Abstract
Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339 peptides representing 132 enzymes in Pseudomonas putida KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for the majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.
Highlights
Liquid chromatography (LC) selected reaction monitoring (SRM, or multiple reaction monitoring – MRM) targeted proteomics is a popular mass spectrometry (MS)-based protein quantification technique (Picotti and Aebersold, 2012; Ebhardt et al, 2015)
We demonstrated that microflow liquid chromatography-selected reaction monitoring (LC-SRM) is a robust, high-throughput targeted proteomic approach with little or no loss of sensitivity relative to nanoflow LC-SRM, and it works well in quantifying metabolic pathway enzymes and providing deep insights into the metabolism of P. putida
The cells were pelleted by centrifugation at 4,500 rpm for 5 min, the supernatant was decanted, and the pellets were washed with ice cold phosphate-buffered saline (PBS) and flash frozen in liquid nitrogen and stored at −80◦C until analysis
Summary
Liquid chromatography (LC) selected reaction monitoring (SRM, or multiple reaction monitoring – MRM) targeted proteomics is a popular mass spectrometry (MS)-based protein quantification technique (Picotti and Aebersold, 2012; Ebhardt et al, 2015). Most targeted proteomics assays are limited to a few dozen proteins per run and the samples. Targeted Proteomics Assays for Pseudomonas putida are often acquired using nanoflow LC-SRM in order to achieve high sensitivity (Picotti et al, 2009; Huttenhain et al, 2012). LC-SRM workflows can monitor hundreds of peptides in a single analysis (Lee et al, 2020), which provides researchers opportunities for deeper exploration into biological systems. The long overhead time (i.e., sample loading, column washing, and equilibrating) of nanoflow LC-SRM can no longer meet the demands of highthroughput studies. Compared to nanoflow LC-SRM, microflow LC-SRM provides higher throughput and better reproducibility, advantages that overshadow its slightly less sensitivity (Bian et al, 2020)
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