Automated Statistical Analysis of Protein Abundance Ratios from Data Generated by Stable-Isotope Dilution and Tandem Mass Spectrometry

TLDR

The study presents ASAPRatio, an algorithm that automatically analyzes protein abundance ratios from two samples and statistically distinguishes proteins with significant changes. ASAPRatio processes LC‑ESI‑MS/MS data of isotopically labeled peptides, applying Savitzky‑Golay smoothing, weighted statistics, and Dixon’s outlier test to compute abundance ratios and associated errors without human intervention. The algorithm proved fast and accurate, correctly identifying core RNA polymerase II components amid copurifying proteins and matching expected values in two test datasets.

Abstract

We describe an algorithm for the automated statistical analysis of protein abundance ratios (ASAPRatio) of proteins contained in two samples. Proteins are labeled with distinct stable-isotope tags and fragmented, and the tagged peptide fragments are separated by liquid chromatography (LC) and analyzed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS). The algorithm utilizes the signals recorded for the different isotopic forms of peptides of identical sequence and numerical and statistical methods, such as Savitzky−Golay smoothing filters, statistics for weighted samples, and Dixon's test for outliers, to evaluate protein abundance ratios and their associated errors. The algorithm also provides a statistical assessment to distinguish proteins of significant abundance changes from a population of proteins of unchanged abundance. To evaluate its performance, two sets of LC-ESI-MS/MS data were analyzed by the ASAPRatio algorithm without human intervention, and the data were related to the expected and manually validated values. The utility of the ASAPRatio program was clearly demonstrated by its speed and the accuracy of the generated protein abundance ratios and by its capability to identify specific core components of the RNA polymerase II transcription complex within a high background of copurifying proteins.