ANALYTICAL BIOCHEMISTRY 70, 110--123 (1976)

Detection of Asparagine and Glutamine in Peptides Sequenced by Dipeptidyl Aminopeptidase I via Gas Ch romatography- Mass Spectrometry M. A. YOUNG AND D. M. DESIDERIO Institute for Lipid Research and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77025 Received October 18, 1974; accepted June 10, 1975 A simple, straightforward method for differentiating asparaginyl (or glutaminyl) dipeptides from aspartyl (or glutamyl) dipeptides is described. The technique involves two separate methods to form methyl esters. The first, employing 0. l N HCl/methanol at room temperature for 4 hr, reveals all dipeptides except nonvolatile Asn- and Gin-containing dipeptides. The second procedure, with 0.1 N HCI/MeOH at 45°C for 16 hr, solvolyses the AsrdGln residues, and new pairs of Asp or Glu dipeptides occur. This approach proved feasible with a synthetic pentadecapeptide, scotophobin, containing A s n - A s n and G l n - G l n as part of its primary structure.

Since McDonald et al. (1) reported that dipeptidyl aminopeptidase I, (DAP I, cathepsin C) removed dipeptides sequentially from a peptide with an unsubstituted amino-terminus, this enzyme evoked widespread interest in its application to peptide sequencing. Following enzymic hydrolysis, the major task was to separate and sequence the dipeptides in the resulting mixture. Paper electrophoresis (2), column chromatography (3), and dansylation procedures (4) were employed to identify the dipeptides. With regard to speed, accuracy, and high sensitivity, gas chromatography-mass spectrometry (gc-ms) has certain advantages over the other techniques. In comparing different methods of derivatization for gas chromatography (gc) (5-7), trifluoroacetyl (TFA) and pentafluoropropionyl (PFP) methyl esters appear most reliable. However, in review of all established methods for preparation of perfluoroacyl esters, asparaginyl and glutaminyl dipeptides cannot be differentiated from aspartyl and glutamyl dipeptides due to solvolysis of the former to the latter. Our objective is to eluciate primary structures ofpolypeptides by means o f D A P I followed by g c - m s to sequence resulting dipeptides. In order to attain our goal it was necessary to establish (i) methylation conditions for dipeptides without Asn of Gin, (ii) conditions for perfluoroacylation of dipeptide methyl esters, and (iii) a new method to form methyl esters of dipeptides containing Ash or Gin, but avoiding solvolysis of the amides. The ability to perform the three above experiments permits one to establish precisely the positions of Asn and Gln in polypeptides. To test llO Copyright© 1976by AcademicPress, Inc. All rightsof reproductionin any form reserved.

PEPTIDE SEQUENCING BY G C - M S

111

this hypothesis a synthetic pentadecapeptide, scotophobin: S e r - A s p Asn - A s n - G l n - G l n - Gly - Lys - Ser - Ala - G l n - Gin - Gly - Gly Tyr-NH2 (8), was employed to illustrate successful discrimination of Asn (or Gin) dipeptides from Asp (or Glu) dipeptides in the NH2-terminal hexapeptide portion. In addition, we investigated the carboxymethylated A chain of porcine insulin and conclude that deamidation had occurred prior to our experiments. During the course of this investigation, we also developed a new method to directly analyse the amino acids Gin, Glu, Asn, and Asp as their trimethylsilyl (TMS)-methylthiazolinone derivatives (9). MATERIALS AND METHODS

Gas chromatography. A Barber-Colman model 5000 gc (Rockford, Ill.) equipped with a flame ionization detector was employed. The U-tube glass column (6 ft × 4 mm) was filled with 2.5% OV-17 on 100-200-mesh Supelcoport (Supelco, Inc., Bellefonte, Pa.), prepared according to Gehrke et al. (10). Packings of 1% Dexsil 300 on Chromosorb W AW D M C S , 100-200 mesh (Analabs, Inc., North Haven, Conn.) and a mixed phase of 2% OV-17/1% OV-210 on Supelcoport, 100-120 mesh (11), were also used. The gc operating conditions were: Injector temperature, 250°C; detector bath, 300°C; flow rate, 33 cm3/min; inlet pressures, nitrogen, 26 psi; air, 40 psi; hydrogen, 16 psi. Gas chromatography-mass spectrometry. An LKB model 9000 gc-ms (LKB Produkter AB, Stockholm-Bromma 1, Sweden) equipped with a 6-ft × 4-mm glass coil column packed with 1% OV-17 (Applied Science Laboratory, Inc., State College, Pa.) was employed. The ionizing current was 60/~A; ion source temperature was 250°C; ionizing voltage used was either 20 or 70 eV and accelerating potential, -3.5 kV. Derivatization and chromatography of dipeptides. Diazomethane was generated by the conventional method (14). The esterification ofdipeptides by CH2N~ was carried out in water solution (15). The tic solvent system was n - B u O H : E t O H : A c O H : H 2 0 , either 8:2:1:3 or 4:1:1:1. Eastman (Rochester, N.Y.) chromatogram sheets (6061, silica gel) were used for tic, and ninhydrin (0.2% in acetone) was used for tic visualization. The plates were examined first at room temperature and again after heating for 0.5 hr at 80°C. Peptide digestion with DAP I. Peptide digestion with D A P I (3 units of enzyme//xmole of peptide) was carried out at pH 5 and 37°C for 4 hr in the presence of E D T A according to Callahan et al. (3). The digest was neutralized to pH 7 and lyophilized. Additional pumping for 3 hr assured absolute dryness. After the residue was esterified with 0.1 N HCI/MeOH, the solvent and excess HC1 were aspirated. Again, additional pumping ensured dryness. The residue was extracted with MeOH:CH2C12 (4:33). The dry residue was perfluoroacylated with 0.5 ml of trifluoroacetic

112

YOUNG AND DESIDERIO

anhydride (TFAA) or pentafluoropropionic anhydride (PFPA) at room temperature. Excess reagent was evaporated by a stream of nitrogen and then pumped for 15 min. The residue was dissolved in dry benzene or dichloromethane for GC or GC-MS analysis. Other materials. D A P I (bovine spleen; lot W-1750, sp act, 18.1 units/mg protein; lot W-1588, sp act, 19.7 units/mg proteins; 0.1 M NaCI-0.1 M acetate buffer, pH 4.5), porcine S -carboxymethylated insulin A chain, and G l n - G l n were obtained from Schwarz/Mann Co. (Orangeburg, N.Y.). P h e - A s p , G l u - G l u , G l n - G l y , G l y - A s n , Trp-Trp, and Ser-Val were purchased from Fox Chemical Co. (Los Angeles, Calif.). Scotophobin was synthesized in this laboratory by the Merrifield solid phase method (8,12). T F A A and P F P A were obtained from Pierce Chemical Co. (Rockford, Ill.). Anhydrous HC1, 99.0% minimum purity, from Matheson Co. (LaPorte, Tex.) and methanol (99.9 mole% purity) from Fisher Scientific Co. (Houston, Tex.) were used. Diazald was purchased from Aldrich Chemical Co. (Milwaukee, Wis.) A stock solution of 5 N HC1 in methanol was prepared by bubbling HC1, after it passed through desiccant and traps, to the anhydrous solvent followed by titration (13). The desired dilute solution was obtained by adding the appropriate amount of methanol. RESULTS 1. Determination of esterification conditions for dipeptides containing no Asn or Gln. P h e - A s p and A l a - S e r served as model compounds. Thin-layer chromatographic results (Fig. 1) illustrate the products found by n-BuOH

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FIG. 1. Thin-layer chromatographic separation of products formed with various esterification conditions: (1) Phe-Asp; (2) esterified Phe-Asp, 0.1 N HCI/MeOH, 25°C+ 30 hr; (3) esterified Phe-Asp; 1 NHCI/MeOH, 25°C, 30 hr; (4) esterified Phe-Asp, 5 NHC1/MeOH, 65°C, 0.5 hr; (5) Ala-Ser; (6) esterified Ala-Ser, 0.1 N HCL/MeOH, 25°C, 30 hr; (7) esterified Ala-Ser, 1 N HC1/MeOH, 25°C, 30 hr; (8) esterified Ala-Ser, 5 N HC1/MeOH, 65°C; 0.5 hr. Estimate of spots (41) 0.05 ~mol; (0) 0.01-0.02 ~mol, (O) 0.005 /xmol.

PEPTIDE SEQUENCING BY GC-MS

113

varying HCI concentration and reaction temperature. The tic plate was developed 7.5 hr and visualized. Optimal yield was obtained and least amount of side products formed with 0.1 N HCI/MeOH at room temperature for 30 hr. In addition, subsequent evidence shows esterification of dipeptides with 0.1 N HC1/MeOH at room temperature is essentially complete in 4 hr. We have found (Fig. 2) that the commonly used diazomethane yields multiple products with dipeptides.

2. Determination of conditions for perfluoroacylation of dipeptide methyl esters. Methyl esters of P h e - A s p and T r p - T r p were model compounds for two extremes of reactivity. P h e - A s p has one (terminal) amino group to be acylated. On the other hand, Trp was reported to be more difficult to acylate because of the indole group hydrogen (16,17). Yields were determined by gc, and the highest peak arbitrarily assigned 100 in both cases. The reaction with T F A A is summarized in Table 1. P F P A acts in a fashion similar to T F A A , except that it is even milder. With T F A A , Phe-Asp-(OMe)2 begins yielding significant side products (6%) after 1.5 hr while P F P A does not, even after 4 hr of reaction. With PFPA, Trp-Trp-OMe reacts similarly, except for the unreacted material that occurs at a broad peak 30°C higher. Table 2 summarizes the data for Trp-Trp-OMe. The above results indicate that 1.5 hr is the optimal perfluoroacylation time with both T F A A and PFPA. As for all other perfluoroacyl methyl esters of dipeptides, the mass spectra of tri-TFA-Trp-Trp-OMe and

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FIG. 2. Thin-layerchromatographic separation of dipeptides esterified dipeptides with either diazomethane or HCL/MeOH; (1) Phe-Asp; (2) esterified Phe-Asp, CH2Nz; (3) esterified Phe-Asp, 0.1 N HCL/MeOH, 25°C, 30 hr; (4) esterified Phe-Asp, 1 N HCI/MeOH,25°C,30hr; (5) Ala-Ser; (6) esterifiedAla-Ser, CH2Nz;(7) esterifiedAla-Ser, 0.1 N HCL/MeOH, 25°C, 30 hr; (8) esterifiedAla-Ser, 1 N HCI/MeOH, 25°C, 30 hr.

114

YOUNG AND DESIDERIO TABLE 1 EFFECT OF TRIFLUOROACETYLATIONREACTION TIME ON THE YIELD OF TRI-TFA-TRP-TRP-OME AND TRI-TFA-PHE-AsP-(OME)za

Reaction time

Tri-TFATrp-Trp-OMe

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0 30 90 95 100

and

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Tri-TFA-PheAsp-(OMe)2

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90 90 95 100 100

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40 40

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YIELD OF TRI-PFP-TRP-TRP-OME

Reaction time

Tri-PFPTrp-Trp-OMe

A a broad peak 30°C higher

15 min 50 min 3 hr

0 60 95

60 50 10

a The peak at such a high temperature (270°C) was still too broad to yield a mass spectrum on the LKB 9000. Presumably this peak was incompletely derivatized material. Yields determined by gc; highest peak set arbitrarily at I00.

tri-PFP-Trp-Trp-OMe, shown in Figs. 3 and 4, respectively, have pronounced parent ions and are easily interpreted. In Fig. 3, NH2-terminal sequence-determining ions (see Scheme 1) occur at m/e 351, 633, 661 and 692 (M+'). Loss from M +" of CH3OH and -COOCHa occur at m/e 660 and 633, respectively. The ion at m/e 226 is due to the side chain while the ion at m/e 298 is due to N ~ - C ~ cleavage. 97:

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116

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Two interesting rearrangement ions occur atm/e 282 and at the base peak m/e 297 and involve the well-known McLafferty rearrangement (18) of a gamma hydrogen: M

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