The phrase “protein and peptide” refers to a relatively pure sample from a bioreactor or purification process. These samples include little or no additional non-protein materials. The generation of free amino acids from an intact protein or peptide is a critical step in the production of useful, accurate amino acid analysis data. To enable this generation, it is necessary to break down or hydrolyze a protein/peptide to its individual amino acid constituents.
In this section, presented are the basic procedures for vapor-phase and liquid-phase protein and peptide hydrolysis, emphasizing the preliminary considerations necessary for optimal analysis.
Additional subsections cover alternative hydrolysis procedures for the analysis of special samples containing amino acids that are not compatible with standard acid (HCl) hydrolysis techniques: tryptophan and cysteine/cystine.
In this section, we focus on acid hydrolysis by HCl, which is the most common method used in preparing amino acid samples. However, for protein samples to be completely hydrolyzed (by any method), several factors must be considered. The neutralizing buffers, as well as any solids present, should be considered in procedural estimations. Also, the rate or extent of hydrolysis varies across the amino acids present in proteins, necessitating time-course studies of the hydrolysis process and proper validation of the overall methods. Proper sample handling during hydrolysis ensures high-quality results. The most common difficulties in this type of analysis generally stem from improper or sloppy technique. For an effective hydrolysis with this method, four questions must first be answered:
The following sections outline the necessary determinations and provide example calculations where appropriate.
Unless sample is limited, there should be between 2 and 25 µg of protein present in the hydrolysis, to minimize the effects of contamination. Regardless of the mode of hydrolysis, 20 µg is recommended.
For a protein that is 5 mg/mL in 150 mM NaCl, the amount of solids (including salts) and the dilution factor will be determined.
The first step is to determine the total amount of solids in the sample. This can be done by multiplying the protein concentration (µg/µL) by the weight concentration of the salt (concentration x MW) for a total amount of solids.
The next step is to determine the dilution needed for the target of 20 µg of protein (in 20 µL). This can be done by multiplying the desired amount/volume by the inverse concentration of the sample. This results in a 1:5 dilution ratio needed for the sample described above.
The total recommended volume for a hydrolysis is 100 µL (when using 6 x 50 mm tubes), with the volume of sample ranging from 10–20 µL. This volume of sample, whether diluted or not, depends on the type of hydrolysis performed and follows these guidelines:
It is important to note, again, that the lower the amount of protein in the sample aliquot the more vulnerable the analysis may be to contamination.
The volume of acid added to the hydrolysis is critical. This is particularly true for liquid- phase hydrolysis. Guidelines for acid volume are below, with an extensive example.
Following the above guidelines, the minimum amount of acid added for a hydrolysis must exceed 25x the buffer concentration and 100x the weight excess of the sample. Therefore, to determine the volume of acid needed, you must calculate the following:
For a protein that is 2.1 mg/mL in 2 mM Na/K2PO4:
The amount of buffer in each tube is determined by multiplying the buffer molar concentration by the number of titratable groups (three for the phosphate buffer) and then adjusting to the total volume of the sample dispensed—in this case, 10 µL.
Each tube will contain 60 nmol of buffer.
Next, the 25-fold excess is determined by multiplying the amount by 25.
This number is the moles of buffer acid required for effective neutralization.
The moles of buffer required for neutralization then must be converted to a volume of acid to add to each tube.
For this sample, a volume of 0.25 µL will effectively neutralize the buffer.
To achieve the 100x excess of acid over the sample, the total amount of solids in the sample must be determined. Total solids will be the protein plus buffer plus any other material present. In this case, the sample is a purified protein.
This determination can be made by multiplying the protein concentration (µg/µL) by the weight concentration of the salt (concentration x MW) for a total amount of solids.
The total solids for this sample calculate to be 24.9 µg.
To determine the target 100-fold excess of acid, multiply the total solids by 100.
Finally, convert the weight excess to a volume of 6 M HCl to add to each tube by multiplying the weight by the weight per mole for the acid by the molarity of the acid.
In this case, 11.4 µL of 6 M HCl will provide a 100-fold excess of acid over the sample.
The final volume of acid to add to each tube is the sum of the volumes needed to neutralize the sample buffer and provide the 100-fold excess:
A volume of 11.65 µL of 6 M HCl is needed to ensure an effective hydrolysis. This volume can be rounded up to a volume that can be transferred accurately.
The use of an internal standard (IS) best compensates for variable hydrolysis of the individual amino acids of the sample. Norvaline (Nva) is a commonly used internal standard.
When using an internal standard:
To determine the amount of IS needed in the starting sample, the calculation works back from the desired amount of IS needed in the sample. As part of this calculation, it’s important to consider the derivatization step.
The total IS is determined by, working backward, multiplying the amount of IS required in the final sample, the dilution factor of the derivatization, and the reconstituted sample in the hydrolysis tube. In this example, 25 pmol of IS is needed in the final derivatized sample, with the sample being diluted 10x during the derivatization and 5x dilution of the sample prior to derivatization. Thus:
This indicates we need 1250 pmol of IS in our hydrolysis sample.
Given the MW (mg/mol) and conversion from µL to mL, we can see that we need 0.14 mg of IS added to each mL of our starting sample.
Vapor-phase hydrolysis is recommended for relatively pure protein or peptide samples containing little or no particulate material. It’s considered the most sensitive approach. A stand-alone, automated, hydrolysis workstation is preferred for this type of hydrolysis. The vacuum control, temperature maintenance, nitrogen flushes, and sample drying required in the procedure are best performed by an automated system such as the Eldex hydrolysis workstation described in Section 4.
Note: For additional reagent information, consult the hydrolysis workstation’s operational manual.
Procedure (based on using an automated workstation):
Note: A brown color in the HCl is common after hydrolysis. It derives from the phenol. Ethanol or acetone works well to remove discoloration from vials and tops.
CAUTION: Hydrolysis problems may be difficult to distinguish from derivatization problems.
Liquid-phase hydrolysis is used when the samples are more complex. In this case, particulate or other foreign matter is present that may interfere with the vapor-phase process. This approach is considered less sensitive overall; but, when performed carefully and precisely, can give good results.
Before starting –
Either weigh samples corresponding to approximately 20 mg protein to the nearest 0.1 mg into hydrolysis tubes or transfer diluted sample to tubes (as calculated in Sections 2.1.3 and 2.1.4). Normally, a total volume of 10 µL is usual. Mix.
Several amino acids are affected by improper hydrolysis. For example:
Analysis of Trp in proteins and peptides is complicated by the instability of this amino acid under normal hydrolysis conditions with 6 N HCl. Alternative hydrolysis procedures can be used to generate intact Trp for analysis:
The MSA reagent must be added directly to the 6 x 50 mm sample tubes (liquid- phase hydrolysis), because the acid is not volatile. The addition of methanol and neutralization after hydrolysis provides good results for tryptophan and does not interfere in any subsequent derivatization process. Figure 2 illustrates the reaction of MSA with protein.
Figure 2. MSA hydrolysis for Trp analysis.
Note: This procedure can also be used for determination of Cys and Met. It converts Cys to Cya and Met to methionine sulfone.
Note: Tyr and Trp are not stable in the performic acid oxidation procedure traditionally used for Cys and Met analysis.
Check for interferences with control blank samples.
■ Interference with Tyr and Val can occur. The cause is unknown. (HCl is preferable for the hydrolysis of Tyr.)
■ Low Met yields are a function of the hydrolysis conditions.
■ Yields of Trp and Met are nonlinear due to the hydrolysis process, not the analysis procedure. As the amount hydrolyzed is reduced, the yields fall; with Met the result is more dramatic.
■ Poor reproducibility of Arg results—cause unknown.
If acid hydrolysis of tryptophan raises stability issues, hydrolysis of protein using base is another alternative.
Figure 3. Sulphur-containing amino acids.
The quantitation of cysteine (Cys) in protein samples is complicated by the instability of this amino acid in standard acid hydrolysis conditions. Unfortunately, unlike with Trp, alternative acid or base hydrolyses are not satisfactory. Two common procedures for Cys analysis involve conversion of the cysteine to more stable derivatives. The first procedure is alkylation of the sulfhydryl group, and the second is an oxidation to the acid-stable sulfonic acid, cysteic or cyanuric acid (Cya).
WARNING: A further complication in Cys analysis is that much of the amino acid is present as the dimer, cystine (Cys2), which must be reduced to cysteine prior to any alkylation procedures.
It is important to note that these specific procedures are performed prior to the standard acid hydrolysis step.
Figure 4. Performic acid oxidation of cystine and cysteine to cysteic acid.
Performic acid is a powerful oxidizing reagent that quantitatively converts cysteine (Cys) and cystine (Cys2) to cysteic acid (Cya) (Figure 4). Literature contains many references to the use of this reagent in a wide variety of conditions and procedures. The following procedure is based upon that of Tarr, G.E., 1986.
Note: This procedure yields the most accurate results for cysteine and methionine.
Note: Tyr and Trp are not stable in this oxidation procedure.
Fiugre 5. Alkylation of cystine and cysteine.
Procedures that alkylate are more selective than performic acid oxidation and result in little or no change to the other amino acids. This makes them better suited to the analysis of the complete protein, as well as other procedures that may follow Cys modification, such as peptide mapping.
In this method, alkylation is presented with 4-vinylpyridine; the general procedure outlined below can also be adapted for several alkylating agents. In principle, a sufficient, reducing reagent must be added to the sample to convert cystine (Cys2) to cysteine (Cys). This is followed by an addition of excess alkylating reagent to the reduced sample (Figure 5).
The following procedure can be used for 1–1000 nmol of protein or peptide.
Note: The total reaction volume can be scaled down by reducing buffer to 0.25 mL, Gu-HCl to 250 mg, DTT to 1 mg, and 4-VP to 2 µL. This is adequate for up to 250 pmol of sample. After alkylation, dilute with 750 µL H20 and proceed.
Note: 8 µL of 4-VP is approximately 74 µmol. Substitution of other alkylating reagents for 4-VP (e.g., iodoacetic acid) in the procedure can be made using the same reagent concentration.
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