWINPEAKS execulatble program, twinpeaks.pl.

To run, type

perl twinpeaks.pl

in the DOS command module (Windows), or shell module on Unix/Linux. On some computers, the extension '.pl' is recognized as a Perl program and the user can directly click on the icon to run the program.

The output files, mcat_pairs.txt and offset_dtas.txt, will appear in the same directory. The most commonly changed variables are at the top of the file under USER PARAMETERS. These are discussed below in 'What the program does'.

What the program does

The program automatically finds pairs of modified and unmodified spectra based on mass differences between the parent ions. The user can change the tolerance (variable) for this match, using:

$parental_ion_tol = 2;

MCAT-modified peptides reproducibly elute slightly later than MCAT-unmodifed peptides following liquid chromatography. The user can adjust the 'lag' tolerated by the program for matching parent ions by adjusting

$scan_tol = 100;

This is the window of scans in which the MCAT-modified parent must appear to be paired with the unmodified parent. Default for this variable is 100.