A high-throughput absolute level quantification of protein-bound amino acids in seeds

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This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi:

10.1002/cppb.20084.

A high-throughput absolute level quantification of protein-bound amino acids in seeds

Abou Yobi and Ruthie Angelovici*

Bond Life Science Center, Division of Biological Sciences, Interdisciplinary Plant Group, University of Missouri, Columbia, Missouri, USA.

*Corresponding author: E-mail: [email protected], tel.: +1 573-882-3440

In the following protocol, we describe a high throughput absolute quantification protocol for 16 protein-bound amino acids (PBAA) that combines a microscale protein hydrolysis step and an absolute quantification step using a multiple reaction monitoring tandem mass spectrometric (LC-MS/MS) detection method. The approach facilitates analysis of a few hundred samples per week by using a 96-well plate extraction setup and avoiding use of additives. Importantly, the method uses only ~3 mg of tissue per sample and includes 12 heavy internal standards to enable the quantification of the absolute levels of PBAAs with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples but is applicable to other plant tissues.

Keywords: Amino acids, protein hydrolysis, seeds, LC-MS/MS

INTRODUCTION

The amino acid (AA) composition of mature, dry seeds is crucial for plant fitness and survival and is rigorously maintained by tight regulation of the transcriptome, proteome, and metabolome (Fatihi et al. 2016; Galili et al. 2016; Wu and Messing 2014). There are two functional AA pools in seeds: protein bound amino acids (PBAAs, ~95%) and free amino

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acids (FAA; ~5%). Their overall content and relative composition play important roles during desiccation, longevity, and early stages of seedling establishment (Angelovici et al. 2011;

Fait et al. 2006; Rajjou et al. 2012; Tan-Wilson and Wilson 2012). For most plants, PBAAs are mainly stored in the form of seed storage proteins, which comprise about 60-70% of a seed’s overall AA content (Flint-Garcia et al. 2009). Storage proteins are very poor in essential amino acids (EAAs), especially in cereal grains, which compromises their nutritional quality(Galili and Amir 2013; Holding and Messing 2013; Shewry et al. 1995).

Since crop seeds, in general, and cereal grains, specifically, are the main source of protein for humans and livestock worldwide, many attempts have been made to alter seed storage protein levels and composition. Knockdown or introduction of transgenic proteins with altered AA composition have had little effect on the overall or relative composition of AA in seeds (Herman 2014; Morton et al. 2016; Withana-Gamage et al. 2013). However, there is substantial natural variation in PBAA in most crop plant seeds, but the key regulators of PBAAs in seeds are still unclear. In the genomic era, new avenues of research are possible if one is able to apply a high throughput quantification of the seed PBAA in large mutant screens or naturally variable populations. To date, the rate limiting factors are the protein hydrolysis step and the lengthy classical detection methods.

Analysis of PBAAs in seeds starts with protein hydrolysis and is followed by hydrolyzed AA detection. The most reliable and commonly used method for protein hydrolysis is acid hydrolysis (Moore 1958), which is slow, labor intensive, and low throughput (Marconi et al. 1995; Otter 2012). The “gold standard” for hydrolyzed AA detection is a high-performance liquid chromatography (HPLC) method that entails a post- column derivatization usually performed by an amino acid analyzer (Smith 2003). However, a single analysis can last more than an hour and is relatively expensive.

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This protocol describes a novel, high-throughput method for PBAA hydrolysis using a 96-plate setup followed by a short, targeted AA analysis using a slightly modified tandem mass spectrometric (UPLC-MS/MS) method that was established previously for FAA detection (Angelovici et al. 2013a; Gu et al. 2010).

BASIC PROTOCOL 1: Protein-bound amino acids extraction and detection procedures

The protocol involves five steps (Fig. 1): (a) acid hydrolysis, (b) preparation of serial dilutions for the standard curve, (c) extraction of amino acids with the extraction buffer EB2, and (d) detection and quantification of AA with UPLC-MSMS. The reagents and solutions section details the preparation of solutions for AA standards and extraction buffers (EB1, EB2).

[*Figure 1 here]

MATERIALS

6N hydrochloric acid solution (6N HCl; Fisher Scientific, cat. no. SA561)

Heavy amino acid internal standards (Suppl. Table 1)

Amino acid standards (Suppl. Table 2; Sigma-Aldrich, St. Louis, MO)

Dithiothreitol (DTT; Fisher, cat. no. R0861).

Perfluoroheptanoic acid (PFHA; Sigma-Aldrich, 342041).

Compressed nitrogen

Acetonitrile (HPLC-grade)

Methanol (HPLC-grade)

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Water (HPLC-grade)

Sub-milligram analytical balance (e.g., Mettler Toledo XS 105; Fisher Scientific, cat. no. 01- 913-892)

Cooler centrifuge equipped with 96-well plate rotors (e.g., Sorvall Legend XTR; Fisher Scientific, cat. no. 75-004-523)

Anti-corrosive Speedvac evaporator equipped with 96-well plate rotors (e.g., Savant SC250 EXP; Fisher Scientific, cat. no. SC250EXP-115) coupled with a refrigerated vapor trap (e.g., Savant RVT5105 Refrigerated Vapor Traps; Fisher Scientific, cat. no. RTV5105-115) and vacuum pump (e.g., VLP120 series; Fisher Scientific, cat. no. 50-870-639)

96-well racks with lids (NOVA Biostorage, part no. MPW51BCPK)

1.1 ml non-coded Screw Cap Tubes V-bottom Bulk (VWR, cat. no. 101975-256)

Disposable, anti-static microspatula (e.g., LevGo, cat. no. 17231B)

3 mm solid glass beads (e.g., Sigma-Aldrich, CLS72683)

3 mm bead dispenser, 96-well (e.g., Qiagen, cat. no. 69973)

High-energy, high-throughput cell disrupter (e.g., Mini-Beadbeater-96 with 1400 to 2400 rpm speed; BioSpec Products, cat. no. 1001)

Oven that can maintain 110°C (e.g., Heratherm Oven OGS60; Fisher Scientific, item no.

T9FB2187511)

Filter plate vacuum manifold, optional (e.g., VWR, cat. no. 16003-836)

Pierceable capband-8 in a capmat format (e.g., Micronic, MP53002)

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Manual decapper (e.g., Micronic, MP54001)

Filter plate, hydrophilic, PTFE, 0.45 µm, clear, non-sterile (e.g., EMD Millipore, cat. no.

MSRLN0450)

96 round-well Plate V bottom microplate, 520 µl (Dot Scientific, cat. no. PC63241-NS6)

96-well full skirt PCR plates (e.g., Denville Scientific, Part no. 1000896)

Pre-slit Silicone mat, round for 96-PCR plate (Midwest Scientific, Inc., part no. MID-MATR- 9-PRT)

50 ml reagent reservoir (e.g., Fisher Scientific, cat. no. 07200127)

8 and 12 multichannel pipettes (200 and 10 µl volumes) and pipette tips

Falcon tubes (50 ml)

Eppendorf tubes (1.5 ml)

Kinetex 2.6 µm C18 100A^o LC column 100 x 2.1 mm (Phenomenex, part no. 00D-4462-AN)

Xevo TQ-S UPLC-MS/MS (Water’s Corporation, MA)

Heat-resistant gloves

Permanent marker

Ice

Heavy amino acid stock solutions (see Reagents and Solutions)

Proteinogenic amino acid stock solutions (see Reagents and Solutions)

Extraction Buffers (see Reagents and Solutions)

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[*Table 1 here]

[*Table 2 here]

Protocol Steps

PERFORM PBAA ACID HYDROLYSIS

1. Weigh out 3 mg of crushed or ground seed and place in 1.1 ml tubes.

For small seeds, like Arabidopsis, crushing seeds by shaking with a beadbeater is sufficient.

For large seeds (e.g., maize and soybean), grind seed to obtain a homogeneous powder prior to weighing. Non-homogenized samples will lead to large technical variations.

2. Place tubes in a 96 well rack following the order: A1 to A12, B1 to B12, and so on 3. Add three 3 mm glass beads to each tube using bead dispenser.

4. In a chemical hood and using a multichannel pipette and a 50-ml reagent reservoir, add 200 µl of 6N HCl to each sample tube and to 3 empty tubes (negative controls).

Always include 3 negative controls in which no sample is added to the 6N HCl to ensure impurities are not contaminating samples.

5. Flash tubes with compressed nitrogen to remove oxygen.

6. Close lids firmly with a capmat to preserve sample during hydrolysis.

7. Shake rack for 4 min using a mini-beadbeater.

8. Spin rack in centrifuge briefly to bring samples down to the bottom of the tubes.

9. Place rack into anti-corrosive, heat-resistant container and close container.

Although capping the 1.1 ml tubes with capmat prevents an HCl leak, samples are placed in a sealed container as an additional safety measure.

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10. Place container in oven preheated to 110°C.

11. Incubate at 110° C for 24 h.

12. Remove container from oven using heat-resistant gloves and cool to room temperature.

13. Remove rack from the container.

14. Centrifuge 15 min at 3700 x g to precipitate debris.

15. Transfer 100 µl of the hydrolyzed sample onto a filter plate using multichannel pipette.

Make sure to maintain the same samples order.

16. Place filter plate on a vacuum manifold, apply vacuum, and recover the hydrolysate with a collection plate.

This filtering step is important because particles can damage the LC-MS/MS or interfere with its proper operation or detection. It is not sufficient to pellet plant tissues and other contaminants via centrifugation prior to injection. If a vacuum manifold is not available, sample filtering can be performed by centrifugation for 15 min. at 3000 x g and room temperature. Higher speeds can damage the filter

membrane.

17. Transfer 10 µl of filtered samples to new 1.1-ml tubes and place in a new rack.

Store remaining filtered samples at -20°C, if you wish to repeat the experiment.

18. Speedvac until samples are fully dried.

It is important to dry completely as any HCl remnants can interfere with detection and quantification.

19. Cover tubes with a capmat and store at 4°C until ready for analysis.

We recommend analyzing samples within 48h

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PREPARE SERIAL DILUTION FOR STANDARD CURVE

20. Prepare heavy and proteinogenic amino acid stock solutions (see Reagents and Solutions).

21. Prepare extraction buffers EB1 and EB2 (see Reagents and Solutions).

22. Transfer 40 µl of each proteinogenic amino acid solution standard mix and 160 µl of EB1 to a tube labeled “200 µM Standard” and mix well by pipetting up and down.

Note: In this step, we dilute all the heavy standard with EB1 by 20% therefore all further dilutions are made with EB2 which is already diluted by 20% (see reagent and solutions).

23. Use EB2 and the initial 200 µM standard to prepare an 8-concentration serial dilution for a standard curve.

We recommend a standard curve with the following concentrations: 0, 0.2, 1, 2, 10, 20, 100, and 200 µM for the 16 proteinogenic amino acids and 0, 0.6, 3, 6, 30, 300, and 600 µM dilutions for Gly. Each point including 0 contains a fixed amount of heavy standard since it is diluted with EB2 (i.e., 8 µM for Ala-d4 and Asp-d3, 12 µM for Gly-d25 µM, and 4 µM for the remaining heavy internal standards). These concentrations can be adapted as needed.

24. Place the standard curve dilutions on ice until ready to transfer to the designated PCR plate.

EXTRACT AMINO ACIDS

25. Re-suspend each pellet with 400 µl of EB2 using multichannel pipette and a 50-ml reservoir.

26. Seal firmly with capmat.

27. Shake 4 min.

28. Centrifuge 15 min at 4°C at 3700 x g.

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29. Transfer 70 µl of each sample to its corresponding well in a 96-well full skirt PCR plate using multichannel pipette.

30. Transfer 70 µl from the standard serial dilution to the PCR plate containing the samples using multichannel pipette.

31. Dilute all samples and standard by half by adding 70 µl EB2 using multichannel pipette.

Note: The final concentration of the standards after dilution are 0, 0.1, 0.5, 1, 5, 10, 50, and 100 µM. These volumes fit the PCR plate volume. This step is necessary to diminish ion suppression and reduce concentrations of interfering contaminants and, thus, improving sensitivity and accuracy of detection.

32. Seal plate with a pre-slit Silicone mat.

33. Store at -80°C until ready for analysis.

Note: Prior to analysis, remove samples from the -80^oC freezer, let thaw, and centrifuge briefly to remove bubbles. We recommend analyzing within 48 h.

DETECT AND QUANTIFY AA USING LC-MS/MS

Several analytical instruments could be used for AA detection after hydrolysis. In this method, we used Xevo TQ-S UPLC-MS/MS (Water’s Corporation, MA) with the following settings:

34. Thaw PCR plate containing samples, prior to injection.

35. Spin briefly at 4°C to remove air bubbles.

36. Adjust LC settings

a. Use a Phenomenex Kinetex 2.6 µm C18 100Å LC column 100 x 2.1 mm (part no.

00D-4462-AN)

b. Set column oven temperature to 30°C.

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c. Set autosampler temperature to 10°C

d. Set injection volume to 10 µL.

e. Set mobile phase to 1 mM PFHA for A and acetonitrile for B and the flow rate to 0.3 mL/min.

The flow gradient of the mobile phase is shown in Table 4.

37. Adjust MS/MS settings

a. Acquire mass spectra using electrospray ionization (ESI) in positive ion mode and multiple reaction monitoring (MRM).

b. Set voltage of capillary, cone, and source offset to 3.17, 18, and 50 V, respectively.

c. Set flow of cone gas and desolvation to 150 L/h and 500 L/h, respectively.

d. Set nebulizer to 7 bar.

e. Set desolvation temperature to 350°C.

f. Set collusion gas flow to 0.15 ml/min.

The detection method is composed of three ESI+ functions (0-2.0, 1.81-3.20, and 2.91-5.80 min). Collision energies and source cone potentials were optimized for each transition using Water’s IntelliStart (see Suppl. Table 5).

38. Inject samples

39. Acquire data using MassLynx (ver. 4.0).

40. Process calibration and quantification of the analytes using QuanLynx software (MassLynx V4.1 SCN919; Water’s Corporation, MA).

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41. Export data to spreadsheet.

42. Back calculate absolute based on total volume and sample weight used to obtain the final concentrations in nmole/mg tissue.

REAGENTS AND SOLUTIONS Heavy amino acid stock solutions

1. Prepare 20 mM stock solutions using 19 µM DTT for each individual heavy standard (Suppl. Table 1), except Gly.

2. Prepare 60 mM stock solution of Gly with 19 µM DTT.

3. Assemble a heavy standard AA mix by combining the appropriate amounts from each individual standard to make the concentration of 200 µM for Ala-d4 and Asp-d3, 600 µM for Gly-d2, and 100 µM for the remaining 9 compounds in the mixture (Suppl.

Table 1).

4. To reach the desired molar concentration for each standard in the mix, adjust the final volume with 19 µM DTT (see an example in the Suppl. Table 3).

5. Aliquot solution according to usage (e.g., 1200 µl).

Two aliquots of 1200 µl are enough for the extraction of a 96-well plate.

6. Label with name and date.

7. Store at -80° C for up to 3 months.

[*Table 3 here]

[*Table 4 here]

[*Table 5 here]

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Proteinogenic amino acid stock solutions

1. Prepare 20 mM stock solutions for each proteinogenic amino standards (Suppl. Table 2), except Gly and Tyr.

2. For Gly, make a 60 mM stock solution.

3. For Tyr, make a 5 mM stock solution.

Tyr does not dissolve well in water at high concentrations.

4. Make a proteinogenic AA standard mix by combining appropriate amounts from each standard to achieve a final concentration of 3 mM for Gly and 1 mM for the

remaining 15 amino acids (see an example in the Suppl. Table 3).

5. Aliquot into small volumes (e.g., 100 µl).

6. Label with name and date.

7. Store at -80°C for no longer than 3 months.

Extraction Buffers

1. To prepare the extraction buffer EB, first calculate the number of samples, standard curve dilutions, and extra sample volume that will be included.

We recommend including a duplicate series of 8 dilutions in the proper range and an extra buffer to account for pipetting errors. We also recommend including an amount that is enough to extract 12 samples) as shown in the formula: N+16 +12 = N+28.

Volumes for 60 samples are calculated in Supplemental Table 3.

2. Prepare extraction buffer in a clean conical tube or bottle as follows and where N = number of samples:

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 Water (ml) = (N+28)*0.456

 10 mM DTT (µl) = (N+28)*0.9

 Heavy standards (ml) = (N+28)*0.024

Stock solution of DTT is 10 mM, and the desired concentration is 19 µM.

3. Place 1 ml of the EB into a clean Eppendorf tube and label “EB1”.

EB1 is used only to make the 200 µM dilution of the initial concentration of the serial dilution for plotting the standard curve.

4. Dilute remaining EB by 20% with HPLC-grade water to make EB2.

Note: Keep both EB1 and EB2 on ice for the duration of the experiment.

COMMENTARY Background information

Despite the availability of cutting edge metabolic analysis instruments and high-throughput FAA analysis from plant tissues (Angelovici et al. 2013b; Gu et al. 2007), releasing AAs from a protein backbone prior to analysis remains challenging. To date, there is no single method that can release all PBAAs (Davidson 1997; Fountoulakis and Lahm 1998). Several hydrolysis methods have been developed. The most prominent method is HCl hydrolysis, which consists of adding 6N HCl to a sample and incubating at 110°C for 20 to 24 h (Moore 1958). Under these conditions, asparagine (Asn) and glutamine (Gln) are completely hydrolyzed to aspartic acid (Asp) and glutamic acid (Glu), respectively; tryptophan (Trp) is completely destroyed; cysteine (Cys) is indirectly measured; and tyrosine (Tyr), serine (Ser),

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and threonine (Thr) are partially hydrolyzed but corrected for(Blackburn 1978). Ser and Tyr are significantly degraded (>30%) under acid hydrolysis (Anders, 2002). Although some AAs, such as alanine (Ala), valine (Val), and isoleucine (Ile), are recovered without noticeable degradation, the breakdown of Ala-Ala, Val-Val, Ala-Val, Ile-Ile, and Ile-Val bonds is not always complete and can require incubation times up to 120 h (Ozols 1990).

A number of variations to this method have been developed. Some methods employ higher incubation temperatures (145 to 166^oC) for shorter periods of times (4 to 16 h) in the presence of an additive, including phenol, beta-mercaptoethanol, performic acid, and thioglycolic acid (Fountoulakis and Lahm 1998). Most of these methods are specifically designed to target AAs that are poorly quantified (i.e., Met and Lys) or completely lost (i.e., Trp and Cys) under normal HCl hydrolysis (Fountoulakis and Lahm 1998 & the references therein). Some of these additives can negatively impact the detection of other AAs (Tsugita and Scheffler 1982). An alternative acid used for protein hydrolysis includes 4 M

methanesulfonic acid (MSA) in the presence of 3-(2-Aminoethyl)-indole or tryptamine (Ingles, 1983). The use of microwaves to hydrolyze proteins has been shown to greatly shorten incubation time(Marconi et al. 1995), but this method requires a great deal of precaution and optimization for each individual protein or sample. It’s also been shown that alkaline hydrolysis is preferred when targeting Trp(Hugli and Moore 1972) since under acid hydrolysis quantification of this amino acid is still challenging even in the presence of additives(Adebiyi et al. 2005).

After protein hydrolysis, several quantification methods can be applied. LC-MS/MS coupled with MRM transitions selected for each compound facilitates fast and accurate absolute quantification of the amino acids, but poorly detects Gly(Gu et al. 2007). Other detection methods can involve pre- or post-column sample derivatization, and the hydrolyzed samples must be analyzed immediately to avoid degradation. Other detection alternatives

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include HPLC, GC-MS, and GC-MS/MS, all of which require a time sensitive sample derivatization or have a less sensitive detection compared to the LC-MS/MS method.

Critical parameters and troubleshooting

The following practices can help ensure a successful amino acid analysis and quantification:

1. All materials used for hydrolysis (e.g., plastic tubes containing samples, mat covering the tubes, rack holding the tubes, Speedvac) must be able to handle a strong acid and not corrode. The Speedvac evaporator also should be equipped with a cold trap to collect any acid residues, which must be disposed as regulated by your institution. Handle 6N HCl in the hood only and avoid touching it with bare hands or spilling it in on surfaces.

2. Do not use 6N HCl past its expiration date, as this can result in partial hydrolysis of a sample.

3. It is essential to have an analytical instrument (e.g., HPLC/UPLC-MSMS) equipped with a 96-well format autosampler. Other instruments, such as the cooler centrifuge and Speedvac, also should also be equipped with 96-well plate and rack rotors.

4. Use multichannel pipettes during all stages of the protocol for a high throughput analysis and the prevention of pipetting errors.

5. Make sure peaks of heavy standards in samples are similar to peaks in standards or choose a different internal standard as this might be due to a co-elution from a

contaminant in the tissue. For example, some tissues produce contaminants that co-elute with Met-d3 (Met*), which makes quantification of Met inaccurate. In such cases, use Ser-d3 (Ser*) instead.

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6. Prepare two serial dilutions and use to plot the standard curve. We recommend injecting a complete series before the first sample and the second series after the last sample to help ascertain whether there were any changes in sensitivity and retention times during the run.

7. Renew the mobile phase A (PFHA) every 2 weeks to avoid a shift in retention time and contaminants.

8. Wash after every 48 samples, before and after each standard, and between treatments to reduce the risk of cross contamination. Run a 20 min wash of absolute methanol at the end of the cycle to clean the column and the UPLC instrument.

9. Use clean columns. A dirty column can cause long retention times and poor detection.

The separation between Leu and Ile can be used as an indicator of the conditions of the column. With a new column, these two compounds separate nearly completely. At 50%

separation, the column is deemed dirty, and cleaning or replacement is required.

10. Use 96-well PCR plates suitable for the LC-MS/MS instrument. Plates not fully aligned with the injection needle can damage the instrument or inject the wrong volume.

11. Use only “Pre-slit” mats to cover 96-well PCR plates, other films can clog the needle.

12. For additional LC-MS/MS-related issues, see (Gu et al. 2007).

13. Although including internal standards, such as norleucine or norvaline, prior to hydrolysis is good practice, the reduced steps in this protocol, the high reproducibly, and the

interference of these two standards with leucine and valine, respectively, render their inclusion unnecessary. According to the 2001 DIONEX technical report, internal standards do not further improve data that are reproducible prior to their inclusion.

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Anticipated Results

To validate this method, we analyzed two concentrations (0.1 and 0.05 mg/ml) of Bovine serum albumin (BSA^;Gold Biotechnology, cat. no. A-420-10) from a 1-mg/ml stock (dissolved in water). We analyzed 12 replicates for each concentration and quantified 16 PBAAs. With the exception of Gly, all AA quantifications were comparable with the expected values deduced from the sequence. The average recovery (observed

concentration/expected) for all AAs, excluding Gly, was ~ 92% (Suppl. Table 6). These results are in the same range of observed values detected by other hydrolysis and quantification studies (Hewitson 2007; Marconi et al. 1995). For example, a microwave hydrolysis of BSA followed by a Beckman 118BL analyzer observed values ranging from 69 to 132% of the expected values (Marconi et al. 1995).

To assess the reproducibility among technical repeats (n=24), we calculated the relative standard deviation (RSD) for each AA, our results indicated an average of ~ 6%

RSD. (See Suppl. Table 6 for detailed analysis.) Because the assay is highly reproducible, it is possible to use recovery assays to adjust the quantification of the absolute levels of AA partially hydrolyzed or poorly detected (Robel and Crane 1972, Darragh, 1996 #321).

However, it is important to use an assay with an appropriate protein. For seeds, one can use a recombinant protein of the main seed storage protein for appropriate calibration and

adjustment.

To assess the reproducibility in various plant tissues, we analyzed 12 technical repeats from Arabidopsis, maize, and soybean seeds and 8 technical replicates from maize leaf tissues. As shown in Suppl. Table 7, the technical repeats had an average RSD of 6.96%

across all the seeds an RSD of 6.58% for maize leaves. Taken together, these results support that the method is highly reliable and reproducible.

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Surprisingly, despite the poor recovery and reproducibility of Gly for BSA, this was not the case in both seed and leaf tissues. The RSD across all seed tissues was 9.63% and 5.89% for maize leaves (Suppl. Table 7). This is probably due to differences in Gly concentrations between the two assays. We found that a low Gly concentrations results in poor recovery and reproducibly, while high Gly concentrations result in the opposite. In the BSA assay, the injected amounts of Gly are much lower than in the analyzed tissue and therefore were poorly quantified.

[*Table 6 here]

[*Table 7 here]

Time considerations

From start to finish, the method takes 2 days. The following is a breakdown of the approximate time needed for each step.

1. 2 h to weigh a plate and add glass beads to each tube using a bead dispenser.

2. 30 min to add 6N HCl, flush with nitrogen, shake, spin down, and place in oven.

3. 24 h for hydrolysis

4. 2 h to cool, filter, and dry samples.

5. 2 h to make extraction buffer, standard preparation, sample extraction, and final dilution.

6. For the LC-MS/MS, 1 h to equilibrate the column, prep the instrument, and prepare sequence table.

7. 10 h to run a full plate.

8. 2 to 6 h for data analysis, depending on experience.

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To take full advantage of the method, analyze two plates at a time, as the time required to analyze two plates at once does not double the time of analyzing one plate.

Acknowledgements

The authors acknowledge Dr. Brian Mooney, Thomas Mawhinney, and Daniel Jones for their valuable advice on protein hydrolysis and LC-MS/MS operation and detection. This study was funded by the University of Missouri-Christopher Bond Life Sciences Center seed fund and the NSF 1355406 (EPSCoR; The Missouri Transect, Climate, Plants, and Community).

Conflict of Interest

Authors declare that there are no conflicts of interest.

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FIGURE LEGENDS

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Figure 1. An overview of the amino acid analysis experimental flow. AA: amino acid; EB: extraction buffer; STD: standard

Tables Table 1

Deuterated MW [Stock] [Stock mix]

Vol (µL) to add

to Used to

or C13 labeled Manufacturer Item No. (g/mol) (mM) (µM) a working mix quantify

DL -Ala-d₄ Cambridge Isotope Labs, Inc. DLM-1276-1 93.09 20 200 180 Ala

DL -Asp-d₃ Cambridge Isotope Labs, Inc. DLM-832-1 136.1 20 200 180 Asp

L -Glu-d₃ Cambridge Isotope Labs, Inc. DLM-3725-0.5 151.14 20 100 90 Glu

Gly-d2 Cambridge Isotope Labs, Inc. DNLM-6862-0.25 78.07 60 600 90 Gly

L -Lys:2HCl-¹³C₆,¹⁵N₂ Cambridge Isotope Labs, Inc. CNLM-291-H-0.1 227 20 100 90 Lys

L -Phe-d₈ Cambridge Isotope Labs, Inc. DLM-372-1 173.19 20 100 90 Phe, Tyr

L -His-d₃ CDN Isotopes D-6679 212.65 20 100 90 His, Arg

L -Leu-d10 CDN Isotopes D-1078 141.24 20 100 90 Leu, Ile

L-Met-d3 CDN Isotopes D-1293 152.23 20 100 90 Met

Ser-d₃ CDN Isotopes D-1583 108.11 20 100 90 Ser, Thr

L -Val-d8 CDN Isotopes D-1076 125.2 20 100 90 Val

L-Pro-¹³C₅-¹⁵N Sigma-Aldrich 608114 121.09 20 100 90 Pro

Total 1260

Preparing 19 µM DTT solution

Water 20 ml

DTT(10 mM stock) 38 µl

Final Std stock solution

19 µM DTT 16740

Heavy STD (column G, line 17) 1260

Total 18000

Aliquat* 1200

*Store at -80^oC

Suppl Table 1. List of the internal standards used for protein-bound amino acid analysis. The Table also shows stocks and needed volumes to make 18 ml of standards, which is enough for 10 plates. Column E shows the stock concentrations for each standard in mM, column F shows the concentration of each standard in the mix, Column G shows the volume needed from each stock solution to make the standard mix, Column H shows the amino acids qunatified using those standards.

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Table 2

C¹² Standard*

MW (g/mol)

[Stock]

(mM)

[Stock mix] (mM)

Vol (µL) to add for the mix**

L-Alanine (Ala) 89.09 20 1 90

L-Arginine (Arg) 210.66 20 1 90

L-Isoleucine (Ile) 131.17 20 1 90

L-Leucine (Leu) 131.17 20 1 90

L-Lysine (Lys) 182.65 20 1 90

L-Metthionine (Met) 149.21 20 1 90

L-Phenyalanine (Phe) 165.19 20 1 90

L-Serine (Ser) 105.09 20 1 90

L-Threonine (Thr) 119.12 20 1 90

L-Valine (Val) 117.15 20 1 90

L-Aspartic acid (Asp) 133.1 20 1 90

L-Glutamic acid (Glu) 147.13 20 1 90

L-Glycine (Gly) 75.07 60 3 90

L-Histidine (His) 209.63 20 1 90

L-Proline (Pro) 115.13 20 1 90

L-Tyrosine (Tyr) 181.19 5 1 360

Total 1710

*All standards are from Sigma-Aldrich (St. Louis, MO)

**Bring the final volume to 1800 µl with 19 µM DTT Make 100 µl aliquots and store at -80^oC

Suppl Table 2. List of the C12 standards used for protein-bound amino acid analysis. The table also shows the stocks and volumes needed to make 17 runs. Column C shows the stock concentrations for each standard in mM, column D shows the concentration of each standard in the mix, Column E shows the volume needed from each stock solution to make the standard mix.

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Table 3

Date:

Experiment:

Number of samples (N) 60 Average sample weight 3

Weighed by ABOU

Extraction Buffer (EB) Preparation Sample Number = N

Standards = 8x2 = 16 Extra = 12

Total = N+16+12 = N + 28

EB

N + 28 88

Component Volume Unit

Water¹ 40.128 ml

DTT² 79.2 µl

Internal standard (IS)³ 1.76 ml

EB 41.97 ml

1.Take 1 ml of EB as EB1

2. EB2

Component Volume Unit

EB-EB1 40.97 ml

Water⁴ 10.24 ml

EB2 51.21 ml

Calculations:

2DTT = (N+28)*0.9

3IS = (N+28)0.024

1Water = (N+28)*0.456

4Water = (EB-1)*(0.25)

Suppl Table 3. An Excel sheet used to calculate the volume of the extraction buffer (EB) needed for each run. Just input the number of the samples (Column B, raw 4) and the volumes of water, DTT, and heavy standards will be calculated automatically. Manual calculations could also be made following the formuals presented in lines 27, 28, 29, and 30.

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Table 4. The mobile phase gradient used for the UPLC Time

(min)

PFHA (A;

1mM)

Acetonitrile

(B) Flow

0 98% 2% 0.3

0.1 80% 20% 0.3

2.3 60% 40% 0.3

3.6 60% 40% 0.3

3.7 98% 2% 0.3

5.8 98% 2% 0.3

Table 5

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Table 6

Suppl Table 5. MSMS-MRM transitions and the conditions used to target individual amino acids. Asteriks represent heavy stnadards.

A.

AA Parent-ion Daughter-ion Dwell (sec) Cone voltage Collusion energy

Gly 76 30 0.02 18 40

Gly* 79 33 0.02 18 20 Cycle time (sec) 0.26

Ala 90 44 0.02 18 15 Retention window (min) 0.0 to 2.0

Ala* 94 48 0.02 10 15 Ionization mode ES+

Ser 106 60 0.02 18 25 Data type MRM data

Ser* 109 63 0.02 18 15 Function type MRM of 13 channels

Pro 116 70 0.02 10 10

Pro* 122 75 0.02 26 15

Thr 120 57 0.02 26 25

Asp 134 74 0.02 18 15

Asp* 137 75 0.02 18 15

Glu 148 84 0.02 18 15

Glu* 151 87 0.02 26 16

B.

Val 118 72 0.02 18 15

Val* 126 80 0.02 18 15 Cycle time (sec) 0.1

Met 150 104 0.02 18 15 Retention window (min) 1.81 to 3.2

Met* 153 107 0.02 18 15 Ionization mode ES+

Tyr 182 136 0.02 18 15 Data type MRM data

Function type MRM of 5 channels

C.

Leu 132 30 0.02 18 15

Ile 132 69 0.02 18 15

Leu* 142 96 0.02 18 15

Lys 147 84 0.02 18 15 Cycle time (sec) 0.2

Lys* 155 90 0.02 18 15 Retention window (min) 0.0-1.8

His 156 110 0.02 26 15 Ionization mode 2.91 to 5.8

His* 159 113 0.02 18 15 Data type MRM data

Phe 166 120 0.02 18 15 Function type MRM of 10 channels

Phe* 174 128 0.02 18 15

Arg 175 70 0.02 26 20

*represents deuterated or C13 labeled isotopes

Function 3

Function 1 (0.00 to 2.0 min)

Function 1

Function 2

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Table 7.

AAExpected Number of ResiduesTheoratical conc. (nmol/mg BSA)Calculated conc. (nmol/mg BSA )% recovery RSD%

Ala 46 696.97 541.50 77.69 6.99

Arg 23 348.48 368.50 105.74 16.20

Asx 54 818.18 806.33 98.55 3.35

Glx 79 1196.97 972.83 81.27 6.71

Gly* 16 242.42 289.64 119.22 189.28

His 17 257.58 240.50 93.37 4.69

Ile 14 212.12 229.50 108.19 10.94

Leu 61 924.24 831.50 89.97 2.18

Lys 59 893.94 839.67 93.93 2.61

Met 4 60.61 61.00 100.65 6.19

Phe 27 409.09 396.33 96.88 2.82

Pro 28 424.24 398.00 93.81 6.27

Ser 28 424.24 384.17 90.55 4.17

Thr 34 515.15 462.00 89.68 4.13

Tyr 20 287.88 194.33 67.51 6.19

Val 36 545.45 491.83 90.17 2.40

Average 91.87 5.72

*Gly was not included in the average calculation of recovery and RSD percentages

Suppl Table 6. A comparison between observed and expecxted values in BSA (recovery). Expected residues are the number of AA present in BSA based on the protein sequence; the theoratical concentration is the amount of each AA in one mg of BSA based on the expected residues; the calculated concentration is the amount of each AA in 1 mg of BSA based on our analysis; the % recovery indicates the

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A. B.

nmole/mg AA Arabidopsis Maize Soybean Arabidopsis Maize Soybean AA mazie leaves

Ala 97.33 129.94 194.88 4.47 8.16 5.85 Ala 142.17 7.85

Arg 61.05 29.86 92.99 2.94 9.36 4.65 Arg 37.48 3.86

Asx 99.30 56.79 309.90 5.04 9.02 6.24 Asx 112.82 6.32

Glx 164.19 163.50 280.22 4.59 8.41 4.18 Glx 109.10 5.15

Gly 233.39 133.86 306.07 12.57 12.14 3.92 Gly 153.01 5.89

His 22.05 17.59 33.37 2.92 5.44 5.70 His 13.15 6.55

Ile 61.31 47.14 156.29 4.37 10.23 6.89 Ile 67.58 6.58

Leu 91.16 136.59 216.14 3.50 9.18 5.92 Leu 107.64 6.45

Lys 59.66 19.63 177.57 3.92 12.54 12.16 Lys 46.64 12.13

Met 17.03 16.87 29.39 4.84 10.77 9.36 Met 19.56 7.31

Phe 46.34 37.60 92.49 10.85 11.08 2.32 Phe 46.32 2.89

Pro 81.12 96.28 151.17 4.58 6.44 6.75 Pro 59.78 7.36

Ser 80.76 61.39 181.04 4.89 9.17 5.75 Ser 71.25 4.66

Thr 72.80 54.62 149.69 3.39 8.33 7.44 Thr 69.68 5.99

Tyr 28.07 22.86 70.37 6.29 11.27 8.70 Tyr 25.18 10.03

Val 76.01 49.77 150.15 3.05 8.65 5.97 Val 77.28 6.30

Average 5.14 9.39 6.36 Average 6.58

nmole/mg seed tissue RSD %

RSD % Suppl Table 7. Protein-bound amino acid (PBAA) analysis from Arabidosis, maize, and Soybean seeds (A) as well as maize leaves (B).

The resultls show the amonut of amino acids in (nmole/mg tissue) as well as the relative standard deviation (RSD%)