TandTRAQ consists of modified code from the GPM, modified iTracker code, and a simple integration script, all written in Perl. It is a command line program to integrate protein identification results from X!Tandem with the quantitation results from the iTracker program.
TandTRAQ will also query, using GPM code, a number of databases in an attempt to return annotations on a particular sequence, as well as relevant links to the database entry.
TandTRAQ requires Perl 5.0 and greater. It also has the following Perl Module Dependencies: CGI, HTTP::Request::Common, and LWP::UserAgent. These can be easily added to an existing Perl instalation via CPAN.
Zip Version or tar.gz version (about 6 megs).
CSF samples were prepared for iTRAQ analysis as recommended by the manufacturer. Briefly, 20 ug of CSF protein was acetone precipitated and resuspended in a small volume of triethylammonium bicarbonate containing SDS and reducing agent. After resuspension and reduction, the proteins were alkylated with XXX and digested via the addition of a 1:50 ratio of sequencing grade trypsin to protein. After digest the samples were dried and resuspended in neat EtOH and labeled with isobaric tags. After labeling the samples were diluted in a large volume of 1% formic acid for SCX cleanup. The samples were batch cleaned on an SCX column and then desalted by SPE on a C18 cartridge in preparation for MDLC.
For multidimensional LC/MS/MS analysis a 150 mm x 5 cm, lab-packed SCX column was prepared for ion-exchange chromatography (Polysulfoethyl A, 5 mm particle size, 300 angstrom) as instructed by the manufacturer. Tryptic peptides from a CSF were injected onto the SCX column and the breakthrough was analysed as the first fraction. Additional pools of peptides were displaced from the ion exchange column via series of injections with increasing ammonium acetate concentration. Injections at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, and 300 mM were used.
Analytical chromatography was achieved using an Eksigent nanoLC (Dublin, CA) to generate a gradient using the following chromatographic conditions; mobile phase A; water, acetonitrile, formic acid, trifluoroacetic acid (95, 4.89, 0.1, 0.01, v/v/v/v) mobile phase B; acetonitrile, isopropanol, water, formic acid, trifluoroacetic acid (80, 10, 9.89, 0.1, 0.01, v/v/v/v/v). Mobile phase B was ramped from 2% to 45% over 40 minutes, increased to 80% in 5 minutes and held for five minutes before being returned to starting conditions. Flow was regulated at 200 nL/min and directed through a 75 mm x 15 cm in-house packed column packed with Astrosil (5 mm particle size, 100 angstrom pore size, C18 reverse phase chemistry, Stellar Phases, Langhorn, PA) coupled to a 5 mM tapered emitter (New Objectives, Woburn, MA). Prior to analytical chromatography, 5 mL of tryptic digest was injected onto a 150 mM x 2 cm sample trap packed with Poros R10 and washed with mobile phase A to remove salts and contaminants after which the trap was switched in-line with the analytical column. Tandem mass spectrometry data was collected using a Qstar XL hybrid time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA) under the following conditions; spray voltage 1800-1900V; TOF-MS scan m/z 400-1600, 0.5 seconds; TOF-MS/MS scan m/z 50-2000, 2.0 seconds, 90 second exclusion; data dependent product ion acquisition of the three most abundant +2 and +3 ions from the TOF-MS scan.
Please report all errors, bugs and feature requests to laderast (at) ohsu (dot) edu.