Reductive desulfuration and mass spectrometric sequencing of sulfur-containing peptides

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chromatography and electrophoresis, and to.ok 145 min for electrophoresis and 20 hr for chroma’tography. The method reported here should lend itself to quantitative determination of the nucleotides extracted from the separated spots, and have application in various projects involving the analysis of complex mixtures of nucleotides. Summcq. Thirteen nucleotides were completely separated from each other in 70 min by high-voltage paper electrophoresis, employing Whatman ET 31 paper and a citrate buffer containing EDTA. REFERENCES 1.

J. J., in “Chromstographic Reviews” (M. Lederer, ed.), Vol. 6, p. 53. Elsevier, Amsterdam/New York, 1964. 2. VANDERHEIDEN, B. S., Anal. Biochem. 22,231 (1968). 3. SERLUPI-CRESCENZI, G., PAOLINI, C., AND LECCIO, T., Anal. Biochem. 23, 263 (1968). SAUPKONNEN,

M. J. SILVER I. ROIJALEWICZ Y. DUGLAS D. PARK Cardeza Foundation of Pharmacology Thomas Jefferson

Philadelphia, Received

and Department University

Pennsylvania 191Or January ,?O, 1970

Reductive

Desulfuration

Sequencing

and

Mass

of Sulfur-Containing

Spectrometric Peptides

The introduction by Lederer’s group of permethylation procedures to increase peptide volatility has greatly advanced the potential of mass spectrometry as a tool for the sequencing of peptides. This subject has been reviewed by Lederer (1). Sulfur-containing peptides offer difficulties in mass spectrometric sequencing in that the spectra of such peptides often display a large number of peaks some of which are derived by (a) elimination of the side chain of methionine by a McLafferty rearrangement and (b) conversion of cysteinyl and cystinyl to dehydroalanyl residues (2). Upon permethylation of methionine peptides Agarwal et al. (3)

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reported the conversion of the methionine side chain to the cyclopropyl derivative, whereas Thomas et al. (1, 4) did not report this reaction, but their derivatives on permethylation appeared to yield sulfonium derivatives. Our experience has shown that AgzO, CHJ permethylation completely converted methionine peptides to other products, among which are compounds of a mass equivalent to either cyclopropyl or propene side chains, whereas the Hakomori permethylation via the procedure of Vilkas and Lederer (5) gave a mixture of unmodified methionyl residues as well as some of the other product. The unmodified methionyl residues fragmented upon electron bombardment mainly by conversion to the equivalent of a glycyl residue by elimination of the side chain by a McLafferty rearrangement as mentioned above. Accordingly, it would appear that removal of sulfur by reductive desulfuration might greatly simplify this problem and the use of Raney nickel was suggested by Lederer (1) and employed by Thomas et al. (4) and Kiryushkin et al. (2). The procedure of the former group (4) involved a 4 hr reflux in ethanol with freshly prepared Raney nickel, whereas the latter group carried out their reaction at 20°C for 2 days in dimethylformamide. Our experience with Raney nickel showed considerable variation in activity in that it was difficult to achieve complete desulfuration under mild conditions in volatile solvents such as water or alcohol. We have preferred to prepare a catalyst by reduction of aqueous solutions of NiCl, with NaBH, similar to that first described by Buisson et al. (6). Such catalysts appear to have a high resistance to fatigue (6), are easy to prepare, and are particularly useful if reductive desulfuration with a deuterated catalyst is required in that D,O and NaBD, can be easily employed. The composition of this type of catalyst is reported as approaching the composition of Ni,B (6). For the assay of this catalyst we convert an aqueous solution of lanthionine to alanine. Sufficiently active catalyst will quantitatively convert lanthionine to alanine in 3 hr at 50°C. Desulfuration of thiol compounds such as cysteine and reduced glutathione is much more rapidly obtained than that of disulfides, cystine, or oxidized glutathione. The reduction of both thiol and disulfide compounds proceeds more rapidly than that of thioethers such as lanthionine and methionine. Our procedure of reductive desulfuration, acylation (in the present we have employed isobutoxycarbonylation but in other peptides we have employed acetylat,ion) and permethylation allows a simple and rapid modification of small levels of peptides, such as can be eluted from fingerprints of peptide digests. A. Preparation of the Catalyst. The preparation unit consists of a

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529

long-stemmed separatory funnel with an L-shaped glass tube pushed through a two-hole rubber plug and placed in the neck of a thick-walled glass cylindrical jar (one tube is connected to a short piece of rubber tubing and clamp, and serves as a pressure valve). This unit is surrounded by an ice bath and placed on a magnetic stirrer. The sodium borohydride solution (5 gm of NaBH, in 100 ml H,O) is added to the jar containing a magnetic stirring bar. The nickelous chloride solution (12 gm NiC1,.6 H,O in 100 ml H,O) is added dropwise and slowly to the stirring sodium borohydride. The pressure release valve is opened only when the reaction becomes too vigorous and should be closed quickly afterward. Three hours is allowed for the reaction and adsorption of the hydrogen gas. After completion of the reaction, the excess NaBH, is destroyed by the addition of 12 N HCl to pH 4. The volume of the mixture is then doubled with distilled H,O, allowing the hydrogenated nickel catalyst to precipitate. After decantation by suction the catalyst is washed three times with distilled H,O by centrifugation and placed in a glass-stoppered bottle under a small volume of distilled water. B. Reductive Desuljuration of Lanthionine. 5 ml of a known concentration (l-3 pmoles/ml) of lanthionine at pH 7.0 is added to a 25 ml glassstoppered Erlenmeyer with about 1.5 gm (wet weight) freshly prepared catalyst. The closed flask is placed in a 50°C Dubnoff shaker and shaken for 3 hr. At the end of this time, the catalyst is centrifuged off and the supernatant removed. The catalyst is treated with 5 ml of hot water to remove absorbed product and centrifuged, the supernatant added to the first supernatant. A rapid qualitative and semiquantitative test of catalyst activity and efficiency of lanthionine reduction is performed by paper chromatography in butanol/acetic acid/water (4: 1: 5). In this solvent, lanthionine stays close to the origin and alanine has an Rf of about 0.3. Quantitative analysis can be performed by automatic amino acid analysis. Satisfactory cat,alyst converts lanthionine quantitatively to two equivalents of alanine in 95 to 100% yield in less than 3 hr. C. Reductive Desuljuration of Model Peptides. The procedure for desulfuration of model peptides duplicates that described for lanthionine except that the flask containing the peptide solution and catalyst is shaken for 16 hr. Amino acid analysis and chromatography of the reduced and hydrolyzed glycyl-methionyl-glycine peptide shows complete loss of methionine with conversion to a-aminobutyric acid; however, amino acid analysis also shows a significant conversion (10%) to the methionine sulfoxide, indicating some oxidation of methionine before complete reductive desulfuration. Perhaps if the reaction were carried out in nitrogen this side reaction could be avoided; however, it did not interfere significantly with the results reported here.

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Oxidized glutathione was quantitatively reduced to ,r-glutamyl-alanylglycine as ascertained by quantitative amino acid analysis after acid hydrolysis. D. IsobutoxycarbonyZation (Iboc). To 5 ml of a solution of the peptidesl is added 70 mg K,CO,. The pH of the solution is kept between 8 and 9. More K,CO, can be added if necessary. While stirring, 3 additions in 30 min intervals of 50 ~1 each of isobutylchloroformate are made. Stirring is continued for 1 hr after the final addition. This overreaction converts the excess isobutylchloroformate to isobutanol, which is removed during the in vacua evaporation. The solution is then transferred to a round-bottom flask and brought to pH 2 with 1 N HCI. The solution is evaporated in vucuo (3040°C) and evaporated three more times from distilled H,O. E. Pemnethylation (Hakomori Method). 30 mg NaH in oil dispersion is rinsed and decanted 3 times with anhydrous ether, while shaking in a test tube. 1 ml dimethyl sulfoxide (DMSO) is added, and the tube is heated and shaken at 50°C until all H, gas evolution ceases. This solution is added to the flask containing the Iboc peptide derivative and distributed homogeneously with a spatula. 1 ml methyl iodide is added to the flask and again distributed homogeneously with a spatula. The flask is now covered and allowed to stand for 1 hr. After this period, 20 ml H,O is added to the flask, followed by 3 extractions with CHCl,, each of 10 ml volume. The CHCl, extracts are combined and washed with distilled H,O. The washed CHCI, is dried over anhydrous NaGSO for 10 min and evaporated (3040°C) in vucuo to dryness. Before mass spectrometry, the material is dissolved in a small volume of dichloromethane and transferred and evaporated in the small glass crucible of the Hitachi direct-inlet mass spectrometer. The compounds were volatile at SO0 to 1OOOC. Results and Discussions. In Figure la, the mass spectrum of isobutoxycarbonylated (Iboc) permethylated oxidized glutathione is given. The spectrum is complex, showing the presence of (Fig. lb) the dehydroalanyl peptide (C) (443, 412, 384, 34O*) as well as other parent molecules. The peaks at 490, 476, 444, 430 suggest that some lanthionyl residues were formed during the derivation procedures (A). The peaks at 491,476,389, 444, 430 and 388* may result from the tripeptide y-glutamyl(S-methylcysteinyl)-glycine (B). Accordingly, it would appear that, under the basic conditions of permethylation, both disulfide cleavage and @ elimination occurred. Further reaction of these products also led to formation of the lanthionyl analog of oxidized glutathione. ‘This procedure is used for both trace levels and up to * The loss of (59 + 44) is typical of Iboe peptide methyl also occurs in the other fragments.

as much as 10 esters.

Loss

mg peptide. of 44 (CO,)

1OOr bp yj Z 8 z 2 -=I F F Q $

8060491

4020I

140

200

300

4ccl

I

1

500

600

m/e

202 (Isa*)

c-OCI C”3

(4

\

/

P

P P ‘CH - CH2 - 0 - c - y - CH -

CH3

CH2

C -

OCH3

%

202 o CH3

f-

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CH*

-

0 -

c”-

N -

.

oc:;)

\ CH

(0)

-

:

cn

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443

FIO. 1. (a) Mass spectrum of isobutoxycarbonylated (Iboc) permethylated oxidized glutathione. (b) Interpretation of fragmentation pattern of isobutoxycarbonylated permethylated oxidized glutathione : The peaks at 490, 476, 444, 430 suggest that some lanthionyl residues (A) were formed during the derivatization procedures. The peaks at 491, 333*, 339 may result from the tripeptide y-glutamyl(S-methylcysteinyl)-glycine (B). The peaks at 443, 412, 384, 340*, 341 show the presence of the dehydroalanyl peptide (C).

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m/e

CH2

-

0 -CH3

FIG. 2. Mass spectrum and interpretation of fragmentation pattern of reductively desulfurized derivative of oxidized glutathione. The spectrum is clearly that of the Iboc permethylated y-glutamyl-alanyl-glycine.

In Figure 2, the mass spectrum of the reductively desulfurized derivative of oxidized glutathione is shown. It is clearly the spectrum of Iboc, permethylated (y-glutamyl-alanyl-glycine) and the sequence is obvious on inspection. In Figure 3a, the mass spectrum of Iboc permethylated glycyl-methionyl-glycine is given. Some parent ions (A in Fig. 3b) containing methionine are present (419, 494, 388, 316*, 372) and their main fragmentation pathway seems to be the loss of CH,=CH-S-CH, and formation of the odd electron ion at 345 (C) by a McLafferty rearrangement. The presence of an odd electron peak at 371 and peaks at 199 and 227 suggests that during either permethylation or electron bombardment conversion of the methionyl side chain to propene or cyclopropyl occurred (B) (3). In Figure 4, the mass spectrum of the reductively desulfurized, Iboc, and permethyIated peptide obtained from glycyl-methionyl-gIycine is cIearly that derived from glycyl-cr-aminobutyryl-glycine. The spectrum is clean, simple, and very easily interpreted by inspection. Clearly, reductive desulfuration of sulfur-containing peptides greatly simplifies their mass spectra and facilitates their sequencing. Upon the reductive desulfuration of peptides alanine results from cysteinyl, cys-

SHORT

533

COMMUNICATIONS

‘OOii2

(a)

345-59-44)

201 ‘II

170

295

’ ‘3

L

200

300 m/e

144

(I)) (A)

C”3 , /

1’) ‘1

:: ‘CH-

CHZ-

O-t-

y-

“‘3

CH2

-C

::

0

c”

0

-

CH3

CH3

371

\ CH 3 ’

B

0

CH3 (V

CH-

CH?-

0-

:: C-

IfCH3

CH2

C-:-N-CHz-C !Hz-/\

-0CH3

tH3 CHz-CH?

199

FIG. 3. (a) Mass spectrum of isobutoxycarbonylated permethylated glycylmethionyl-glycine. (b) Interpretation of fragmentation pattern of isobutoxycarbonylated permethylated glycyl-methionyl-glycine: Ions at 419, 494, 388, 317, 316*, 372 derived from the methioninyl peptide are present (A). The presence of an odd electron peak at 371 and peaks at 199, 227 suggest the conversion of the methionyl side chain to a propene or cyclopropyl group (Bl. The formation of the odd electron ion at 346 (Cl indicates elimination of the side chain of methionine by a McLafferty rearrangement.

112 IOO”t51 %Of2Q 4 X’ r 342 % 228 243 2M 2ll 11200 199 111,,I 1, ,I 1t1 1 3.58

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SHORT

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60-

40-

282

373

~

314

I

300

140

I

400

m/e

FIG. 4. Mass spectrum and interpretation of fragmentation pattern of reductively desulfurized Iboc permethylated derivative of glycyl-methionyl-glycine. The maw spectrum is clearly that derived from glycyl-waminobutyryl-glycine.

tinyl, or lanthionyl residues, and could be confused with alanine already present in the peptides. Introduction of deuterium rather than hydrogen during reductive desulfuration, as suggested by Lederer (1)) could differentiate between these alanine residues and, accordingly, could be routinely employed. The type of Ni catalyst we employ here, in addition to its advantages in our hands of reactivity over the Raney-type catalyst, can be easily prepared in its deuterated form. Summary. The difficulties presented by sulfur-containing amino acids in mass spectrometric sequencing of peptides can be eliminated by reductive desulfuration in the presence of an easily prepared and efficient NizB catalyst. The procedure described consisting of reductive desulfuration, isobutoxycarbonylation and permethylation allows simple and rapid modification of small levels of peptides, and advances the potential of mass spectrometry as a tool in peptide sequencing procedures. RZFERENCES 1. LEDERER, E., 2. KIRYUSHKIN, SHEMYAKIN,

Pure

Appl.

A. A., M.

M.,

Chem. GORLENRO, Ezperientia

17, 489

A., 25,

(1968). ROSINO~, 913 (1969).

B. V.,

OWHINNIROV,

A.,

AND

SHORT 3. AGARWAL, SHEPPARD, 4. THOMAS,

K. L., JOHNSTONE, R. A., KENNER, R. C., Nature 219, 498 (1968). D. W., D’As, B. C., GERO, S. P., AND

Gommun. 5. VILKAS, 6. BUISSON,

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COMMUNICATIONS

G. W.,

D. S.,

MILLINGTON,

E., &&em.

LEDERER,

AND

Biophys. Res.

32, 519 (1968).

E., AND P. R.,

LEDERER, E., Tetrahedron AND JOSEPH, N., Compt.

Letters, No. 26, 3089 (1968). Rend. 232, 627 (1951). MERCEDES ALEXANDER EDWARD OLGA PAUL

Department of Biochemistry and the Unit Albert Einstein College of Medicine Bronx, New York 10461 Received February 18, 19i+O

Another

Simple

Equilibrium

for

Research

A.

PAZ

BERNATH HENSON

0. M.

BLUMENFELD GALLOP

in Aging

Dialysis

Apparatus

An equilibrium dialysis apparatus was recently described in this journal by Nye (1). We have independently developed an apparatus that performs a similar function, but has several distinct advantages: (A) It can easily utilize celIs with volumes as small as 0.025 ml or as large 8,s0.500 ml. (B) The volume ratio of inner to outer cells can be readily varied from 1:l to 1:20. (C) It can be quickly built from inexpensive materials without access to machine tools. (Construction requires only a razor blade, a cork borer, and a glass cutter.) The apparatus’ consists of a sandwich of two sheets of inert Silastic silicone rubber cemented to strips of l/4” plate glass with Dow Corning Aquarium Cement. The construction of one half-cell is shown in Figure 1. Two halves are used to clamp a sheet of dialysis membrane. The wells are formed by cutting carefully spaced circles with a cork borer from the silicone rubber before cementing it to the glass. The volume of the cells depends upon the diameter of cork borer used, and it is apparent that the ratio of the “sample” chamber to “buffer” chamber can be easily varied. ’ Patent

applied

for.