Abstract
Over the past 10 years, the bioenergy field has realized significant achievements that have encouraged many follow on efforts centered on biosynthetic production of fuel-like compounds. Key to the success of these efforts has been transformational developments in feedstock characterization and metabolic engineering of biofuel-producing microbes. Lagging far behind these advancements are analytical methods to characterize and quantify systems of interest to the bioenergy field. In particular, the utilization of proteomics, while valuable for identifying novel enzymes and diagnosing problems associated with biofuel-producing microbes, is limited by a lack of robustness and limited throughput. Nano-flow liquid chromatography coupled to high-mass accuracy, high-resolution mass spectrometers has become the dominant approach for the analysis of complex proteomic samples, yet such assays still require dedicated experts for data acquisition, analysis, and instrument upkeep. The recent adoption of standard flow chromatography (ca. 0.5 mL/min) for targeted proteomics has highlighted the robust nature and increased throughput of this approach for sample analysis. Consequently, we assessed the applicability of standard flow liquid chromatography for shotgun proteomics using samples from Escherichia coli and Arabidopsis thaliana, organisms commonly used as model systems for lignocellulosic biofuels research. Employing 120 min gradients with standard flow chromatography, we were able to routinely identify nearly 800 proteins from E. coli samples; while for samples from Arabidopsis, over 1,000 proteins could be reliably identified. An examination of identified peptides indicated that the method was suitable for reproducible applications in shotgun proteomics. Standard flow liquid chromatography for shotgun proteomics provides a robust approach for the analysis of complex samples. To the best of our knowledge, this study represents the first attempt to validate the standard flow approach for shotgun proteomics.
Highlights
Advances in biofuels research focusing on feedstock characterization and engineering (Persil-Cetinkol et al, 2012; DeMartini et al, 2013; Shen et al, 2013; Eudes et al, 2014) as well as the genetic manipulation of microbes (Alper et al, 2006; Tyo et al, 2007; Keasling, 2008; Lee et al, 2008) have progressed significantly in the last few years
This work prompted us to assess the utility of standard flow liquid chromatography (LC) for shotgun proteomic methods related to biotechnology
APPLICATION OF STANDARD FLOW liquid chromatography-mass spectrometry (LC-mass spectrometry (MS))/MS WITH PROKARYOTIC SAMPLES Initial experiments were performed on E. coli, a well-characterized organism with minimal proteomic complexity
Summary
Advances in biofuels research focusing on feedstock characterization and engineering (Persil-Cetinkol et al, 2012; DeMartini et al, 2013; Shen et al, 2013; Eudes et al, 2014) as well as the genetic manipulation of microbes (Alper et al, 2006; Tyo et al, 2007; Keasling, 2008; Lee et al, 2008) have progressed significantly in the last few years. An important component for biotechnological research is sample throughput supported by a robust analytical platform. Recent advances in proteomics and metabolomics have focused on liquid chromatography-mass spectrometry (LC-MS) methods by increasing their sensitivity to aid discovery-based research efforts. This is most evident with the development of nano-LC couple to high-resolution mass spectrometers; yet, this technology is yet to mature into a robust platform capable of consistently analyzing hundreds of samples per week. We published a high throughput targeted proteomic toolkit based on standard flow chromatography coupled to mass spectrometry (MS) to help address these issues for Escherichia coli; a significant amount of methods development was necessary. This work prompted us to assess the utility of standard flow liquid chromatography (LC) for shotgun proteomic methods related to biotechnology
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