Increased selectivity, analytical precision, and throughput in targeted proteomics

Abstract

Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring (SRM) mode are characterized by a high degree of selectivity and low limit of detection; however the concurrent analysis of multiple analytes occurs at the expense of sensitivity due to reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented, in which SRM is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions are acquired in a data dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique is applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We show a limit of detection down to 10s of amol injected, and a throughput exceeding 6000 transitions in one 60 minute experiment. The technique is integrated into a linear workflow including experimental design, data acquisition, and data evaluation enabling large scale proteomic studies.