A Novel Approach to Measure Crop Plant Protein Expression

Crop development to improve yield or disease resistance has been explored for centuries and the technologies to measure these improvements have subsequently become complex. The use of transgenes in crop plants is a more technically advanced approach than traditional breeding and the success of this approach is best assessed using modern techniques that accurately quantify the desired traits. Here, we applied targeted liquid chromatography–mass spectrometry (LC–MS) using synthetic stable isotope–labeled peptides to identify and quantify the relative levels of transgenic to native protein. The methodology was developed using rice plants in which mRNA expression and phenotypic effect of the transgene had been validated. Relative quantification of transgenic barley alanine aminotransferase (AlaAT) used targeted LC–MS of tryptic protein fragments. We chose the LC–MS method as a superior technique to directly measure protein levels because other methods such as western blot analysis and RNA were unable to distinguish the minor amino acid differences between the transgenic and native proteins. Establishment of this methodology is a first step toward using LC–MS as a predictive tool to quantify the value of genetically engineered plants before the high investment of a full field trial.

The improvement of crop plants for yield, insect resistance, and abiotic stress tolerance is a continuous process in agriculture. In addition to traditional breeding, genetic engineering offers an approach to introduce changes with a targeted adjustment in a plant’s ability to grow. While the ultimate goal in crop improvement is often focused on increasing yield, the tools to measure the biological changes in the plant vary. Selecting the right tool to quantify the improvement hinges on many factors but all other things being equal, the most important consideration is the balance of time and cost.

We detected and quantified transgenic proteotypic peptides using stable isotope–labeled peptides as internal standards and spiked them into rice leaf samples to accurately quantify the endogenous levels of transgenic protein. This workflow is similar to other targeted proteomic workflows for the identification of biomarkers and low-level endogenous proteins in complex matrices (6,7).

Our goal was to measure the amount of transgenic barley AlaAT protein against the amount of native AlaAT across multiple rice lines. If the technique proves to be a robust and accurate means to measure protein levels, in the future we could apply LC–MS to evaluate the potential performance of a rice line before the investment in space, time, and resources for a field trial. By correlating yield improvements to transgenic protein levels in the greenhouse or growth chamber, we could significantly reduce the number of transformation lines that require field testing. The first step in this process is to establish the methodology with plant lines that have been evaluated in the field and determine whether LC–MS is a suitable tool for measuring protein levels. The work we describe here established the necessary resources to differentiate transgenic from native protein using LC–MS as a primary tool.

Experimental Methods

Tissue Collection

Table I: Targeted peptides of barley alanine aminotransferase.

Total Protein

Quantification and Normalization

Protein samples were prepared using the NucleoSpin RNA/Protein kit and quantified using the Protein Quantification Assay (both from Macherey-Nagel) according to manufacturer instruction. All protein samples were analyzed by 4–12% Bis-Tris sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in MES buffer (Invitrogen).

Protein Gel Electrophoresis

A 5-μL volume of Kaleidoscope Prestained Standard (Bio-Rad) and 70 μg of each total protein sample were loaded onto a NuPAGE Novex 4–12% Bis-Tris Gel 1.5 mm, 10-well precast polyacrylamide gel (Invitrogen). The protein bands were visualized using the Colloidal Blue Staining Kit (Invitrogen).

Excision of Barley AlaAT Protein Bands

The gel was cut into approximately 1-mm pieces for each sample between the BSA band (<78 kDa) and the carbonic anhydrase band (>45.7 kDa), a section that contains the target barley AlaAT protein. Blank lanes were used as controls. Gel pieces were stored separately at -20 °C in 1.5-mL siliconized Eppendorf tubes.

Peptides and Digest

The bands corresponding to AlaAT were excised, destained, reduced with tris (2-carboxyethyl)phosphine (TCEP), and alkylated with iodoacetamide before digestion using an in-gel tryptic digestion kit (Thermo Fisher Scientific). Quantification of AlaAT in transgenic rice used targeted LC–MS of four peptides. We previously discovered which peptides could be readily detected in the transgenic rice lines. Peptides were selected based on results from transgenic rice samples and are described in Table I. Liquid chromatography– multiple reaction monitoring-mass spectrometry (LC–MRM-MS) was used to determine which of the specific proteotypic peptides generated clear MRM signals in the endogenous matrix of background peptides. The peptides were also selected based on unique sequence identity for barley over the rice native AlaAT. Four synthetic heavy peptides (Table I, Thermo Fisher Scientific) were mixed with sequencing-grade porcine trypsin (Promega) just before its addition to excised gel pieces. Protein was digested using a protein-to-enzyme ratio of approximately 50:1 and incubated at 37 °C for 16 h. The amount of each stable isotope (13C/15N)–labeled peptide added to each gel sample was 1500 fmol. For each analysis, one-half of the recovered tryptic digest was analyzed.

Figure 1: Protein sequence alignment of the barley and rice AlaATs.

Chromatography and Mass Spectrometry

Chromatography of peptides used a Paradigm MDLC MS4 LC pump and a 150 mm × 0.2 mm, 3-μm dp, 200-Å C18AQ column (Michrom Bioresources). Peptides were eluted using a 2-μL/min flow rate and a gradient of acetonitrile (solvent B) in 0.1% formic acid (solvent A) as follows: 5–40% B over 50 min, 40–80% B over 1 min, hold at 80% B for 1 min, 80–5% B over 1 min, and hold at 5% B for 14 min.

An LCQ Deca XP-plus ion-trap mass spectrometer (Thermo Scientific) equipped with a Michrom Advance Spray Source was used for MS-MS analysis in positiveion electrospray mode. The source spray voltage was 1200 kV and the capillary temperature was 200 °C. The MS-MS filters, instrument conditions, and voltages were optimized for each targeted peptide by direct infusion and LC–MS analyses of the heavy synthetic peptides. LC–MS data were integrated and processed using Xcalibur software (Thermo Scientific).