Multiplexed Protein Quantification in Maize Leaves by Liquid Chromatography Coupled with Tandem Mass Spectrometry: An Alternative Tool to Immunoassays for Target Protein Analysis in Genetically Engineered Crops

Abstract

A multiplexing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method to quantify three proteins in maize leaves was developed and validated. For each protein, a hybrid Q-TRAP mass spectrometer was operated in the information-dependent acquisition (IDA) mode to select optimal potential signature peptides. The respective signature peptides were then further optimized and quantified as protein surrogates by multiple reaction monitoring (MRM). Leaf crude extracts were subject to microwave-assisted trypsin digestion for 30 min and then injected directly onto a high-performance liquid chromatography (HPLC) column without further separation or enrichment. The minimum sample process enabled us to achieve high recovery and good reproducibility, with a throughput of 200 samples per day. Using recombinant proteins as standards, a linear dynamic quantitative range of 2 orders of magnitude was obtained (correlation coefficient > 0.997) with good accuracy (deviation from nominal concentration < 15%) for all three proteins. Our study demonstrates that LC-MS/MS can be used as an alternative to immunoassays to quantify multiple low abundant proteins in genetically engineered crops.

A laser ablation ICP‐MS based method for multiplexed immunoblot analysis: applications to manganese‐dependent protein dynamics of photosystem II in barley (Hordeum vulgare L.)

“…Comparison of the LA-ICP-MS and ESI-LC-MS/MS techniques SRM is typically applied for validating changes in multiple protein abundances measured by untargeted proteomics, e.g. DDA (Thingholm et al, 2011;Kroksveen et al, 2013), whereas immunoblotting is commonly used when a smaller number of proteins need validation or when mass spectrometry instrumentation is unavailable (Hu and Owens, 2011;Aebersold et al, 2013;Kume et al, 2014). In the present study, DDA and SRM data were compared with LA-ICP-MS data in order to investigate the potential of the latter technique for targeted and multiplexed protein analysis.…”

Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette

“…HPPD protein concentration in young soybean leaves was measured using liquid chromatography-tandem mass spectrometry according to Hu and Owens (2011) with some modifications. Leaf samples were collected from the youngest expanded trifoliate leaf.…”

MS‐Based Methodologies for Transgenic Foods Development and Characterization

“…The combination of iTRAQ labeling with a fractionation step using SCX chromatography as a previous step to nLC-ESI-QTOF-MS enabled the quantitative detection of 0.5% GM soya in seed mixtures. The suitability of LC-ESI-QTRAP-MS for simultaneous identification and quantification of three transgenic proteins in maize leaves has been recently reported (Hu and Owens, 2011). The analysis of tryptic-digested extracts under linear ion-trap mode provided a linear dynamic quantitative range of two orders of magnitude (correlation coefficient >0.997) with good accuracy (deviation from nominal concentration <15%) for the recombinant proteins.…”