Anal. Chem. 1995,67,1644-1645
Recognition of the Carboxy=TerminalPeptide in Cyanogen Bromide Digests of Proteins Constance M. Murphy and Catherine FenseIau* Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Bahimore, Maryland 2 1228
A method is reported that allows carbow-terminal peptides to be recognized in cyanogen bromide cleavage reactions. Recognition is based on mass spectrometric analysis of the peptide products before and after reaction with acidic methanol. Peptides generated from intemal positions in the protein are terminated by homoserine lactone, which undergoes methanolysis to add 32 mass units. The peptide containing the original carboxyl terminus is converted to a methyl ester, with a mass increment of 14 Da.
Table 1. Molecular Weights for Some Cyanogen Bromide Dlgest Fragments from Equine Liver Olutathione STransferase
mass
peptide sequence“ YIEVIADLGEm’ FQQVPmVEIDGm’ DEKSLAEAFWFR EPI RWLLAAAGVEFEEK FIKSAEDLDKLRNDGSL”
nonmeth- dif- multiples derivatized ylated ference of 14 1314.7 1561.0
1203.6
1277.6 1374.6 1616.8
3942
4087
74 60 56
no no yes
145
no
m, homoserine; m’, homoserine lactone. Sequences from ref 7. In the course of mapping and sequencing proteins, it is often important to identify the carboxy-terminal region of the protein. This facilitates interpretation of map and sequence information and helps reveal ragged ends and posttranslational processing. The application of mass spectrometry has allowed the develop ment of new strategies to distinguish the C-terminal peptide in mixtures of peptides derived from proteolysis. One approach, introduced by Rose and co-workers’ and successfully used by other^,^-^ calls for enzymatic cleavage of the protein of interest in a 1:l mixture of water and lsO-labeledwater. In the hydrolytic reaction, both l6O and l8O are incorporated into the a-carboxyl groups of newly released peptide fragments; however, the original carboxyl terminus in the protein does not incorporate oxygen from the solvent. When these digest products are examined by mass spectrometry, two molecular ions separated in mass by 2 Da are recorded for all the peptides except the original C-terminal peptide. This strategy has not been used with cyanogen bromide cleavages where product ion formation is more complex. In this report we demonstrate that esterification can be used with mass spectrometryto identify the Gterminal peptide released by cleavage digestion of a protein with cyanogen bromide. This analysis utilizes molecular weight determinations before and after esterification and is based on differences in the molecular weights of the products formed by reactions of carboxylic acids and of lactones with, for example, methanol. Reaction with cyanogen bromide generates peptide products that are terminated in homoserine, homoserine lactone, or a mixture. The product reflects the acid used and the length of reaction. The use of 70% (1) Rose, IC;Savoy, L . A ; Simona, M. G.; Offord, R E.; Wingfield, P. Biochem. J. 1988,250,253-259. (2) Vestling, M. M.; Hua, S.; Murphy, C. M.; Smith, P. B. W.; Fenselau, C. C. /. Protein Chem. 1990,9, 320-321. (3) Medzihradszky, K. F.; Gibson, B. W.; Kaur, S.; Yu, 2.; Medzihradszky, D.; Burlingame. k L.; Bass, N. M. EUY./. Biochem. 1992,203, 327-339. (4) Oda, Y.; Herrmann, J.; Gitt, M. A.; Turck, C. W.; Burlingame, A L.; Barondes, S. H.; Leffler, H. J. Bid. Chem. 1993,268, 5929-5939.
1644 Analytical Chemistv, Vol. 67,No. 9,May 7, 7995
trifluoroacetic acid, recommended to eliminate formylation or acetylation as a side reaction? leads to formation of homoserine lactone as the dominant terminal residue! When treated with acidic methanol, the lactones open with the addition of methanol (32 Da) to produce hydroxy methyl esters. In contrast, methyl esterification of sidechain acid groups and terminal carboxylate groups results in the addition of multiples of 14 to the molecular weight. A terminal peptide ending in an amide group would be increased by multiples of 14 associated with sidechain carboxylates. An example of the application of the strategy is illustrated in Table 1, which lists the molecular weights of four peptides released in a study of the unknown sequence of equine liver glutathione Stransferase? The protein was incubated at a concentration of approximately 25 mg/mL in 70%trifluoroacetic acid with a %fold excess of cyanogen bromide. The reaction was stirred for 24 h under argon, and the products were fractionated by reverse-phase HPLC. A second column in Table 1 contains molecular weights of several peptides derivatized after reaction with a 10016 v/v mixture of methanol and acetyl chloride for 1h at room temperaturee8 Mass differences are noted in the table, and the carboxyl-terminal peptide can be readily distinguished because its mass is increased by multiples of 14 Da. Information is also obtained on the number of acidic side chains in each peptide. The second peptide in the column contains homoserine, the product of an incomplete reactiongJOby CNBr with a residue (5) Goodlett, D. R; Armstrong, F. B.: Creech, R J.; van Breemen, R B. Anal. Biochem. 1990,186,116-120. (6) Lee, T. D.; Shively, J. E. Methods Enrymol. 1990,193,361-374. (7) Murphy, C. M. Ph.D. Dissertation, University of Maryland Baltimore County, Baltimore, MD, 1994. (8) Hunt, D. F.; Yates, J. R, 111; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. S C ~U.S.A. . 1986,83,6233-6237.
0003-2700/95/0367-1644$9.00/0 0 1995 American Chemical Society
identifed as methionine by other methods. Fast atom bombardment was used on a sector instrument to analyze the three smaller peptides, and laser desorption on a time-of-fight mass SPckometer provided molecular weights for the peptide and its derivative of around 4000. Cyanogen bromide products often are heavier polypeptides, in which the 4 mass unit cipher can be more readily distinguished than the 2 mass unit increment offered by l80. (9) Schroeder,W. A; Shelton,J. B.; Shelton,J. RArch. Biochem. Biophys. 1969, 130,551-556. (10) Dogen, N.; LaPresle, C. Biochem. J 1979,177, 251-257.
ACKNOWLEDGMENT
This work was supported by grants from the National Science Foundation and the National Instihtes of Health (GM21248). Received for review November 18, 1994. February 23, 1995.@
Accepted
AC941119R Abstract published in Advance ACS Abstracts, April 1, 1995.
Analytical Chemistv, Vol. 67,No. 9,May 7, 7995
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