Nanoscale two-dimensional liquid chromatography tandem mass spectrometry of rice leaf and root proteins
In this chapter, we present a detailed experimental method for the large-scale identification of proteins in rice leaf and root tissues using 2D liquid chromatography tandem mass spectrometry. The method is realized on a nanoscale flow rate dual-phase column (strong cation exchange/reversed-phase) with electrospray emission injectors, a technique known by the acronym Mudpit (Multidimensional Protein Identification). This experiment is based on the "Guide to Plant Proteomics Experiments" (France), H. Tillemment, M. Zivi, C. Damerweiler, V. Mitschine, eds.
Operation method
Nanoscale two-dimensional liquid chromatography tandem mass spectrometry of rice leaf and root proteins
Materials and Instruments
Reversed phase loading materials Move 3.1 Leaf and root tissue sampling and protein precipitation For more product details, please visit Aladdin Scientific website.
Buffer HPLC grade acetonitrile HPLC grade methanol Calcium chloride solution Ammonium bicarbonate solution
Ion Talk Tandem Mass Spectrometer
( 1 ) Cut unsenesced green leaves from the middle of the plant and freeze them immediately in a self-sealing plastic bag. If freezing facilities are not available, they can be placed on ice and then stored at -20°C. The leaves should be stored at -20°C for a long time.
( 2 ) Dig up the plant root system and take it about an inch below the green stem segment. Shake the root system and quickly place it in 3 large beakers in sequence to wash away the soil before freezing in a self-sealing plastic bag or storing temporarily on ice.
( 3 ) Weigh 2.5 g of frozen leaves and roots with stalks and other material removed, cut the sample with pre-cooled scissors and then , place in a pre-cooled ceramic mortar with liquid nitrogen and grind to a powder, several additions of liquid nitrogen are required to keep the tissue frozen.
( 4 ) Place the frozen leaf or root powder into a 40 ml centrifuge tube, add 25 ml of leaf resuspension solution, shake to mix, make sure all the powder is resuspended, and then let stand at -20°C for 45 min, then shake to mix well and centrifuge at 35,000 g for 15 min.
( 5 ) Remove the supernatant with a glass pipette, taking care not to disturb the precipitate. Wash the precipitate with an equal volume of EDTA. Shake to disperse the precipitate, let stand on ice for 5 min, and centrifuge at 35,000 g for 15 min. Repeat at least twice until the leaf protein precipitate is no longer green.
( 6 ) Freeze-dry the precipitate to obtain a light brown material containing proteins, cell walls, and fibers.
3.2 Preparation of protein samples from TCA/acetone precipitated powder
( 1 ) For each sample, weigh 20 mg of the TCA/acetone precipitated powder (see 21.3.1 for preparation) into a capped 1.5 ml centrifuge tube.
( 2 ) Add 1 ml of 100 mmol/L Tris-HCl (pH 8.5) to each tube, vortex for 1 min, centrifuge at 18000 g for 10 min, and discard the supernatant (see Note 1).
( 3 ) Add 500 μl of 8 mol/L urea to each tube, vortex for 5 min, ultrasonic water bath for 10 min, and then rotate for 30 min.
( 4 ) Centrifuge at 18,000 g for 10 min and transfer about 400 μl of supernatant containing 0.1-0.5 μg/μl of protein (see Note 2) to another tube.
( 5 ) Repeat steps 3 and 4 with 8 mol/L urea, combine the supernatants and adjust the volume to 600 μl.
3.3 Protease Digestion of Extracted Proteins
( 1 ) Add 5 μl of exonuclease lysine protease working solution to a total volume of 600 μl of protein extraction solution and digest overnight at 37°C on a rotary shaker (see Note 3).
( 2 ) Add 2300 μl of 100 mmol/L ammonium bicarbonate, 30 μl of 100 mmol/L calcium chloride, 60 μl of acetonitrile, and 10 μl of trypsin working solution to each sample. The final concentrations were 1.6 mol/L urea, 76 mmol/L ammonium bicarbonate, 1 mmol/L calcium chloride, 2% ( V/V) acetonitrile, and 3.3 ng/L trypsin.
( 3 ) Digestion was carried out on a rotary shaker at 37°C for 24~48 h. 60 μl of formic acid was added to reach a final concentration of 2%, and the samples were stored at -20°C for preparation.
( 4 ) Equilibrate the C18 SeP-Pak column with 15 ml of 90% acetonitrile solution containing 0.1% formic acid, and then wash the column four times with 20 ml of double-distilled water containing 0.1% formic acid.
( 5 ) Aspirate 3 ml of protease digest onto the equilibrated C18 Sep-Pak column, collect the effluent, and repeat the slow pass twice. The column is washed with 20 ml of double-distilled water containing 0.1% formic acid to elute salts and other loosely bound materials. The peptides were eluted from the C18 Sep-Pak column with 3 ml of 90% acetonitrile solution containing 0.1% formic acid.
( 6 ) Concentrate the above eluate to about 500 μl using a vacuum drying concentrator. 16000 r/min centrifugation for 10 min, if there is still precipitate, take the supernatant and remove the precipitate. Continue to concentrate the sample to 50 μl using a vacuum drying concentrator and dilute to 200 μl with double-distilled water containing 0.1% formic acid.
( 7 ) Equilibrate the C18 Spec- Plus solid phase extraction pipette tip with 300 μl of 90% acetonitrile solution containing 0.1% formic acid, and then rinse five times with 300 μl of double-distilled water containing 0.1% formic acid.
( 8 ) Aspirate 200 μl of peptide solution into the equilibrated C18 Spec-Plus solid phase extraction pipette tip, collect the effluent, and repeat the slow pass twice (see Note 4). Rinse the column 5 times with 300 μl of double-distilled water containing 0.1% formic acid, and then wash out the peptides with 300 μl of 90% acetonitrile solution containing 0.1% formic acid.
( 9 ) Concentrate the above eluate to about 10 μl using a vacuum drying concentrator, and this solution can be directly sampled onto a dual-phase Mudpit chromatography column.
3.4 Preparation of a two-dimensional (SCX/RP) nano HPLC column with spray emitter
( 1 ) Preparation of the column: A 25 cm long fused silica capillary with an inner diameter of 100 μm and an outer diameter of 360 μm is pushed into a laser puller, leaving a 3-8 μm fused silica capillary head. This allows it to be used as both an ion source needle and a chromatography column connected to the inlet of a mass spectrometer [ 25 ].
( 2 ) A microcentrifuge tube containing filling material suspended in methanol solution was placed in a pressure chamber and capped. The blunt end of the capillary tube was connected to the top cap by screwing a metal hoop to the same depth as the filler material. After tightening the metal hoop, the gas chamber was pressurized to 500 psi, and the filler material suspended in methanol solution was pushed into the chromatography column, and when the filler material was loaded to the 3-8 μm tip of the capillary tube, the methanol flowed through the column to the top of the column, and 4 cm of Strong Cation Exchange (SCX) filler was loaded into the capillary tube first, followed by 8 cm of countercation exchange (CX) filler, which was then loaded to the top of the column. The capillary is loaded first with 4 cm of strong cation exchange (SCX) filler and then with 8 cm of reversed-phase chromatography (C-18 ) filler.
( 3 ) The packed column is connected to high-performance liquid chromatography (HPLC) with a PEEK T-tube connector. The gold wire electrode of this T-tube connector (connected to the high-voltage power supply of the mass spectrometer) provides a voltage upstream of the chromatography column, forming a liquid-phase connection with the column's elution solution.
( 4 ) Wash the chromatography column with HPLC buffer B for 1 h at a flow rate of 1 μl/min to completely fill the column packing.
( 5 ) Equilibrate the column with HPLC Buffer A for 1 h at a flow rate of 1 μ/min before use.
3.5 Separation of peptides by two-dimensional nano-escalation high performance liquid chromatography
Column-switching separation is an on-line separation method in which a biphasic column is continuously eluted by HPLC buffer to allow the eluate to enter the mass spectrometer to ensure that no sample is lost during sample loading, salt elution, and rinsing of the column. The first phase separates peptides based on charge properties and the second phase is based on hydrophobic properties.
( 1 ) Inject 10 μl of sample into a 10 μl autosampling loop to complete loading of the peptides onto the bed of the strong cation (SCX) column (see Note 5).
a. Push the peptide into the autosampling ring by injecting 5% Buffer B into the ring at a flow rate of 10 μl/min, a process that takes approximately 25 min.
b. Then reduce the flow rate to 400 nl/min and inject for 35 min to push the sample all the way through and rinse the column.
c. Any peptides passing through the cation exchange medium will bind to the reversed phase medium, and any peptides passing through both resin media will be detected and fragmented in the mass spectrometer, the full run time taking approximately 70 min.
( 2 ) Reversed phase gradient elution with 5%~50% Buffer B at 400 nl/min for 60 min. followed by gradient elution with 50%~98% Buffer B for 5 min and elution with 98% Buffer B for 5 min, so that any residual peptides will be eluted. The column was rinsed quickly (1 min) with 5% buffer B, then increased the rinse flow rate to 1 μl/min for 24 min, and re-equilibrated the column for a total run time of 95 min (see Note 6).
( 3 ) Elute the SCX column with 250 mmol/L ammonium acetate solution at progressively increasing concentrations for 4 min, with each salt elution accompanied by a reversed-phase gradient elution (described in Step 2) to transfer peptides from the ion-exchange medium to the reversed-phase medium. Those peptides that are loosely bound to the column can be transferred in a low concentration of Buffer C, while tightly bound peptides require a higher concentration of Buffer C or Buffer D. The peptides that are loosely bound to the column can be transferred to the reverse-phase medium.
a. First elute with 5% Buffer B for 5 min at a flow rate of 400 μl/min.
b. Elute the column with X% Buffer C for 4 min, X = 10% in the first salt elution.
c. Elute the column with 5% Buffer B for 7 min, followed by a reversed-phase gradient elution of 5% to 50% Buffer B for 60 min.
The residual peptide will be eluted after eluting the column with a gradient of 50%~98% Buffer B for 5 min and then 98% Buffer B for 4 min. d. The residual peptide will be eluted after eluting the column with a gradient of 5%~98% Buffer B for 5 min and then 98% Buffer B for 4 min.
e. Rapidly rinse the column with 5% Buffer B, and then, increase the flow rate to 1 μl/min for 24 min to re-equilibrate the column.
f. The column is then eluted with Buffer C at different percentage concentrations (X = 20, 30, 40, 50, 60, 70, 80, 90, 100) in steps of 105 min for each of the 10 steps.
( 4 ) When the 250 mmol/L ammonium acetate solution salt elution is completed, the final step of salt elution is to replace the previously mentioned X% buffer C with 50% buffer D to ensure that all peptides tightly bound to the SCX column are transferred to the reversed-phase medium. A rapid gradient change elution and 20 min elution with 98% Buffer B maximizes the elution of tightly bound peptides from the reversed-phase media. The total run time is 105 min. Table 21-1 shows the chromatographic procedure for Steps 3 and 4.
3.6 Data-dependent tandem mass spectrometry analysis using Thermo Electron's HPLC-ion trap mass spectrometer LCQ DecaXP Plus
( 1 ) Basic parameters of the instrument: mass spectrometry detection scanning range: m/z 400~1500; 5 micro-scans, injection time: 500 ms, mass spectrometry signal sensitivity: 1 X 105; cleavage separation selection width: 2.7 Da; the default value of peptide carrying charge: MS/MS = 2; spray voltage: 1.8 kV; dynamic exclusion: 7 min window, repeat counting = 2 , recording time = 0.5 min; the number of peptides in a window: 0.5 min; the number of peptides in the window: 0.5 min; the number of peptides in the window: 0.5 min. Recording time = 0.5 min; Exclusion mass: low = 0.8 amu, peak = 1.5 amu; Collision energy normalized = 35%, excitation Q = 0.25, collision time = 30 ms; The 3 strongest ions were selected for cleavage in the order of n = 3, 2, 1.
( 2 ) Using these parameter settings, when the program is set to read the tandem mass spectrometry signals of peptides eluting from the chromatography column , the mass spectrometer continuously acquires data throughout the 13 steps in the HPLC described earlier. Dynamic exclusion is used to ensure that the abundance of peptides, and the isotopes associated with them, are not cleaved repeatedly, thereby maximizing the dispersion of different peptide ions that are selected for cleavage.
3.7 Identification of proteins using data retrieval
Unreadable tandem mass spectrometry profiles were searched and compared on protein sequence databases for the identification of peptides after protease digestion, and the identification information of these peptides was used to assemble protein identification information.
( 1 ) Search the complete set of tandem mass spectra against the appropriate protein sequence database using one of the following software programs:
Sequest (Themo Hectron Inc.) [26, 27], Mascot (Matrix Sciences Inc., London, UK) [ 28, 29] or Xtandem ( open software, available from the Manitoba Proteomics Center http : " www. proteom& ca/ opensource, html). 
( 2 ) There are many endogenous proteases present in the preparation of most biological samples, so non-trypsin-digested peptides can also occur during digestion. Therefore, it is best not to specifically point out the full trypsin-digested peptide fragments when setting up the parameters in the search library, so as to avoid the obvious loss of information.
( 3 ) The peptide identification data need to be sorted and filtered to remove as much irrelevant information as possible. Unfortunately, there is no clearly defined criterion that guarantees that all peptides output from a data retrieval program are correct, nor is it possible to exclude incorrect peptides. A review article describing the parameters of data retrieval will show that there is little unity of opinion on this issue. The best that can be done is to optimize and refine a set of published parameters in the same laboratory, using the same instrumentation, and digesting samples with known proteins. The accuracy of peptide judgments can also be improved by searching for statistical significance evaluations [ 32, 33] or by repeated searches of an inverse protein sequence database [31, 34, 35].
( 4 ) One issue that remains controversial is whether Mudpit data reports include protein identifications based on single peptide identifications. In general, about half of the protein identifications in Mudpit experiments are based on single peptide information, so this is an important issue to consider. Of course, some protein identifications are very accurate because a small protein may have only one available trypsin-hydrolyzed peptide, or some modification obscures the identification of other peptides, or although many trypsin-hydrolyzed peptides are produced, only one has been correctly selected for cleavage. In addition, protein identifications based on single peptides are available in most Mudpit databases. Small variations in database search parameters can severely affect the search results, and the chances of generating a false-positive identification are high. Protein identifications should ideally report the number of proteins based on information from two or more peptide identifications, as well as the number of two different profiles containing single peptide identifications [36] (see 21.3.8).
3.8 Example of results: Mudpit analysis of leaf and root tissues prepared from mature rice
1. Plant growth and collection Rice (Oryzasahm, cv. Nipponbare) seeds were planted in a greenhouse with 12 h of light per day, a controlled light temperature of 29°C for 12 h, a dark temperature of 21°C for 12 h, and maintained at 30% humidity, and the plants were grown in small pots containing 50% Sim-shine mix and 50% nitro-humus, and were sprouted for 50 days. Leaf and root material was taken after 50 days of germination.
2. Protein extraction, sample preparation and Mudpit analysis
The leaves and roots were ground to powder in liquid nitrogen using mortar and pestle. Proteins are extracted by the TCA/acetone method (see 21. 3.1), the extracted proteins are digested by the methods of 3.2 and 3.3, and the peptide mixtures are analyzed according to the methods described in 3.4-3.7.
3. Mass spectrometry database search and analysis
All tandem mass spectrometry data comparisons were searched against a database of rice protein sequences from NCBI's public resources, supplemented by a file of intrinsic contaminants including trypsin, the intracellular protease lysine-C, keratin, albumin, casein, and other common laboratory contaminants.
Data were retrieved using Xtandem software and analyzed using the Proteomics Global Analysis Machine web tool (www.thegpmorg). Xtandem software retrieval parameters were set: fragment ion single isotope mass error: 0.5 Da; parent ion single isotope mass error: 2.0 std. Da; spectral dynamic range: 100; total peaks in spectrum: 50; Maximum effective threshold >0.1. oxidation-induced variable modification of methionine by 16 Da is acceptable.
4. Experimental Results of Mudpit Analysis of Rice Leaves and Roots Tables 21-2 to 21-4 show the results of Mudpit analysis of rice leaves and roots. The total number of proteins identified in each experiment is listed in Table 21-2; they were categorized into three groups: two or more identified peptides; two or more identified peptides or one identified peptide with a prevalence less than 0.001; and one or more identified peptides with a prevalence less than 0.1. These identified protein quantities were further categorized as common to roots and leaves, as well as categories specific to each of them. Table 21-2 also includes information on the number of MS/MS spectra retrieved in each experiment, as well as the number of peptides in each category as described above. 
The results of these experiments clearly illustrate the following points.
( 1 ) Hundreds of proteins can be identified from a single plant tissue material in a single experiment.
( 2 ) Retrieving all the spectra obtained from the two experiments over 91000 MS/MS and applying the minimum criteria, we identified 578 proteins in leaves and 538 in roots, including 1005 nonredundant protein identifications.
( 3 ) These numbers were reduced to 329, 303, and 549, respectively, after applying two or more more more stringent criteria for peptide identification.
( 4 ) The number of protein identifications based on a single identified peptide was 43% of the total, which is in line with (or slightly lower than) previously published datasets where percentages have been specifically reported [ 12, 36, 38]. The number of proteins identified from these two experiments is in excellent agreement, especially when not taking into account the reproducibility in the Mudpit analysis technique as a focal point for Mudpi, however, the total number of non-redundant proteins identified shows only 10% ~15% overlap in both datasets, which just goes to show the tissue-specificity of the protein expression.
Another interesting feature of these results is that although the number of proteins identified in leaves and roots was similar, more than twice as many peptides were identified in leaves as in roots. This is due to the fact that a large number of peptides identified from leaves were derived from RuBisCo, as shown in Table 21-3.
Table 21-3 lists the top 25 proteins identified from rice leaves by Mudpit analysis, sorted according to the Xtandem protein prevalence. Table 21-4 lists the top 25 proteins identified from rice roots by Mudpit analysis, also sorted by Xtande protein predictions. The most abundant proteins in leaf and root tissues are listed in Tables 21-3 and 21-4, and these proteins are also the major proteins in these tissues and are highly expressed. Many isoforms of RuBisCo, ATP synthase, photosystem protein complexes, and several other chloroplast-specific proteins are present in leaf tissues. Roots contain a large number of carbohydrate metabolizing enzymes such as sucrose- UDP glucosyltransferase 2, propylphosphate isomerase, ascorbate peroxidase and glyceraldehyde triphosphate dehydrogenase. Interestingly: the roots also contain high levels of expressed disease process related proteins that may play an important role in the response mechanisms to biotic and abiotic stresses in the soil.