A software toolkit and interface for performing stable isotope labeling and top3 quantification using Progenesis LC-MS

Abstract

Numerous software packages exist to provide support for quantifying peptides and proteins from mass spectrometry (MS) data. However, many support only a subset of experimental methods or instrument types, meaning that laboratories often have to use multiple software packages. The Progenesis LC-MS software package from Nonlinear Dynamics is a software solution for label-free quantitation. However, many laboratories using Progenesis also wish to employ stable isotope-based methods that are not natively supported in Progenesis. We have developed a Java programming interface that can use the output files produced by Progenesis, allowing the basic MS features quantified across replicates to be used in a range of different experimental methods. We have developed post-processing software (the Progenesis Post-Processor) to embed Progenesis in the analysis of stable isotope labeling data and top3 pseudo-absolute quantitation. We have also created export ability to the new data standard, mzQuantML, produced by the Proteomics Standards Initiative to facilitate the development and standardization process. The software is provided to users with a simple graphical user interface for accessing the different features. The underlying programming interface may also be used by Java developers to develop other routines for analyzing data produced by Progenesis.

FIG. 1.

Screenshot of Progenesis Post-Processor used for SILAC analysis.

FIG. 2.

A box plot of the first replicate of the SILAC data set. The x-axis displays the expected ratios of proteins in each run (calculated from the median peptide ratio, using all peptides matched to a given protein), and the y-axis displays the observed log₁₀ ratios. The dotted line displays the expected values.

FIG. 3.

A box plot displaying the observed values of absolute abundance (fmol/μL), calculated by the top3 protocol (“_Top3”), average of the total abundance (“_Ave”), and total abundance (“_Total”), in conditions B–E for 41 UPS proteins. Expected values and summary statistics are displayed in Table 2.