Created: 1st September 1999, last updated: 12th November 1999, © 1999 ABRF
Sheng Tang, Jacek Mozdzanowski, and Kalyan R. Anumula
SmithKline Beecham Pharmaceuticals, Research and Development, King of Prussia, PA
Reprinted from the electronic version of JBT available at http://www.abrf.org/JBT/JBT.html accession #0016.
We developed a novel chemistry for C-terminal sequencing of proteins based on derivatization with acetylisothiocyanate to yield amino acid thiohydantoins (TH-AAs), and it was used for manual sequencing of biopharmaceutical products on a routine basis. This simple chemistry was automated using a ABI 473A N-terminal sequencer. All reagents (R1, trimethylsilylisothiocyanate; R3, alkaline thiocyanate for cleavage) and solvents required for sequencing were accommodated on the sequencer, which was modified to deliver liquid R2 (acetyl chloride) to the reaction vessel. The conversion flask was used for preparing the TH-AAs for analysis by on-line HPLC using a graphitized carbon (Hypercarb) column. Results obtained with model proteins and recombinant protein drugs suggest that at least three residues from the C terminus can be easily determined. The C-terminal heterogeneity in more than five types of recombinant immunoglobulin G was determined, and the differences in Gly-Lys ratios were consistent with changes observed in the isoelectric focusing profile of these antibodies. Because the chemistry uses only four reagents delivered to the reaction vessel and three to the conversion flask, we believe that the automated protocol can be easily adapted to any existing N-terminal sequencer. (J Biomol Tech 1999;10:144-148)
Key words: Carboxyl-terminal sequencing, protein chemistry, acetylisothiocyanate, amino acid thiohydantoin.
Address correspondence and reprint requests to Kalyan R. Anumula, SmithKline Beecham Pharmaceuticals, Research and Development, King of Prussia, PA 19406 (email: Kalyan_Anumula-1@SBPHRD.com).
The amino-terminal sequence analysis of protein and peptide based on the Edman degradation has been widely used for protein structure determination. As an orthogonal method, C-terminal sequencing can provide structure information on proteins with blocked N-termini by means of natural modification. It also provides information on the posttranslational processing at the carboxyl terminus of gene products and facilitates the production of more specific probes for gene cloning. Although a number of methods for C-terminal sequencing have been reported, the thiocyanate method that was first described in 1926 by Schlack1 has been the subject of many studies (reviewed by Inglis2).
The thiocyanate method involves an activation step using acetic anhydride to activate the carboxylic acid at the C terminus of protein or peptide to yield an oxazolinone, followed by a derivatization step using an isothiocyanate donor reagent to convert the protein or peptide to a peptidylthiohydantoin.2 The derivatized amino acid is then cleaved by an acid or base to yield a shortened peptide or protein and a thiohydantoin amino acid (TH-AA). Although this chemistry is similar to the Edman chemistry, it is difficult to find suitable activation, derivatization, and cleaving reagents that do not modify or cleave the protein test article extensively. Although a number of modifications to the thiocyanate method were reported, diphenylphosphoro-isothiocyanatidate (DPP-ITC) was introduced as an efficient reagent for C-terminal sequence analysis.3 In this method, the protein or peptide sample is treated with diisopropylethylamine to convert the C-terminal carboxylic acid into a carboxylate salt, and then the sample is treated with DPP-ITC, followed with pyridine to form a peptidyl isothiocyanate. Cleavage of the derivatized amino acid is performed with sodium trimethylsilanolate solution.3
We developed a simple, novel chemistry for C-terminal sequencing of protein or peptides. In this approach, acetylisothiocyanate (AITC) is used in acidic conditions as a derivatization reagent to convert the C-terminal amino acid to a thiohydantoin, and an alkaline potassium thiocyanate (KSCN) solution is used to cleave the TH-AAs from proteins.4,5 The AITC chemistry has been used in manual sequencing of biopharmaceutical products on a routine basis for several years in our laboratory. We have automated this chemistry using an Applied BioSystems 473A N-terminal sequencer. The TH-AAs cleaved from the proteins were analyzed by on-line high-performance liquid chromatography (HPLC) using a graphitized carbon (Hypercarb) column. TH-AA standards from Hewlett Packard (Wilmington, DE) were used for identification and quantitation. However, TH-AA standards can be prepared easily from the amino acids by treatment with AITC. This report describes the method, operating conditions for the ABI 473A sequencer, and sequencing results from two representative proteins.
Acetyl chloride (Ac-Cl), triethylamine (TEA) and beta-lactoglobulin (beta-Lac) were from Sigma Chemical Company (St. Louis, MO). Acetic anhydride (Ac2O), acetic acid, KSCN, 4-methylmorpholine, and trimethylsilylisothiocyanate (TMS-ITC) were obtained from Aldrich (Milwaukee, WI). C-terminal thiohydantoin amino acid standards in acetonitrile (TH-Std) were obtained from Hewlett Packard. All other chemicals used were HPLC or reagent grade. Proteins were purchased from Sigma and dissolved in water or 1% TFA/ water.
Protein samples (1 to 5 nmol) were coupled onto the Sequelon-DITC membrane disk (Millipore, Bedford, MA) according to the manufacturer's suggestions, and only a portion of the disk was installed in the glass membrane block for sequencing. Sequencing was performed on an ABI model 473A protein sequencer using a newly created sequencing cycle. Briefly, the cycle consisted of the following major steps: the sample was acetylated with R2 (acetyl chloride/acetic anhydride/acetic acid mixture) at 60°C for 10 minutes; the C-terminal residues were derivatized to TH-AAs with multiple sequential delivery of R2 and R1 (TMS-ITC) at 60°C for three times for 7 minutes; and after washing the membrane with S2 (methanol), the C-terminal TH-AAs were cleaved from the protein with R3 (KSCN in sodium phosphate solution, pH 11) at 60°C for two times for 5 minutes. The cleavage solution containing TH-AAs was transferred to conversion flask (25°C), neutralized with X1 (3% acetic acid in methanol), dried, and dissolved in R4 (HPLC solvent A). The solution (20 µL, 17%) was injected for on-line HPLC analysis. After washing the membrane with S2 (methanol), the steps described previously were repeated during the second sequencing cycle. Table 1 describes the reagents and solvents used in the sequencer.
TABLE 1
Reagents and Solvents Used for C-terminal Sequencing
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| Bottle Position |
Reagent and Solvent | Purpose | ||
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| R1 | 10% TMS-ITC in acetonitrile | Derivatize the C terminus | ||
| R2 | 30% acetyl chloride, 5% acetic anhydride, and 5% acetic acid in acetonitrile |
Activate the C terminus, mixing with R1 to derivatize the C terminus |
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| R3 | 0.1 M KSCN, in 0.1 M sodium phosphate (pH 11) and contains 30% methanol (v/v) |
Cleave TH-AAs | ||
| R4 | HPLC solvent A | Dissolve flask contents, transfer to HPLC | ||
| X1 | 3% acetic acid in methanol | Neutralize cleavage solution | ||
| S4 | 20% acetonitrile in water | Wash the conversion flask | ||
| S2 | Methanol | Wash the reaction cartridge | ||
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TMS-ITC, trimethylsilylisothiocyanate; KSCN, potassium thiocyanate; TH-AAs, amino acid thiohydantoins.
TH-AAs released from the C termini of proteins were analyzed with on-line HPLC using an ABI 473A sequencer. The TH-AAs were separated on a Shandon Hypercarb graphitized carbon column (2.1 X 100 mm, 7 µm) from Alltech Associates (Waukegan, IL) at 50°C using a flow rate of 0.2 mL/min. The following gradient was used: 7% B for 2 minutes, 7% to 58% B over 23 minutes, and then 80% B wash over 9 minutes. Solvent A was 0.1% TFA with 0.2% 1-hexanesulfonate in water, and solvent B was acetonitrile.
The column effluent was monitored at 265 nm and 319 nm (for dehydro-TH-Ser and TH-Thr) using two ultraviolet (UV) detectors connected in series. TH-AA standard mixture from Hewlett Packard was dried and reconstituted with buffer A. Acetylated TH-Lys standard was made from Lys amino acid (in excess) treated with a mixture of R1 and R2 at 60°C for 20 minutes.
The automated C-terminal sequencing method described is based on the novel AITC chemistry.4,5 The overall reaction has two stages (Fig. 1). In the first stage, protein or peptides treated with a mixture of acetyl chloride and TMS-ITC in acetonitrile. At this stage, the protein C termini are activated with acetyl chloride in acidic conditions to yield a stable protein carboxyl chloride. At the same time, the acetyl chloride is also reacted with TMS-ITC to form AITC, which reacts with C termini to yield the TH derivatives. AITC (a fresh mixture of acetyl chloride and TMS-ITC) is used directly in the manual method for convenience. Because this reagent mixture is unstable, the components are installed separately on the ABI 473A sequencer. The R1 and R2 reagents are delivered separately and sequentially in a repeated manner to achieve mixing of R1 and R2 on the protein or peptide sample.
FIGURE 1. Reaction scheme for derivatization of protein C-terminal amino acid using acetylisothiocyanate and acetylchloride.
In the second stage, the TH-AAs are cleaved from the C termini of the proteins by use of an alkaline thiocyanate (KSCN, pH 11). The yield of TH-AAs is nearly quantitative in the first cycle because of the high efficiency of this chemistry. Because the sequencing chemistry is similar to the Edman degradation, the AITC chemistry can be easily adapted to any N-terminal sequencer.
Figure 2 shows the separation of 19 common amino acids using the graphite column. For each amino acid thiohydantoin, except TH-Ile, a single peak with a characteristic retention time was observed. TH-Ile yielded two peaks. Lysine residue yielded an acetylated thiohydantoin derivative in the current sequencing chemistry, and it eluted immediately after the TH-Lys with the free epsilon-amino group. Cysteine and serine residues yield the same dehydro-thiohydantoin derivative, and it had an absorption maximum at 319 nm. TH-Thr also dehydrated under these conditions and was detected at 319 nm. TH-Trp is eluted in the wash in the current HPLC run.
FIGURE 2. The TH-AA standard mixture (25 pmol each) was separated on a graphitized carbon column and detected at 265 and 319 nm. N-acetylated TH-Lys is indicated by the Ac-Lys. Artifact peaks presented in this mixture are indicated by an asterisk. Elution of unmodified Cys derivative is also indicated.
We used the ABI model 473A Protein Sequencer with a slight modification for the automation of AITC chemistry. The delivery lines of the cartridge holder were exchanged such that the reagent solutions and gas flowed from the bottom to the top. The line of the R2 was also modified such that the R2 a delivered as a liquid. The total time of the cycle is about 1 hour. To ensure efficiency of the sequencing chemistry, all the steps--including load, deliver, transfer, and dry functions--in the cycle must be timed and optimized. During the sequencing analysis, R1 and R2 (derivatizing reagents) were delivered separately and sequentially in a repeated manner to achieve mixing thoroughly on the protein sample. The times for derivatization and cleavage steps are usually about 20 and 10 minutes, respectively.
A diverse range of proteins have been analyzed using this automated method, and in our experience, three residues from the C termini of the proteins can be easily determined. More extensive sequencing was limited by sample washout of >50% at each cleavage step.
Figures 3 and 4 show the results of sequencing analysis of lysozyme and recombinant immunoglobulin G (rIgG). The expected Lys from the C terminus of rIgG is found in trace amounts because of extensive C-terminal processing (typical for IgGs). Other model proteins analyzed by this procedure are described in Table 2.
FIGURE 3. Automated C-terminal sequencing of lysozyme (approximately 0.4 nmol, without reduction and alkylation). Cycle 1 (Leu), cycle 2 (Arg), and cycle 3 (Cys) are identified. Leucine recovery in the first cycle is nearly quantitative, and that of Arg in the second cycle is 25%. Cysteine is detected at 319 nm. Artifact peaks are indicated by an asterisk.
FIGURE 4. Automated C-terminal sequencing (first cycle) of a rIgG sample (1 nmol) immobilized on Sequelon-DITC membrane disk. The expected C-terminal residues, Lys and Gly from the heavy chain and Cys from the light chain, are identified. Glycine recovery is nearly quantitative. The artifact peak is indicated by an asterisk.
TABLE 2
Summary of Proteins Analyzed by C-terminal Sequencing
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| Protein | Amount Analyzed |
Expected Sequence |
Determined Sequence |
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| Lysozyme | 0.4 nmol | -Gly-Cys-Arg-Leu | -Cys-Arg-Leu | |||
| Lactalbumin | 0.4 nmol | -Cys-Glu-Lys-Leu | -Glu-Lys-Leu | |||
| rIgGs | 1.0 nmol | H-chain-SPGK | -Gly-Lys | |||
| (>5 types) | L-chain-TEC/S | -C/S | ||||
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All the results were reproducible using an ABI 473A sequencer. The sequencing chemistry uses only four reagents (R1, R2, R3, and S2) to be delivered to the reaction cartridge and three reagents (R4, X1, and S4) to the conversion flask to prepare the TH-AAs for the HPLC analysis. We therefore believe that this chemistry can be adapted to any existing N-terminal sequencer.
We have developed a sequencing chemistry based on novel acetylisothiocyanate degradation of C-terminal amino acids. An ABI 473A N-terminal sequencer was used to demonstrate the automation of this chemistry, but the chemistry can be used in other sequencers designed for N-terminal sequencing. A new HPLC method using graphitized carbon (Hypercarb) column was able to identify 19 TH-AAs, except tryptophan, which eluted in the column wash. Typically, three residues from the C terminus of the proteins are easily identified. Extended sequencing is not currently possible because of rather heavy (>50%) washouts during the cleavage steps. Further optimization of the sequencing method should allow identification of more than three residues.
1. Schlack P, Kumpf W. Uber eine neue Methode zue Ermitt-lung der Konstitution con Peptiden. Z Physiol Chem 1926;154:125-170.
2. Inglis AS. Chemical procedures for C-terminal sequencing of peptides and proteins. Anal Biochem 1991; 195:183-196.
3. Bailey JM, Nikfarjam F, Shenoy N, Shively JE. Automated carboxy-terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate. Protein Sci 1992;1:1622-1633.
4. Anumula KR. Method for carboxy terminal protein or peptide sequencing. US patent #5,641,685 (1997).
5. Anumula KR, Tang S. Novel chemistry for sequencing of proteins from carboxyl terminus yields a simple method. FASEB J 1995;9:A1477.