Abstract
Recent improvements in the mass accuracy and resolution of mass spectrometers have led to renewed interest in label-free quantification using data from the primary mass spectrum (MS1) acquired from data-dependent proteomics experiments. The capacity for higher specificity quantification of peptides from samples enriched for proteins of biological interest offers distinct advantages for hypothesis generating experiments relative to immunoassay detection methods or prespecified peptide ions measured by multiple reaction monitoring (MRM) approaches. Here we describe an evaluation of different methods to post-process peptide level quantification information to support protein level inference. We characterize the methods by examining their ability to recover a known dilution of a standard protein in background matrices of varying complexity. Additionally, the MS1 quantification results are compared to a standard, targeted, MRM approach on the same samples under equivalent instrument conditions. We show the existence of multiple peptides with MS1 quantification sensitivity similar to the best MRM peptides for each of the background matrices studied. Based on these results we provide recommendations on preferred approaches to leveraging quantitative measurements of multiple peptides to improve protein level inference.
Highlights
The ability to identify and quantify hundreds or even thousands of peptides in a single data dependent LC/MS analysis of a proteolytic digest from a complex mixture such as serum or plasma established mass spectrometry-based proteomics as a tool of choice for biomarker discovery research
This report serves as an extension to our previously described approach [1] to label-free relative peptide and protein quantification for discovery, hypothesis generating, experiments using previous generation of nominal mass accuracy hardware
This extension includes the following aspects which we have found to have significant improvement over the first generation of label free quantification
Summary
The ability to identify and quantify hundreds or even thousands of peptides in a single data dependent LC/MS analysis of a proteolytic digest from a complex mixture such as serum or plasma established mass spectrometry-based proteomics as a tool of choice for biomarker discovery research. These are the ultimate hypothesis-neutral experiments that have the promise of finding solutions for major gaps in drug discovery and other biological inquiries. From these analyses we propose a relatively simple yet powerful method for mass spectrometry proteomics quantification that can be implemented in a relatively straightforward manner
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