Mass spectrometric signature of S-prenylated cysteine peptides

ANALYTICAL

BIOCHEMISTRY

193,173-177

Mass Spectrometric Cysteine Peptides

(1991)

Signature of S-Prenylated

Albert A. Tuinman,*p’ Deborah A. Thomas,* Fred Naider,? and Jeffrey M. Beckert

Kelsey

D. Cook,*

Chu-Biao

Xue,?

Departments of *Chemistry and SMicrobiology, University of Tennessee, Knoxville, Tennessee and TDepartment of Chemistry, College of Staten Island, Staten Island. New York 10301

Received

September

21, 1990

The fast atom bombardment mass spectra of peptides containing S-prenylated cysteine display signature fragmentations characteristic of this modified amino acid. The fragmentation is independent of the nature of the cysteine carbonyl substituent, easily differentiates prenyl from nonprenyl alkylation, and readily identifies the oligomer count of the prenyl. This screening method, which requires little time, effort, or material (compared with previous analysis methods based on chemical degradation), greatly facilitates the identification of these prenylated proteins. 0 1991 Academic Press,

Inc.

Protein prenylation is a recently discovered posttranslational modification that has important ramifications for protein structure and function (1,2). For example, farnesylation of the RAS’ oncogene product is required for its membrane association and subsequent oncogenesis of the cell overexpressing the RAS protein (3). A large percentage of pancreatic and colon tumors express the RAS protein (4,5), and a number of other cellular proteins, whose function is unknown, also contain prenyl substituents (6-8). One of the best-characterized farnesylated peptides is the a-factor (HTyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-AlaCys(S-farnesyl)-OCH,) of Saccharomyces cereuisiae (9), which is required for sexual conjugation and is produced by the MATa haploid cell (10). This pheromone (11) and a number of its analogs have been synthesized for 1 To whom correspondence ’ Abbreviations used: RAS, MS, fast atom bombardment tic acid; TG, thioglycerol; M quasi-molecular ion; MA, Me, and C.

should be addressed. rat sarcoma; MAT, mating type; FABmass spectrometry; TFA, trifluoroace+ H+, the protonated molecule = the MC, masses of the fragments A, B,

0003.2697/91$3.00

Copyright All rights

37996;

0 1991 by Academic Press, of reproduction in any form

structure-function studies. During characterization of the synthetic compounds, it became apparent that the mass spectra revealed a signature of the farnesyl modification. In this report we describe how this signature can be used to identify the farnesyl group or other prenyl groups on peptides and proteins. MATERIALS

AND

METHODS

Analogs were synthesized by solution-phase and/or solid-phase procedures (11,12). They were homogeneous as judged by TLC, HPLC, amino acid analysis, and 400-MHz ‘H NMR. The mass spectra were acquired at 8 kV accelerating potential and resolution (ml Am) 2000 on a VG ZAB-EQ mass spectrometer. The Ion Tech atom gun was operated with xenon at 9 kV and discharge current at 0.2 to 0.4 mA to decrease chemical noise and prolong signal longevity (13). Samples were prepared for MS by manually spreading ca. 2-5 pg of dried peptide on the FAB target and overlaying with l-2 ~1 of a freshly prepared solution of 1% trifluoroacetic acid (TFA) in thioglycerol (TG). This method provided longer signal lifetime, lower source pressure, and more abundant peptide ions than the water/acetonitrile/ TFA/TG mixture recommended by Anderegg et al. (9). Data were recorded on a VG 11-2505 data system operating in the MCA mode (a computer simulation of true multi-channel acquisition). RESULTS

AND

DISCUSSION

The fast atom bombardment mass spectrum (FABMS) of synthetic a-factor shows the protonated molecular ion (M + H+, mass 1630) as the most intense peak in the high mass range (Fig. la). Fragment clusters B and C of Fig. la are attributable to the loss of the farnesyl moiety, with and without the thioether link. Verification that these peaks result from loss of the prenyl moiety is 173

Inc. reserved.

TUINMAN

lee-

ET

AL.

YIIKGVFWL)PH

BE. 68 _ 204

INi-

YIIKGVFWDPA--N

BE. 68.

lee

1

YIIKGVFWDPH

1658

WH3

I

48.

m/z FIG.

1.

FAB-MS

of a-factor

(a) and the analogous

geranyl

provided by the spectra of the geranyl (Fig. lb) and isoprenyl (Fig. lc) analogs. In each of the latter spectra, fragment clusters appear at the same masses as B and C in Fig. la, despite the lower mass of the protonated molecular ion (marked A in each spectrum). Further confirmation that clusters B and C result from the fragmentation of the protonated molecular ion (rather than from sample contamination by unprenylated material) was derived from tandem mass spectra (14,15) of com-

a-factor

(b), isoprenyl

a-factor

(c), and cetyl

a-factor

(d).

pounds 2,5,7, and 9 (Table 1) wherein B and C are the prominent fragments (these results will be presented elsewhere). Plausible fragmentation mechanisms to account for the formation of the clusters marked B and C are given in Fig. 2. The transition A + B of Fig. 2 requires the presence of a &r-double bond on the hydrocarbon chain, with at least one hydrogen at a cis-oriented d-carbon. Significantly, lack of one of these features (the

IDENTIFICATION

OF

S-PRENYLATED TABLE

Tabulated

Spectra

CYSTEINE

175

PEPTIDES

1

of the a-Factor Derivatives Masses

Structure”

and (intensities)b

Compound

X

Y

R

A’

Bd

C

D’

1 2

UP Ala

OMe OMe

Far Far

1630 (100) 411 (21)

1426 (12) 207 (100)

1392 (5) 173 (9)

1614 (10) 395 (2)

3

UP

OMe

Ger

1562 (100)

1426 (14)

1392 (8)

1646 (12)

4 5

UP Ala

OMe OMe

Pre Pre

1494 (100) 275 (100)

1426 (6) 207 (94)

1392 (3) 173 (21)

1478 (12) Non@

6 7

UP Ala

Open Open

Far Far

1686 (100) 467 (23)

1482 (16) 263 (100)

1448 (7) 229 (22)

1671 (10) Non@

8 9

UP Ala

OPre OPre

Far Far

1684 (100) 465 (30)

1480 (10) 261 (53)

1447 (5) Non@

1669 (9) Nones

10

UP

NH,

Far

1615 (100)

1411 (15)

1377 (5)

1600 (12)

11

UP

OMe

Me

1440 (100)

1428 (?)”

1393 (3)

1425 (8)

12

UP

OMe

Cet

1650 (100)

1426 (3)

1392 (5)

1635 (15)

13

UP

OMe

BZ

1516 (100)

1426 (8)

1392 (2)

1500 (9)

Other

192 (47)h 1616 (8)’ 397 (53)’ 193 (100)’

’ Each structure corresponds to A’ of Fig. 2, with the substituents X, Y, and R’ as indicated: UP = undecapeptide (the residues H-Tyr-IleIle-Lys-Gly-Val-Phe-Trp-AspPro-Ala-); Ala = H-alanine; Me = methyl (CH,); Pen = isopentyl (C,H,,); Pre = isoprenyl (C,H,); Ger = geranyl (C,,H,,); Far = farnesyl (C,,H,,); Bz = benzyl (C,H,); Cet = cetyl (n-C,,H,,). b The code letters represent peak clusters of the protonated molecular ion (A), the signature fragments (B and C), and the loss of methyl/ methane (D) (see Fig. 1). Mass numbers in Da; intensities in arbitrary units, normalized to the most abundant ion in the high mass region of the spectrum. ’ Isotope distribution in the quasi-molecular ion clusters corresponded well with theory; e.g., calculation of the methyl ester geranyl thioether (compound 2) with elementary composition C,H,,,N,,O,,S predicts relative intensities 100/98/55/22 for the masses 1562-1565 (compare 100/94/56/27, Fig. lb). d The listed mass is the most intense peak in the cluster and corresponds to the loss of the thioether hydrocarbon according to Fig. 2 A -+ B. However, cluster shapes do not correspond to the calculated isotope ratios (see Results and Discussion). ’ The listed mass is the most intense peak in the cluster. Cluster shapes do not correspond to the isotope ratios calculated for products C of Fig. 2 (see Results and Discussion). ’ The listed mass is the most intense of the cluster. Cluster shapes diverge significantly from theoretical, and the D-cluster probably consists of A-CH, and A-CH,. For the undecapeptides, the D-cluster intensities are fairly consistent, and constitute a good benchmark for comparing the intensities of the B- and C-clusters. #No distinct cluster discernable above the peak-at-every-mass background typical of FAB-MS. ’ Corresponds to the loss C,H,, and C,,H,,. ’ Corresponds to loss of C,H,. j Corresponds to loss of C&H,, and C,H,. k The B-cluster, if present, would overlap the D-cluster.

P,y-double bond) in the cetyl analog, produces a spectrum without a prominent B-cluster (Fig. Id). For the B-cluster to indicate a prenyl loss, the mass loss (MA - MB) must be a multiple of the isoprene monomer mass (C,H,; 68 Da). The C-clusters of Figs. la-d are derived by cleavage of the cysteine C-S bond (A’ --* C, Fig. 2). The mass difference (MB - Mo = 34 Da) signals a possible thioether linkage. The transition A’ + C is not dependent on the presence of a &y-double bond in the alkyl chain, and therefore the C-cluster in Fig. Id is of an intensity comparable to those in Figs. la-c. In summary, if the FAB-MS of a peptide (protonated molecular ion mass MA) displays prominent fragment clusters B and C (signature clusters) such that (MA - MB

= n X 68) and (MB - Mc = 34), the possibility of an S-prenylated cysteine is strongly indicated. Signature ions appear as peak clusters rather than single peaks due to natural isotope abundances, primarily 13C and 34S. If the fragmentation reaction A + B were “clean,” the B-cluster profiles would be similar to those of the A-clusters in Fig. 1. The observed distortion of the B-cluster envelope indicates a competing reaction (probably homolytic cleavage of the S-R bond) leading to a radical cation of one lower mass than B. The overlap of the cluster from this second fragment type with that of fragment B causes the distorted B-cluster profiles observed in Fig. 1. An analogous argument explains the even greater distortion of the C-clusters, with ho-

176

TUINMAN

+X-N

ET

C 'Y

red

AL.

HZ c\s

H

Hs

H

k

B k 0 +x-NH

"-p Hz

\

P

‘y

+

HSR’

I! H2

R’

A’

'C J

C

FIG. 2. Postulated mechanisms for generation of the deprenylated ions B and C from the quasi-molecular matrix is expected to protonate a nitrogen of the peptide chain (represented as X’). Loss of the prenyl participation of the charge site, in a thermally induced “charge remote fragmentation” (18).

molytic cleavage of the cysteine CH,-S bond competing with A’ + C. In some spectra (e.g., 8 and 11 of Table 1) the distortion makes h4n - 33 (rather than MB - 34) the most intense peak in the C-cluster. Although the loss of 204 Da in the FAB-MS of a-factor has previously been observed and interpreted to indicate a farnesyl moiety (9), that observation was not pursued to establish its general applicability as a screen for prenyl substitution. The scope and general utility of the signature fragments as an analytical screen for prenylated cysteines are addressed below, utilizing the data in Table 1. Cysteine carbonyl substituent variations. Compounds 1,6,8, and 10 of Table 1 represent changes in the cysteine carbonyl substituent of A (=A’), with all other structural parameters unaltered. Significantly, the relative intensities of fragments B and C differ little as a function of changes in that substituent. Even the transition from an ester (1,6, and 8) to an amide linkage (10) does not alter the signature fragment pattern. By extrapolating this observation to N-substituted cysteine amides, we anticipate that the characteristic signature fragments will be observed for prenyl cysteines, even when that residue does not occupy the C-terminal position of the peptide. The ester-linked isoprenyl moiety of compound 8 displays noticeable loss of C,H, appearing as a cluster between A and B with the most prominent mass at 1616. This loss presumably arises via a fragmentation similar to A + B of Fig. 2, but occurring at the ester oxygen rather than the thioether sulfur. Although the resulting 68 Da mass loss mimics part of the S-prenylated cys-

ion A = A’. The acidic FAB moiety probably occurs without

teine “signature,” the lack of an accompanying C-cluster 34 Da below the supposed B-cluster indicates that the link is not via sulfur, thus avoiding misinterpretation. Nonprenyl cysteine thioethers. Entries 1, 3, 4, and 11-13 of Table 1 represent different hydrocarbons forming cysteine thioethers, while all other structural features remain unchanged. As noted above, the cetyl group of 12 does not meet the criteria for fragmentation A + B of Fig. 2. The B-cluster attributed to loss of the cetyl moiety is therefore barely discernable above the noise (Fig. Id). On the other hand, the benzyl thioether 13 loses C,H,, even though the structural requirements for the Fig. 2 mechanism are not met by the protonated molecular ion. A different mechanism must be operative in this case: possibly loss of a benzyl carbene. Note that the resulting fragment peak at 1426 cannot be mistakenly attributed to loss of a prenyl moiety because the mass difference (MA - MB = 90) is not a multiple of 68 Da. Effect of peptide chain length. Two appropriately substituted oligopeptide backbones were available from the synthesis of a-factor analogs: the dipeptide H-AlaCys-OY (2,5,7, and 9) and the dodecapeptide H-TyrIle-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys-OY (all other compounds in Table 1). The signature B-clusters, and in most cases the C-clusters, are considerably stronger (relative to the protonated molecular ions) for the dipeptides than for the dodecapeptides. Based on this very limited number of observations, it appears that the prenyl signature fragments are stronger for shorter peptide backbones.

IDENTIFICATION

OF

S-PRENYLATED

Conclusions. Hitherto, identification and differentiation of S-prenyl cysteine derivatives have been lengthy and complicated processes involving degradation of the thioether with Raney nickel, purification of the reaction mixtures, and identification of the hydrocarbon fraction by gas chromatography/mass spectrometry (GC-MS). An alternative chemical degradation (alkylative cleavage of the thioether to produce an alkenol, followed by purification, derivitization, and GCMS) is at least as cumbersome (9,16) and requires substantial amounts of material (e.g., 165 pg of sample was used to identify the farnesyl group in a-factor (9)). Also, undesired side reactions in the degradation steps of each of these sequences can lead to product mixtures which complicate interpretation of the results (6,9,17). In contrast, the preliminary identification of S-prenylated cysteines by signature recognition in the FAB mass spectrum is fast and requires only 2-5 pg of material for the samples analyzed. The prenyl oligomers differ in mass by 68 Da per monomer, and there is apparently (see Fig. 1) no fragmentation within the oligomer. Identification of the signature fragment(s) therefore unambiguously indicates both the presence and the oligomer count of the prenyl group. We are not aware of other protein modifications which could mimic this signature, certainly palmitoylation or myristoylation is not expected to do so. Spectra of sufficient quality to identify the signature B- and C-clusters were achieved without special instrumentation beyond the “standard’ magnetic sector mass spectrometer equipped for FAB. For smaller peptides (tlOO0 Da) quadrupole instruments would probably suffice. For this signature analysis to work optimally with newly isolated proteins, it will probably be necessary to generate peptide fragments by some well-characterized enzymatic or chemical method, such as trypsinization or cyanogen bromide treatment. After separation of peptide fragments by HPLC, the most hydrophobic fractions can readily be analyzed by FAB-MS for their signature profiles. Although S-prenylated-cystein-containing peptides generated in this manner may have that residue in a position other than the C-terminal position, we nonetheless anticipate observation of the relevant signature fragments. This anticipation is based on data from the available model compounds, as indicated above under Cystein carbonyl substituent variations. Extending the technique to apply to mixtures of peptides (derived, e.g., by digestion of proteins) would constitute an improvement in the procedure by circumventing the costly separation step mentioned above. However, weak (ca. lo-2095 relative abundances) signature fragment clusters could be obscured by the more intense peptide molecular ion clusters. Nonetheless,

CYSTEINE

177

PEPTIDES

such analyses may be possible with a high degree of specificity by using tandem mass spectrometry to examine one peptide molecular ion at a time and by determining the presence of signature product ions after collision induced dissociation (14,15). Preliminary MS/MS experiments on the prenylated dipeptides 2,5,7, and 9 indicate the viability of such an approach, with the major collision-induced fragments being of type B and C (cf. Results and Discussion). ACKNOWLEDGMENTS The UTK Chemistry Mass Spectrometry Center is funded by the Science Alliance, a State of Tennessee Center of Excellence. The NSF Chemical Instrumentation Program also contributed to the acquisition of the ZAB-EQ (grant CHE-86-09251). This work was supported in part by Grants GM-22086 and GM-22087 from the National Institute of General Medical Sciences and CHE 88-22787 from the National Science Foundation. We thank Dr. J. D. Reynolds for acquiring some of the spectra.

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