MALDI TOF Mass spectrometry and tandem MS/MS

Retrieving Data from Proteus LIMS LabLink:

When we have finished analyzing your proteomic samples, the results are posted in the Proteus LabLink LIMS system for you to retrieve. You can get these by logging onto the system at https://proteuslablink.med.psu.edu/lablink and exporting/downloading the appropriate files to your computer. For non-gel spot samples (where 1D and 2D LC separations have been used, e.g., MudPit and iTRAQ experiments), our very stringent false discovery rate estimations (FDR) will show you how many confident protein and peptide IDs there are for your sample(s), where the lowest ranking protein ID on that ID list still has less than a 5% chance of being a False Positive ID; the less stringent ProteinPilot Unused scores will show how many proteins are ID'd at p<0.05, based on the Paragon algorithm (contained in the ProteinPilot program) which we use for analysis (You can read more about the Paragon Algorithm and how it works in the published work about the algorithm at Mol Cell Proteomics. 2007 Sep;6(9):1638-55. Epub 2007 May 27. The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra. Shilov IV, Seymour SL, Patel AA, Loboda A, Tang WH, Keating SP, Hunter CL, Nuwaysir LM, Schaeffer DA. Similarly, you can read more about the PSPEP FDR algorithm and how it works at Calculating False Discovery Rates (FDR).

For these 1D and 2D LC experiments, the sample results in the Proteus LabLink LIMS consist of (1). a .group file (the detailed analysis results of proteins and peptides ID'd, and relative quantitation ratios for iTRAQ experiments); (2) an Excel document which has the results of the PSPEP false discovery rate estimation (FDR); and (3) several .txt files which contain the peptide and protein ID information and scores exported from the .group file. All the .txt files can be imported into Excel directly by opening them from within Excel and following the text import wizard that pops up.

If your results are from an iTRAQ experiment, these .txt files will also include a separate protein ID file for each iTRAQ reagent as the denominator of the calculated iTRAQ ratio, i.e., one file has the 113 reagent amounts as the denominator of the iTRAQ ratio and therefore contains the 114/113, 115/113, 116/113 ratios, plus the p-value associated with each ratio to show which changes are statistically significant. A second file has the 114 reagent amounts as the denominator of the iTRAQ ratio and therefore contains the 113/114 (obviously the inverse of the 114/113 ratio), 115/114, 116/114 ratios, etc.

The .group file has the most detailed information in it, all the .txt exported results and more. The .group file can be viewed interactively by downloading the "Trial" version of the Protein Pilot program (Windows computers only), the full version of which produced the .group file, at https://licensing.appliedbiosystems.com/download/index. Although this is not clearly explained on the ABI web site, installing the trial version and simply not entering any activation code will allow you to use the program as a viewer and look at your results in the .group file (you can also download the trial and enter the 30-day activation code they send you - at the end of the trial period, the software will still continue to function as a viewer). Using the ProteinPilot program as a .group file viewer, for example, you can see color-coded indications of which proteins have significantly changed between different samples.

The Excel file contains the estimate of the number of ID'd proteins (and peptides, under different tabs) in your analysis that have an estimated false discovery rates (FDR) below 1%, 5%, 10% etc. We use the 5% Local FDR number, as this is a very stringent criterion in which the LOWEST ranking protein (peptide) has a 5% chance of being a false positive, and all ID'd proteins with higher scores than that one have progressively lower possibilities of being false positives as you go up your protein ID list. You can read more about how the FDR is calculated, why we use the Local (as opposed to Global) FDR estimate, and the literature reference for the PSPEP program that is used to calculate these on our WEB pages shown  here.