Liquid Chromatographic Analysis and Mass Spectrometric

Jun 29, 2012 - Ioanna Ntai , Luca Fornelli , Caroline J. DeHart , Josiah E. Hutton , Peter F. Doubleday , Richard D. LeDuc , Alexandra J. van Nispen ,...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/ac

Liquid Chromatographic Analysis and Mass Spectrometric Identification of Farnesylated Peptides Marina Wotske,† Yaowen Wu,‡ and Dirk A. Wolters*,† †

Department of Analytical Chemistry, Ruhr-University of Bochum, Bochum, Germany Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany

‡

S Supporting Information *

ABSTRACT: Farnesylation involves the post-translational attachment of a 15 carbon unit to the C-terminus of proteins, thus allowing them to incorporate into membranes. The farnesylation reaction requires farnesyldiphosphate as the farnesyl group donor and is catalyzed by the farnesyltransferase. Some of the most familiar farnesylated proteins belong to the Ras protein superfamily, well-known oncoproteins. As Ras proteins require the membrane localization for the transduction of extracellular signals, farnesyltransferase inhibitors are discussed as chemotherapeutic agents. Despite the importance of this post-translational modification, farnesylated peptides have been investigated rarely by means of high-pressure liquid chromatography in combination with mass spectrometry. In this study, we examined the liquid chromatographic separation of farnesylated peptides with the help of the multidimensional protein identification technology. The peptides were further ionized by electrospray ionization and subsequently analyzed by tandem mass spectrometry. We demonstrated that farnesylated peptides are more strongly retained by reversed phase than nonfarnesylated peptides. This allowed for the identification of farnesylated peptides, if spiked into complex peptide samples. In some cases the farnesyl group was apparently split off from the peptide during the ionization process, and tandem mass spectra often revealed a neutral loss of the farnesyl moiety.

P

100−200 different types of prenylated proteins. However, a much smaller number has been identified so far, of which the majority were geranylgeranylated.7 Examples of farnesylated proteins are the nuclear lamins A and B, the α-subunit of the heterotrimeric G protein transducin, and all three isoforms of the Ras GTP (guanosine triphosphate)binding proteins. Known geranylgeranylated proteins include the members of the Rho protein subfamily, whereas the members of the Rab protein subfamily are usually doubly geranylgeranylated. The hydrophobic prenyl groups serve as membrane anchors and thus allow the interaction of Ras proteins with receptor tyrosine kinases and the transduction of extracellular signals, for instance. In the case of Rab proteins, the hydrophobic residues facilitate the interaction with GDI (guanosine dissociation inhibitor), which extracts Rab proteins from the plasma membrane.8 Three distinct protein prenyltransferases that attach prenyl groups to the C-termini of proteins have been reported so far. The protein farnesyltransferase (FTase) transfers a farnesyl group from farnesyldiphosphate (FPP) to the cysteine of the CaaX box. The protein geranylgeranyltransferase type-I

renylation is the post-translational modification of a specific set of eukaryotic proteins. Thereby, a farnesyl or a geranylgeranyl group is bound to the C-terminus of proteins. Both the farnesyl and the geranylgeranyl group belong to the class of isoprenoids, which are lipids that are built of five-carbon isoprene units. The farnesyl group consists of three isoprenoid units, whereas the geranylgeranyl group exhibits four isoprenoids units, thus resulting in an elemental composition of C15H25 (204 Da) and C20H33 (272 Da), respectively. Prenylatable proteins usually have a C-terminal consensus sequence, called the CaaX box, where C is cysteine, a is mostly an aliphatic amino acid, and X is any amino acid. The prenyl group is attached via a thioether linkage to the cysteine of the CaaX box. The aaX portion of the CaaX box significantly determines the nature of the prenylation reaction.1,2 Besides being farnesylated or geranylgeranylated, proteins can also be doubly geranylgeranylated. The latter do not possess a typical CaaX box; instead the modified cysteines can be located next to each other or separated by one or two amino acids. The position of the two cysteines within the last four amino acids is not predefined either.3,4 In 1978, Sakagami and co-workers were the first to describe prenylated peptides from Tremella mesenterica.5,6 Meanwhile, prenylated proteins were found in almost all eukaryotes. Mammalian cells are supposed to contain approximately © 2012 American Chemical Society

Received: May 25, 2012 Accepted: June 29, 2012 Published: June 29, 2012 6848

dx.doi.org/10.1021/ac301437m | Anal. Chem. 2012, 84, 6848−6855

Analytical Chemistry

Article

Figure 1. Base peak chromatograms from the analysis of 5 pmol of Rab1B-CVIM. These chromatograms refer to the 125 mM ammonium acetate salt step: (A) full MS; (B) XIC for the peptide IDSTPVKPAGGGCVIM; (C) XIC for the peptide IDSTPVKPAGGGCVIM(Ox); (D) XIC for the peptide IDSTPVKPAGGGC(Far)VIM; (E) XIC for the peptide IDSTPVKPAGGGC(Far)VIM(Ox). Far is farnesyl, NI is normalized intensity, Ox is oxidation, and RT is retention time.

(GGTase-I) catalyzes the analogous reaction with geranylgeranyldiphosphate (GGPP) as the geranylgeranyl group donor. The protein geranylgeranyltransferase type-II (GGTase-II), also called Rab geranylgeranyltransferase (RabGGTase), transfers usually two geranylgeranyl groups to the C-termini of Rab proteins exclusively.9−14 This reaction requires the Rab escort protein (REP), and only the Rab:REP complex is recognized by the RabGGTase.15 Farnesylated and geranylgeranylated proteins are further processed by the endoproteolytic cleavage of the aaX sequence and the subsequent methylation of the α-carboxyl group of the prenylated cysteine. Some farnesylated proteins, such as H- and N-Ras, receive a second lipid chain, a thioetherlinked palmitoyl group.16 The members of the Ras protein superfamily are the most common oncoproteins. Mutational activation of the Ras subfamily occurs in approximately 20% of human cancers. 17 Overexpression of Rab and Rho subfamilies was observed in tumors.18−20 Since Ras proteins need a farnesyl group for association with membranes and thus for the transduction of extracellular signals, farnesyltransferase inhibitors (FTIs) have been a promising strategy of anticancer therapeutics developed since 1990.21−23 They either compete with the substrates, CaaX box or FPP, or coordinate to catalytic zinc ions. Well-established FTIs are R115777 (tipifarnib), SCH66336 (lonafarnib), and BMS214662. Other inhibitors are L-778123, which inhibits both FTases and GGTases-I,24,25 and BMS1−4, which inhibit FTases and GGTases-II.26

Though protein prenylation has been vigorously studied over the past 20 years, systematic mass spectrometric analyses are rare. Hoffman and Kast analyzed two synthetic farnesylated peptides by means of ESI (electrospray ionization) and MALDI (matrixassisted laser desorption/ionization) MS/MS (tandem mass spectrometry). They discovered that upon fragmentation farnesylated peptides produce a neutral loss and/or a marker ion that equate the farnesyl group (204 Da) or the protonated farnesyl group (205 Da), respectively. Moreover, they identified farnesyl fragment ions at m/z 135, 149, and 163 that could further help to examine fragment ion spectra of possibly farnesylated peptides.27 The aim of our study was to determine whether or not farnesylated peptides can be separated from unfarnesylated peptides via MudPIT (multidimensional protein identification technology). Three FTase substrates, namely, Rab1B-CVIM, Rab3A-CVIM, and Rab6A-CVIM, were used for this purpose. In addition, digests of these proteins were separately spiked into a complex mixture, in this case increasing amounts of HeLa cell lysate. In all three cases farnesylated peptides were retained more strongly by the reversed-phase (RP) material than their unfarnesylated counterparts and therefore eluted later. This allows for the analysis of farnesylated peptides in complex mixtures, as the gradient can be adapted in such a way that an effective separation of farnesylated peptides is possible. Our approach made it possible to identify farnesylated peptides in increasing protein amounts (up to 50 μg) obtained from HeLa cells. 6849

dx.doi.org/10.1021/ac301437m | Anal. Chem. 2012, 84, 6848−6855

Analytical Chemistry

Article

Figure 2. Base peak chromatograms from the analyses of 5 pmol of Rab1B-CVIM in increasing amounts of HeLa cell proteins. These chromatograms refer to the 125 mM ammonium acetate salt step. The asterisks indicate the retention times of the farnesylated C-terminal peptide (blue) and the farnesylated C-terminal peptide with an oxidized methionine (green): (A) 5 μg of HeLa proteins; (B) 10 μg of HeLa proteins; (C) 20 μg of HeLa proteins; (D) 50 μg of HeLa proteins.

■

EXPERIMENTAL SECTION Biological Materials. Rab1B-CVIM, Rab3A-CVIM, Rab6ACVIM (see the Supporting Information for the amino acid sequences), and FTase were expressed in BL21(DE3) cells and purified by Ni−NTA affinity chromatography and gel filtration. HeLa cells were a kind gift from the Department of Biochemistry I, Ruhr-University of Bochum, Germany. AspN and trypsin were purchased from Promega (Madison, U.S.A.). DNase (deoxyribonuclease) I was obtained from Roche (Basel, Switzerland). Chemical Materials. Complete protease inhibitor tablets are commercially available from Roche (Basel, Switzerland). RapiGest SF surfactant was purchased from Waters Corporation (Milford, U.S.A.). Chromatographic Materials. Luna C18(2), particle size 3 μm, pore size 100 Å, from Phenomenex (Torrance, U.S.A.) was used as the RP material, and polysulfethyl A, particle size 5 μm, pore size 200 Å, from PolyLC (Columbia, U.S.A.) acted as the

strong cation-exchange (SCX) material. Deactivated fused-silica capillaries with an inner diameter (i.d.) of 100 μm were obtained from Polymicro Technologies (Phoenix, U.S.A.), and deactivated fused-silica capillaries with an i.d. of 180 μm were purchased from SMS (Idstein, Germany). Prenylation Reaction. The prenylation reaction was carried out as described by Nguyen et al.28 HeLa Cell Lysis. The HeLa cells were resuspended in lysis buffer (prenylation buffer with DNase I and complete protease inhibitors) and lysed by sonication. The lysate was then centrifuged for 10 min at 4500 rcf. Subsequently, the proteins were precipitated with the 10-fold volume of ice-cold ethanol at −28 °C overnight. The protein concentration was determined with the BCA (bicinchoninic acid) protein assay kit from Thermo Fisher Scientific (Waltham, U.S.A.). Protein Digestion. The proteins were digested with AspN (Rab3A-CVIM and Rab6A-CVIM) (1:20 w/w) or trypsin 6850

dx.doi.org/10.1021/ac301437m | Anal. Chem. 2012, 84, 6848−6855

Analytical Chemistry

Article

Figure 3. MS/MS spectrum of the doubly charged peptide IDSTPVKPAGGGC(Far)VIM. The singly charged b-ions are marked yellow, and the singly charged y-ions are marked blue. The neutral loss corresponding ion is located at 773.1171 m/z.

proteins were added, and the buffer B increase was reduced to 1.43% min−1. ESI-MS/MS. The HPLC pump was connected to an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, U.S.A.). Each MS run consisted of MS scans from 400 to 2000 m/z followed by MS/MS scans of the top 20 ions, which were fragmented using CID (collision-induced dissociation). The MS scans were performed in the Orbitrap using a resolution of 60 000, whereas the MS/MS scans were carried out in the ion trap. The mass window for the dynamic exclusion was set to ±10 ppm. The time for the dynamic exclusion was set to 100 ms. The isolation mass width was adjusted to 2 m/z. The mass spectrometer was operated in the positive ion mode. Data Analysis. The MS/MS data were searched with the Sequest algorithm implemented in the Thermo Proteome Discoverer 1.2 software (Thermo Fisher Scientific, Waltham, U.S.A.) against a database with entries of the three proteins and the FTase. The precursor and the fragment mass tolerances were set to ±10 ppm and ±1 Da, respectively. A maximum of two missed cleavage sites was allowed for the search. The oxidations of cysteine and methionine, as well as the farnesylation of cysteine, were included as variable modifications. The search results were filtered with a false discovery rate of 1% and a peptide probability of ≥20.

(Rab1B-CVIM and HeLa cell lysate) (1:50 w/w), respectively. A solution of 50 mM ammonium hydrogen carbonate with 0.05% RapiGest SF surfactant was used as the digest buffer. The digest batches were incubated under constant shaking at 37 °C overnight and subsequently acidified with 100% formic acid to pH 1. MudPIT. The MudPIT chromatography was performed according to Washburn et al.29 Modifications included the use of two different capillaries. The first capillary with an i.d. of 100 μm was packed with 10 cm RP material. A second capillary with an i.d. of 180 μm was first packed with 5 cm of SCX material and then with 3 cm of RP material. The capillaries were coupled via a nanofilter assembly with a 1 μm Ti frit (Upchurch Scientific, Oak Harbor, U.S.A.). The column was then connected to a quaternary Accela HPLC (high-performance/pressure liquid chromatography) pump (Thermo Fisher Scientific, Waltham, U.S.A.), and the buffer flow through the column was adjusted to 250−300 nL min−1. Each analysis started with an RP gradient generated with buffer B, followed by a 5 min long ammonium acetate salt step, and then again by an RP gradient. The steepness of the RP gradient and the number of the salt steps were adapted to the complexity and the chromatographic characteristics of the samples. Buffer A consisted of 0.1% formic acid and 2% acetonitrile, buffer B consisted of 0.1% formic acid and 80% acetonitrile, and the buffers C and D contained 250 mM and 1.5 M of ammonium acetate, respectively. First, each of the three proteins was analyzed without the addition of HeLa proteins. These analyses were run in triplicate. Afterward, the proteins were spiked into increasing amounts of HeLa proteins, whose quantities were 5, 10, 20, and finally 50 μg. In the case of Rab1BCVIM, 5 pmol (0.11 μg) was used for the analyses. The RP gradients exhibited an increase of 1.43% buffer B/min. The ammonium acetate concentrations for the salt steps were as follows: 125 mM, 250 mM, and 1.5 M. The same gradients were applied to the analyses with HeLa proteins. Amounts of 10 pmol (0.25 μg) of Rab3A-CVIM and 5 pmol (0.12 μg) of Rab6A-CVIM were used in each of the runs. The buffer B increase for both proteins was 3.33% min−1, and only one salt step with 1.5 M ammonium acetate was implemented. Two additional salt steps (125 and 250 mM) were included, when HeLa

■

RESULTS AND DISCUSSION Rab1B-CVIM. All three Rab proteins that were analyzed in this study represent mutants that make them suitable substrates for the FTase. The digests of these proteins were separated by means of MudPIT, where polysulfethyl A was used as SCX and C18 as reversed-phase HPLC resin. Due to the cleavage with trypsin the C-terminal peptide of Rab1B-CVIM exhibited the sequence IDSTPVKPAGGGCVIM. It was mostly doubly charged, and the methionine was oxidized in approximately 50% of the cases. Typically the peptide eluted after the 125 mM ammonium acetate salt step. Figure 1 shows exemplarily the base peak chromatograms of one of the three initial runs. Figure 1A depicts the base peak chromatogram of all m/z values, whereas parts B−E of Figure 1 display the XICs (extracted ion chromatograms) of the doubly protonated C-terminal peptide with an oxidized methionine (Figure 1, parts C

6851

dx.doi.org/10.1021/ac301437m | Anal. Chem. 2012, 84, 6848−6855

Analytical Chemistry

Article

Figure 4. Base peak chromatograms from the analyses of 10 pmol of Rab3A-CVIM in increasing amounts of HeLa cell proteins. These chromatograms refer to the 1.5 M ammonium acetate salt step. The asterisks indicate the retention times of the farnesylated C-terminal peptide (blue) and the farnesylated C-terminal peptide with an oxidized methionine (green): (A) 5 μg of HeLa proteins; (B) 10 μg of HeLa proteins; (C) 20 μg of HeLa proteins; (D) 50 μg of HeLa proteins. The peptide DQQAPPHKC(Far)VIM(Ox) could not be detected in this case.

and E) and a farnesylated cysteine (Figure 1, parts D and E), respectively. For the nonfarnesylated peptides the monoisotopic m/z values of 772.89472 and 780.89218 (oxidized methionine) were used to generate the XICs, whereas for the farnesylated peptides the m/z values of 874.98862 and 882.98608 (oxidized methionine) were employed. In each case the mass tolerance accounted for ±10 ppm. Figure 1A shows that the most intensive ions eluted between minutes 30 and 75. As expected, the farnesylated peptides eluted relatively late (57.85 and 61.08 min) due to the strong interaction of the hydrophobic farnesyl group with the RP material. It first seems that the nonfarnesylated peptides (Figure 1, parts B and C) eluted at the same time as their farnesylated counterparts. However, two other intensive peaks can be recognized at an earlier stage of the chromatogram, namely, after 40.18 and 42.79 min. These two retention times would meet our expectation for nonfarnesylated peptides. One explanation for the peaks at 57.87 and 61.12 min in parts B and C of Figure 1 would be

that the farnesyl group is split off the peptide during the ionization process. The relatively low intensity of these peaks suggests that this event is rather uncommon. The identical peak forms between parts B and D and C and E of Figure 1 back the theory of the farnesyl neutral loss during the ESI procedure. This phenomenon was observed in all three replicates of Rab1B-CVIM (see Supporting Information Figures S-1 and S-2). The low intensities of the peaks at 40.18 and 42.79 min in Figure 1, parts B and C, are an indicator for the almost quantitative conversion of the Rab1B-CVIM proteins during the farnesylation process. After the three initial runs the protein was spiked into increasing amounts of HeLa cell proteins. The base peak chromatograms of these measurements are depicted in Figure 2. The amount of HeLa proteins was increased from 5 (Figure 2A) to 10 (Figure 2B), 20 (Figure 2C), and finally 50 μg (Figure 2D). The asterisks indicate the retention times of the farnesylated peptide with an oxidized methionine (green) and the farnesylated 6852

dx.doi.org/10.1021/ac301437m | Anal. Chem. 2012, 84, 6848−6855

Analytical Chemistry

Article

Figure 5. Base peak chromatograms from the analysis of 5 pmol of Rab6A-CVIM in increasing amounts of HeLa cell proteins. These chromatograms refer to the 1.5 M ammonium acetate salt step. The asterisks indicate the retention times of the farnesylated C-terminal peptide (blue) and the farnesylated C-terminal peptide with an oxidized methionine (green): (A) 5 μg of HeLa proteins; (B) 10 μg of HeLa proteins; (C) 20 μg of HeLa proteins; (D) 50 μg of HeLa proteins. The peptide DIKLEKPQEQPVSEKC(Far)VIM(Ox) was not observed in this case.

was about half the intensity of the most intensive fragment ion in the MS/MS spectrum. A marker ion that equates the protonated farnesyl group could not be detected in this case, because it lies outside of the measuring range. Rab3A-CVIM. Analogous to the measurements with Rab1BCVIM, Rab3A-CVIM was initially analyzed without the addition of HeLa proteins. As stated in the Experimental Section, 10 pmol of this protein was used to achieve similar relative intensities as in the case of Rab1B-CVIM. The reason for this circumstance is the use of a different protease, namely, AspN, instead of trypsin. AspN seems to be sterically hindered by the farnesyl group and thus often misses the cleavage site next to the C-terminus. This results in a mixture of different farnesylated C-terminal peptides and the farnesylated C-terminal peptide without any missed cleavage sites (DQQAPPHKCVIM) being less abundant.

peptide with the unmodified methionine (blue). The ions eluted roughly between minutes 25 and 65, the farnesylated peptides being usually the last eluting ions. These experiments show that the identification of low quantities of farnesylated peptides in a complex mixture is possible without an enrichment step prior to the HPLC− MS analysis. Aside from the liquid chromatographic characteristics we also investigated the mass spectrometric attributes of farnesylated peptides. Figure 3 shows an MS/MS spectrum of the doubly protonated peptide IDSTPVKPAGGGC(Far)VIM. The singly charged b-ions are annotated in yellow, and the singly charged y-ions are annotated in blue. According to Hoffman and Kast farnesylated peptides produce a neutral loss and/or a marker ion upon fragmentation.27 The neutral loss corresponding ion [M + 2H − Far]2+ can be observed at 773.1171 m/z. Usually this ion 6853

dx.doi.org/10.1021/ac301437m | Anal. Chem. 2012, 84, 6848−6855

Analytical Chemistry

■

The peptide DQQAPPHKCVIM was mostly doubly charged and sometimes oxidized at the methionine residue. As a consequence, the two farnesylated peptide species amounted to 785.92837 (unmodified methionine) and 793.92583 m/z (oxidized methionine). The farnesylated peptides typically eluted after the 1.5 M ammonium acetate salt step. Again the phenomenon of the farnesyl neutral loss during the ESI procedure could be observed in all three replicates (see Supporting Information Figures S-3−S-5). Figure 4 shows the base peak chromatograms from the analyses of 10 pmol of Rab3A-CVIM in increasing amounts of HeLa cell proteins. The farnesylated peptides eluted relatively late and long after the majority of other peptides. No farnesylated peptides with an oxidized methionine were identified after the addition of 50 μg of HeLa proteins (Figure 4D). The MS/MS spectrum is displayed in Supporting Information Figure S-6. Again a possible marker ion lies outside of the measuring range. The neutral loss corresponding ion can be seen at 684.0593 m/z. Rab6A-CVIM. Other than in the case of Rab3A-CVIM, 5 pmol of Rab6A-CVIM was sufficient for the analyses, though both proteins were digested with the same protease. The expected C-terminal peptide of Rab6A-CVIM (DIKLEKPQEQPVSEKCVIM) is longer than the expected C-terminal peptide of Rab3A-CVIM, and therefore, AspN is obviously not sterically hindered by the farnesyl moiety any longer. The peptide DIKLEKPQEQPVSEKCVIM was generally triply charged and sometimes had an oxidized methionine, thus resulting in m/z values of 806.78493 and 812.11657 (oxidized methionine). These peptides typically eluted after the 1.5 M ammonium acetate salt step. The farnesyl neutral loss phenomenon during the ionization process can be viewed in Supporting Information Figures S-7−S-9. The results of the spiking experiments are depicted in Figure 5. Similarly to Rab3ACVIM, the farnesylated peptides can be observed in the final phase of the elution. The peptide DIKLEKPQEQPVSEKC(Far)VIM(Ox) could not be identified in the case of 50 μg of HeLa proteins (Figure 5D). Supporting Information Figure S-10 shows the tandem mass spectrum for the peptide DIKLEKPQEQPVSEKC(Far)VIM. The neutral loss corresponding ion can be seen at 739.0054 m/z. A potential marker ion lies outside of the measuring range.

Article

ASSOCIATED CONTENT

S Supporting Information *

Amino acid sequences of Rab1B-, Rab3A-, and Rab6A-CVIM, base peak chromatograms from the analyses of Rab1B-, Rab3A-, and Rab6A-CVIM without the addition of HeLa cell proteins, and tandem mass spectra of the farnesylated peptides from Rab3A- and Rab6A-CVIM. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +49 (0) 234-32-25463. Fax: +49 (0) 234-32-14742. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We are grateful to the German Research Foundation (DFG) for generous funding in the SFB 642 initiative. We also thank Professor Dr. Rolf Heumann from the Ruhr-University of Bochum for the kind gift of HeLa cells.

■

REFERENCES

(1) Yokoyama, K.; Goodwin, G. W.; Ghomashchi, F.; Glomset, J. A.; Gelb, M. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88 (12), 5302−5206. (2) Reid, T. S.; Terry, K. L.; Casey, P. J.; Beese, L. S. J. Mol. Biol. 2004, 343 (2), 417−433. (3) Farnsworth, C. C.; Seabra, M. C.; Ericsson, L. H.; Gelb, M. H.; Glomset, J. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91 (25), 11963−11967. (4) Gelb, M. H.; Reiss, Y.; Ghomashchi, F.; Farnsworth, C. C. Bioorg. Med. Chem. Lett. 1995, 5 (8), 881−886. (5) Sakagami, Y.; Isogai, A.; Suzuki, A.; Tamura, S.; Tsuchiya, E.; Fukui, S. Agric. Biol. Chem. 1978, 42 (5), 1093−1094. (6) Tsuchiya, E.; Fukui, S.; Kamiya, Y.; Sakagami, Y.; Fujino, M. Biochem. Biophys. Res. Commun. 1978, 85 (1), 459−463. (7) Gelb, M. H.; Brunsveld, L.; Hrycyna, C. A.; Michaelis, S.; Tamanoi, F.; Van Voorhis, W. C.; Waldmann, H. Nat. Chem. Biol. 2006, 2 (10), 518−528. (8) Pylypenko, O.; Rak, A.; Durek, T.; Kushnir, S.; Dursina, B. E.; Thomae, N. H.; Constantinescu, A. T.; Brunsveld, L.; Watzke, A.; Waldmann, H.; Goody, R. S.; Alexandrov, K. EMBO J. 2006, 25 (1), 13− 23. (9) Yokoyama, K.; Goodwin, G. W.; Ghomashchi, F.; Glomset, J.; Gelb, M. H. Biochem. Soc. Trans. 1992, 20 (2), 489−494. (10) Casey, P. J.; Seabra, M. C. J. Biol. Chem. 1996, 271 (10), 5289− 5292. (11) Taylor, J. S.; Reid, T. S.; Terry, K. L.; Casey, P. J.; Beese, L. S. EMBO J. 2003, 22 (22), 5963−5974. (12) Long, S. B.; Casey, P. J.; Beese, L. S. Nature 2002, 419 (6907), 645−650. (13) Zhang, H.; Seabra, M. C.; Deisenhofer, J. Structure 2000, 8 (3), 241−251. (14) Hicks, K. A.; Hartman, H. L.; Fierke, C. A. Biochemistry 2005, 44 (46), 15325−15333. (15) Leung, K. F.; Baron, R.; Seabra, M. C. J. Lipid Res. 2006, 47 (3), 467−475. (16) Hancock, J. F. Nat. Rev. Mol. Cell Biol. 2003, 4 (5), 373−384. (17) Bos, J. L. Cancer Res. 1989, 49 (17), 4682−4689. (18) Sahai, E.; Marshall, C. J. Nat. Rev. Cancer 2002, 2 (2), 133−142. (19) Cheng, K. W.; Lahad, J. P.; Kuo, W. L.; Lapuk, A.; Yamada, K.; Auersperg, N.; Liu, J.; Smith-McCune, K.; Lu, K. H.; Fishman, D.; Gray, J. W.; Mills, G. B. Nat. Med. 2004, 10 (11), 1251−1256. (20) He, H.; Dai, F.; Yu, L.; She, X.; Zhao, Y.; Jiang, J.; Chen, X.; Zhao, S. Gene Expression 2002, 10 (5−6), 231−242. (21) Tamanoi, F.; Gau, C. L.; Jiang, C.; Edamatsu, H.; KatoStankiewicz, J. Cell. Mol. Life Sci. 2001, 58 (11), 1636−1649.

■

CONCLUSIONS We analyzed the digests of three different farnesylated proteins by means of HPLC−ESI-MS/MS. Farnesylated peptides elute later than nonfarnesylated peptides and therefore can be identified by mass spectrometry, even if being low abundant. This enables the detection of farnesylated peptides in complex peptide samples containing up to 50 μg of digested HeLa cell proteins. Apparently, the farnesyl group was occasionally split off the peptide during the ionization process. In addition, farnesylated peptides produced a farnesyl neutral loss upon fragmentation. Our study provides data for the fundamental understanding of the behavior of farnesylated peptides in HPLC−ESI-MS/MS analyses and is therefore the basis for further experiments, such as the elucidation of the prenylome in response to inhibitor stimulation. Although other enrichment steps have been proven to be powerful approaches for the analyses of prenylated GTPases,28,30 an unbiased direct analysis might be beneficial in the future. 6854

dx.doi.org/10.1021/ac301437m | Anal. Chem. 2012, 84, 6848−6855

Analytical Chemistry

Article

(22) Basso, A.; Kirshmeier, P.; Bishop, W. R. J. Lipid Res. 2006, 47, 15− 31. (23) Reid, T. S.; Beese, L. S. Biochemistry 2004, 43 (22), 6877−6884. (24) Lobell, R. B.; Liu, D.; Buser, C. A.; Davide, J. P.; DePuy, E.; Hamilton, K.; Koblan, K. S.; Lee, Y.; Mosser, S.; Motzel, S. L.; Abbruzzese, J. L.; Fuchs, C. S.; Rowinsky, E. K.; Rubin, E. H.; Sharma, S.; Deutsch, P. J.; Mazina, K. E.; Morrison, B. W.; Wildonger, L.; Yao, S. L.; Kohl, N. E. Mol. Cancer Ther. 2002, 1 (9), 747−758. (25) Reid, T. S.; Long, S. B.; Beese, L. S. Biochemistry 2004, 43 (28), 9000−9008. (26) Lackner, M. R.; Kindt, R. M.; Carroll, P. M.; Brown, K.; Cancilla, M. R.; Chen, C.; de Silva, H.; Franke, Y.; Guan, B.; Heuer, T.; Hung, T.; Keegan, K.; Lee, J. M.; Manne, V.; O’Brien, C.; Parry, D.; Perez-Villar, J. J.; Reddy, R. K.; Xiao, H.; Zhan, H.; Cockett, M.; Plowman, G.; Fitzgerald, K.; Costa, M.; Ross-Macdonald, P. Cancer Cell 2005, 7 (4), 325−336. (27) Hoffman, M. D.; Kast, J. J. Mass Spectrom. 2006, 41 (2), 229−241. (28) Nguyen, U. T.; Guo, Z.; Delon, C.; Wu, Y.; Deraeve, C.; Franzel, B.; Bon, R. S.; Blankenfeldt, W.; Goody, R. S.; Waldmann, H.; Wolters, D.; Alexandrov, K. Nat. Chem. Biol. 2009, 5 (4), 227−235. (29) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19 (3), 242−247. (30) Kho, Y.; Kim, S. C.; Jiang, C.; Barma, D.; Kwon, S. W.; Cheng, J.; Jaunbergs, J.; Weinbaum, C.; Tamanoi, F.; Falck, J.; Zhao, Y. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (34), 12479−12484.

6855

dx.doi.org/10.1021/ac301437m | Anal. Chem. 2012, 84, 6848−6855