Total soluble protein extraction from recombinant CTAG soybean seeds. TIMING 1-2 h for one sample
1| Using a coffee grinder, grind the soybean seeds into a fine powder. Using an analytical balance, weigh out 100 mg of powder and store the remaining powder in a vacuum bag at -80 °C for up to 1 year.
2| Place the weighed sample into a 2 mL capped centrifuge tube. Add 1 mL of petroleum ether and slowly vortex the sample for 15 min. Discard the supernatant and repeat the step twice (2X). Troubleshooting: Gently drop the solution out to avoid powder losses.
3| Allow the petroleum ether to evaporate for 10 min. Add 1 mL of the extraction buffer and slowly vortex the sample at room temperature for 10 min.
4| Leave the sample on the centrifuge for 5 min at 5000 r.min-1 at 4 °C. Transfer the supernatant to a new centrifuge tube. At this step, it can be stored at -20°C for one week. Pause point
Protein concentration TIMING 1-2 h
5| For each 200 μL of sample, add 800 μL of cold acetone to the centrifuge tube. Vortex thoroughly and keep at -20 °C for 1 h, vortexing every 15 min.
6| Centrifuge the sample for 10 min at 13000 rpm. Discard the supernatant and allow the pellet to dry at room temperature for 30 min. Critical Step Do not overdry the pellet or it may become instable and partially insoluble.
7| Carefully dissolve the pellet with 500 μL of 50 mM NH4HCO3. Quantify it using the Quant-iT™ Protein Assay Kit (Invitrogen) and dilute it with 50 mM NH4HCO3 to a 1 μg.μl-1 concentration. At this point, the sample can be stored at -20 °C for one week. Critical Step For quantification purposes, the fluorometer must be calibrated for the correct protein dosage.
Sample preparation for nanoUPLC-MSE acquisition TIMING 2 d
8| Place 50 μL of the 1 μg.μl-1 sample in a capped microcentrifuge tube.
9| Add 10 μL of 50 mM NH4HCO3.
10| Add 25 μL of the surfactant solution and vortex. Critical step The surfactant solution must be applied only if the sample is placed in the ammonium bicarbonate buffer at an alkaline pH. At an acidic pH, the surfactant will be depredated, and the solution’s kinetic energy will be reduced prior to digestion, resulting in more missed cleavages and bigger peptide fragments. .?Troubleshooting
11| Place the tube in a dry bath set at 80 °C. Heat for 15 min. Critical step: Ensure the dry bath is set to the correct temperature before heating the sample.
12| Remove the tube from the dry bath. Perform a short spin; then add 2.5 μL of the reduction solution and vortex slightly.
13| Place the tube in a dry bath set at 60 °C and heat for 30 minutes. Critical step: Ensure the dry bath is set to the correct temperature before heating the sample.
14| Remove from the dry bath, allow the tube to cool to room temperature and then centrifuge it. Add 2.5 μL of the alkylation solution and vortex slightly.
15| Place the sample in the dark at room temperature and allow 30 minutes of reaction time.
16| Add 10 μL of the digestion solution and vortex slightly. Digest the sample at 37°C in a dry bath overnight. This produces a 1:100 wt:wt ratio of enzyme:protein.
17| Following digestion, to precipitate the surfactant, add 10 μL of hydrolysation solution and vortex. Then centrifuge the samples at 14000 rpm at 6 °C for 30 minutes. Transfer the supernatant to a Waters Total Recovery vial. Critical step The surfactant must be fully precipitated to ensure proper dissolution of the protein prior to injection in the chromatograph and to avoid contamination during MSE acquisition. Ensure the centrifugation step is well controlled to avoid the injection of precipitation residues into the nanoUPLC system. Troubleshooting.
18| Add 5 μL of ADH and then add 85 μL of the nanoLC-MSE solution. The final concentration of the protein is 250 ng.μL-1 and that of ADH is 25 fmol.μL-1. The final volume is 200 μL. Store at -80 °C up to 6 months. Critical step: Correctly pipetting these solutions is crucial for a good protein quantification by PLGS; therefore, it is critical to keep the counts/fmol stoichiometric ratio between the sum of the ion intensity and the concentration for a standard protein (manual response factor). It is desirable to use a manual response factor instead of the concentration amount of the internal standard protein for the best quantification analysis.
NanoUPLC-MSE acquisition TIMING 1 d
19| The nanoACQUITY™ UPLC™ system was configured as follows: the samples were initially transferred with an aqueous 0.1% formic acid solution to trap the column with a flow rate of 15 μL.min-1 for 1 min with a 5 μL loop.
CRITICAL STEP: To acquire data with the system, some considerations must be made upon installation and engineering the setup. The initial instrument setup is critical. For this purpose and for system qualification, 1 μg of the E. coli digestion standard was acquired during installation. The E. coli sample was spiked with rabbit phosphorylase B for a final concentration of 40 fmol.μL-1 on the column. The expected dynamic range was measured and the specifications were applied to reach a minimum of 2-3 orders of magnitude for the Synapt HDMS first generation mass spectrometer. After system qualification completion, the samples were left running in the MSE positive mode with a nano-electrospray source.
20| The peptides were separated with a gradient of 5–40 % mobile phase B over 90 min at a flow rate of 600 nL.min-1, followed by a 10 min rinse with 85% of mobile phase B.
21| The column was re-equilibrated at the initial conditions for 10 min. The column temperature was maintained at 35 °C. The lock mass was delivered from the auxiliary pump of the nanoACQUITY pump with a constant flow rate of 150 nL.min-1 at a concentration of 200 fmol of GFP solution (Sigma-Aldrich, USA) to the reference sprayer of the mass spectrometer NanoLockSpray™ source. ?Troubleshooting: The column diameter is critical to achieve the best resolving power and increase the peak capacity. For optimum loading for 75 μm inner diameter columns, consider using 250 to 500 ng of protein digest and 200 to 400 nL.min-1; for 100 μm columns, use 440 to 880 ng of digest and 400 to 600 nL.min-1; for 150 μm columns, use 1 to 2 μg of digest and 800 nL.min-1 to 1.2 uL.min-1; and for 300 μm columns, use 4 to 8 ug and 4 to 5 uL.min-1 with an analytical ESI source. If the analysis is with a common 2D SCX or 2D with dilution, the amount of sample injected can be multiplied by the fraction number to keep the column capacity at a maximum.
22| All samples were analysed in triplicate using a Synapt HDMS™ first generation mass spectrometer. For all measurements, the mass spectrometer operated in the “V-mode” of analysis with a typical resolving power of at least 10000 full-width half-maximum (FWHM) and a sampling rate of 10 to 20 points across the chromatography peak to provide good quantification and peak representation into the chromatogram.
23| All analyses were performed using the positive nano-electrospray ion mode (nanoESI+).
24| The time-of-flight analyser of the mass spectrometer was externally calibrated with GFP b+ and y+ ions from m/z 50 to 1990 with the data post acquisition lock mass corrected using the GFP monoisotopic precursor ion of [M + 2H] 2+ = 785.8426.
25| The reference sprayer was sampled with a frequency of 30 s.
26| The nanoUPLC-MSE data were collected in an alternating low energy and elevated energy mode of acquisition. The continuum spectra acquisition time in each mode was 1.5 s of scan time with at least 10 points per peak on the chromatogram.
27| In the low energy MS mode, the data were collected at a constant collision energy of 3 eV.
28| In the elevated energy MS mode, the collision energy was increased from 12 to 45 eV during each 1.5 s spectrum.
29| The radiofrequency applied to the quadrupole mass analyser was adjusted such that ions from m/z 50 to 2000 were efficiently transmitted.
Data Processing and Protein Identification TIMING 1 d
30| The MS data obtained from the nanoUPLC-MSE were processed and searched using the ProteinLynxGlobalServer (PLGS) version 2.4v configured as follows. Sequences from Glycine max were downloaded from UniProt54. In PLGS, a new databank named “GLYCINE” was created, and the file containing amino acid sequences was appended. The protein identifications were obtained with the embedded ion accounting algorithm of the software and by searching the database with MassPREP™ Protein Digestion Standards (MPDS) inside as an UniProtKB/Swiss-Prot sequences (Phosphorylase - P00489 - PHS2_RABIT, Bovine Hemoglobin - P02070 - HBB_BOVIN, ADH - P00330 - ADH1_YEAST, BSA - P02769 - ALBU_BOVIN) and a CTAG-P78358 protein appended to the database. CRITICAL STEP: The database must be correctly loaded into the PLGS. The identifications and quantitative data packaging were generated using dedicated algorithms42, 55 and searching against a species-specific database56. Refer to the software manual on how to proceed with the input method into the databank administration tool. ?Troubleshooting.
31| In PLGS, a new workflow was created for Electrospray-MSE analysis by setting the data bank to “GLYCINE” and setting the peptide and fragment tolerance to automatic. The minimum fragment ion matches per peptide was set to 3. The minimum fragment ion matches per protein was set to 7. The minimum peptide matches per protein was set to 1. The maximum protein mass was set to 600 kDa. Trypsin was chosen as the primary digest reagent, allowing 1 missed cleavage. Carbamidomethyl-C and the oxidation of M were set to fixed and variable modification, respectively. N-linked and O-linked options were set as variable glycosylation modification, the calibration protein was set to P00330 (corresponding to ADH sequence in database) and the calibration protein concentration was set to 25 fmol.uL-1. CRITICAL STEP: These configurations will determine the protein identification processes and may vary from sample to sample. Changes in specificity and selectivity can vary because the minimum fragment ion matches per peptide was set to 3 and can be as low as 1; the minimum fragment ion matches per protein was set to 7 and can be as low as 5; and the minimum peptide matches per protein was set to 1. The maximum protein mass was set to 600 kDa; if the EST database was used, this can be increased to at least 1000 kDa. For standard concentration assignments, it is preferable to use the manual response to keep the counts/fmol ratio within a minimum coefficient of variation (CV).
32| In PLGS, a new data preparation was created for Electrospray-MSE analysis by setting the chromatographic peak width and MS TOF resolution in automatic mode. The lock mass for charge 2 was set to m/z 785.8426 (corresponding to GFP mass), and the lock mass windows were set to ±0.25 Da. The low and elevated energy thresholds were set to 250.0 and 100.0 counts, respectively. The retention time windows were set to automatic, and 1500 counts were applied to the intensity threshold. CRITICAL STEP: Ensure the m/z value of GFP and the charge state set are correctly assigned to avoid error in the PLGS processing. Check the instrument calibration prior to analysis. If the interval window is more than 0.4 Da for GFP, calibrate the instrument. ?Troubleshooting
33| In PLGS, open a new project. Add 3 new original samples, named SOYCTAG L3, SOYCTAG L37, and SoyCN, which correspond to the lineage 3, 37 of the recombinant CTAG in soybean and non-transgenic soybean samples to be analysed and compared, respectively. If more samples need to be compared, add more original sample tags.
34| In PLGS, add a new microlitre plate named CTAG. For each sample, add the original raw data from the acquisition, the data preparation file and the workflow file to a vial position. After the files are combined, raw data processing is possible. Tables 2 and 3 indicate a typical result. CRITICAL STEP: Ion detection, clustering, and normalisation were performed in PLGS with ExpressionE software license installed (Waters, Manchester, UK). The intensity measurements are typically adjusted, i.e., deisotoped and charge state-reduced EMRTs that replicate throughout the complete experiment for analysis at the EMRT cluster level. The components are typically clustered together with a 10 ppm mass precision and a 0.25-min time tolerance or sufficient value to achieve at least 15 points per peak. The alignment of elevated energy ions with low energy precursor peptide ions is conducted with an approximate precision of 0.05 min. To analyse the protein identification and quantification level, the observed intensity measurements are normalised to the intensity measurement of the identified peptides of the digested internal standard, as described elsewhere56.
35| For expression analysis, add a new “expression analysis” in PLGS, placing the samples created in step 33 into separate groups. In the quantification analysis, use the normalisation in proteins, selecting ADH protein in the table. The results are shown in Fig. 4.
Troubleshooting advice can be found in Table 3.