Low-Energy Collision-Induced Dissociation Fragmentation Analysis of

The development of methods to chemically modify and isolate cysteinyl-residue-containing peptides (Cys-peptides) for LC−MS/MS analysis has generated...
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Anal. Chem. 2002, 74, 2284-2292

Low-Energy Collision-Induced Dissociation Fragmentation Analysis of Cysteinyl-Modified Peptides Oleg V. Borisov, Michael B. Goshe, Thomas P. Conrads,# V. Sergey Rakov, Timothy D. Veenstra,*,# and Richard D. Smith*

Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, MSIN: K8-98, Richland, Washington 99352

The development of methods to chemically modify and isolate cysteinyl-residue-containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotopecoded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolatespecific biotin group to modify and isolate Cys-containing peptides in conjunction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of Deinococcus radiodurans, the presence of these label-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys residue, and to differentiate identical Cys-peptides labeled with either ICAT-d0 or ICAT-d8. Proteomics can formally be defined as the complete characterization of the entire protein complement expressed by a cell at a given time under a specific set of conditions; however, most individual proteomic studies focus on characterizing one or two specific aspects of a cell’s proteome. Some of the more prevalent characteristics being studied include the identification of the expressed proteins,1-3 sites of posttranslational modifications,4,5 # Present address: NCI Biomedical Proteomics Program/Analytical Chemistry Laboratory, National Cancer Institute at Frederick, P.O. Box B, Frederick MD 21702-1201. (1) Zivy, M.; de Vienne, D. Plant Mol. Biol. 2000, 44, 575-580.

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and quantitation of relative protein abundances between two different proteomes.6-8 Identification of the expressed proteins within a proteome, and the posttranslationally modified sites within them, is primarily accomplished by initially fractionating the proteins and then using mass spectrometry (MS) for identification. Most strategies of identification by MS rely on the analysis of enzymatically produced peptides followed by either peptide mapping or tandem MS (MS/MS) to obtain sequence information for a single peptide.9,10 Peptide mapping is generally limited to cases where the observed peptide masses are known to originate from a single isolated protein and requires several peptide masses to unambiguously identify a protein with the typically achieved mass measurement accuracies. In the case of MS/MS, however, the sequence information obtained for a single peptide is often sufficient for unambiguous protein identification. The use of MS/ MS enables the identification of peptides from complex mixtures of proteins and does not require the exhaustive separation necessary for peptide mapping. As mentioned above, the quantitation of relative protein abundances between two proteome samples is an active area in proteomics. In conventional two-dimensional polyacrylamide gel electrophoresis analysis of proteomes, two distinct proteome samples are separated on two separate gels, and the differences in protein abundances are measured by comparing the intensities of the Coomassie or silver-stained protein spots.11,12 Recently, other techniques involving metabolic labeling of proteins with an (2) Taylor, R. S.; Wu, C. C.; Hays, L. G.; Eng, J. K.; Yates, J. R.; Howell, K. E. Electrophoresis 2000, 21, 3441-3459. (3) Kaji, H.; Tsuji, T.; Mawuenyega, K. G.; Wakamiya, A.; Taoka, M.; Isobe, T. Electrophoresis 2000, 21, 1755-1765. (4) Hanisch, F. G.; Jovanovic, M.; Peter-Katalinic, J. Anal. Biochem. 2001, 290, 47-59. (5) Yan, J. X.; Sanchez, J. C.; Binz, P. A.; Williams, K. L.; Hochstrasser, D. F. Electrophoresis 1999, 20, 749-754. (6) Gygi, S. P.; Rist, B.; Aebersold, R. Curr. Opin. Biotechnol. 2000, 11, 396401. (7) Seow, T. K.; Ong, S. E.; Liang, R. C. M. Y.; Ren, E. C.; Chan, L.; Ou, K.; Chung, M. C. M. Electrophoresis 2000, 21, 1787-1813. (8) Yu, L. R.; Zeng, R.; Shao, X. X.; Wang, N.; Xu, Y. H.; Xia, Q. C. Electrophoresis 2000, 21, 3058-3068. (9) Conrads, T. P.; Anderson, G. A.; Veenstra, T. D.; Pasa-Tolic, L.; Smith, R. D. Anal. Chem. 2000, 72, 3349-3354. (10) Appella, E.; Arnott, D.; Sakaguchi, K.; Wirth, P. J. EXS 2000, 88, 1-27. (11) Celis, J. E.; Kruhoffer, M.; Gromova, I.; Frederiksen, C.; Ostergaard, M.; Thykjaer, T.; Gromov, P.; Yu, J.; Palsdottir, H.; Magnusson, N.; Orntoft, T. F. FEBS Lett. 2000, 480, 2-16. 10.1021/ac010974p CCC: $22.00

© 2002 American Chemical Society Published on Web 04/09/2002

isotopically defined medium have been developed.13-15 These methods are attractive because two isotopically distinct versions of each peptide are used in the analysis to effectively provide an internal standard to measure relative protein abundance. The isotope-coded affinity tag (ICAT) method developed by Aebersold and co-workers differs from metabolic labeling in that the stable isotope is incorporated within the Cys-labeling reagent.16 The ICAT method involves the affinity isolation of Cys-containing peptides that have been chemically modified with a molecule that contains a linker arm connecting a thiolate-reactive group to a biotin moiety. We have recently developed a method that combines metabolic labeling with a Cys-peptide capture and affinity isolation reagent, (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetylPEO-biotin), which is structurally similar to the ICAT reagent.14 Because both methods label Cys residues and use immobilized avidin to isolate the biotinylated Cys-peptides, the complexity of the proteome mixture is decreased and also potentially provides a useful identification constraint, since only Cys-peptides are modified and isolated. An important consideration when using any kind of covalent modifying reagent in combination with mass spectrometry and database searching is the completeness of the reaction (i.e., is every targeted site modified) as well as the effect on the fragmentation of the derivatized peptides and the modifying reagent itself. Peptides have predictable and well-characterized fragmentation patterns, and most identification software, such as SEQUEST,17 use algorithms based on the observation of these product ions. Although the ICAT procedure has generated much publicity in proteomics, there are relatively few publications detailing many of its aspects regarding peptide identification. For these Cys modifications to be useful for MS analysis, they should have a minimal effect on the overall fragmentation of the modified peptides and not interfere in peptide identification using automated database searching. To further characterize the behavior of the ICAT and acetyl-PEO-biotin tags under varying CID conditions and how it affects peptide identification using database searching, we have investigated the fragmentation of several acetyl-PEO-biotin-modified peptides originating from the derivatization of individual Cys-peptides, a single protein (ribonuclease A), and a proteome sample from Deinococcus radiodurans. The results of these experiments revealed that several label-specific product ions could be used to ascertain whether a peptide contains the Cys modification. The ramifications of using these labelspecific product ions as markers to distinguish between modified and unmodified peptides using Cys-capture reagents for proteomewide analyses is also discussed. (12) Jungblut, P. R.; Bumann, D.; Haas, G.; Zimny-Arndt, U.; Holland, P.; Lamer, S.; Siejak, F.; Aebischer, A.; Meyer, T. F. Mol. Microbiol. 2000, 36, 710725. (13) Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6591-6596. (14) Conrads, T. P.; Alving, K.; Veenstra, T. D.; Belov, M. E.; Anderson, G. A.; Anderson, D. J.; Lipton, M. S.; Pasˇa-Tolic´, L.; Udesth, H. R.; Chrisler, W. B.; Thrall, B. D.; Smith, R. D. Anal. Chem. 2001, 73, 2132-2139. (15) Gao, H. Y.; Shen, Y. F.; Veenstra, T. D.; Harkewicz, R.; Anderson, G. A.; Bruce, J. E.; Pasˇa-Tolic´, L.; Smith, R. D. J. Micro. Sep. 2000, 12, 383-390. (16) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (17) Yates, J. R.; Eng, J. K.; McCormack, A. L. Anal. Chem. 1995, 67, 32023210.

EXPERIMENTAL PROCEDURES Materials. The materials used in all experiments were obtained from commercially available sources and used without further purification unless otherwise noted. Laminin was obtained from Perseptive Biosystems (Foster City, CA), six Cys-containing peptides were obtained from Bachem (Torrance, CA). Ribonuclease A (RNase A) was obtained from Sigma (St. Louis, MO). (+)-Biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetylPEO-biotin) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP‚HCl) were obtained from Pierce (Rockford, IL). Acetonitrile (HPLC grade), methanol, and glacial acetic acid (ACS reagent grade) were purchased from Aldrich (Milwaukee, WI), and trifluoroacetic acid (TFA) (HPLC grade) was purchased from Sigma (St. Louis, MO). Water was purified using a Barnstead Nanopure Infinity water purification system (Dubuque, IA). Labeling Cys-Peptides and RNase A with Iodoacetyl-PEObiotin. Lyophilized Cys-peptides were dissolved in 50 mM NH4HCO3, pH 8.2. Cysteinyl residues were labeled using a 3 molar excess of iodoacetyl-PEO-biotin with constant stirring in the dark for 90 min at ambient temperature. After biotinylation, the labeled peptides were immediately frozen, lyophilized, and stored at -20 °C. Further purification of the biotinylated peptides was performed using Microcon-SCX microconcentrators (Millipore, Bedford, MA) employing a strong cation-exchange membrane. A stock solution of 4 mg/mL of RNase A was prepared by dissolving lyophilized protein in 0.1 M NH4HCO3, pH 8.2, containing 6 M guanidine hydrochloride (GdnHCl). Disulfides were reduced by adding a 5 molar excess of TCEP‚HCl over cysteinyl residues and incubating the sample for 1 h at 37 °C. One-half of the sample (2 mg) was biotinylated using a 5 molar excess of iodoacetyl-PEO-biotin with constant stirring in the dark for 90 min at ambient temperature; the other half remained unlabeled. Buffer exchange into 0.1 M NH4HCO3, pH 8.2, was performed by gel filtration using prepacked PD-10 columns containing Sephadex G-25 (Amersham Pharmacia Biotech, Piscataway, NJ). The samples were then digested with trypsin (Promega, Madison, WI) using a 1:100 (w/w) trypsin-to-protein ratio at 37 °C overnight. Both samples were subsequently dried down and stored at -20 °C. Growth of D. radiodurans. D. radiodurans R1 was cultured in TGY medium (2.5 g tryptone, 0.5 g glucose, 0.5 g yeast extract in 0.5 L of water) and grown at 30 °C with shaking at 225 rpm. At an OD600 nm of 1.0, the cells were harvested by centrifugation at 10000g for 30 s. The cells were resuspended in 200 µL of PBS (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2) and lysed by bead beating in the presence of 0.1 mm zirconium beads for three cycles of 60 s at 5000 rpm. Between cycles of bead beating, the samples were kept on ice for 60 s. The cell lysate was recovered and centrifuged at 10000g for 10 min to remove any cell debris. Labeling and Affinity Isolation of Cys-Peptides of D. radiodurans. The soluble proteins extracted from D. radiodurans were desalted into 50 mM Tris‚HCl, 5 mM EDTA, pH 8.4, and the protein concentration was measured using the Biuret assay. GdnHCl was added to a final concentration of 6 M, and the samples were reduced by adding tributylphosphine (5 mM) with a 1 h incubation at 37 °C. The protein sample was separated into two equal aliquots and labeled with ICAT-d0 or ICAT-d8, as described. ICAT was added to each sample in a 5-fold excess over Analytical Chemistry, Vol. 74, No. 10, May 15, 2002

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cysteinyl residues (calculated assuming 1 mg of protein equals 30 nmol and 6 Cys residues/protein). The reaction mixture was incubated with stirring for 90 min in the dark. Both ICAT derivatized samples were pooled and then desalted into 0.1 M NH4HCO3, 5 mM EDTA, pH 8.4, and digested with trypsin (1:50 trypsin-to-protein ratio) overnight at 37 °C. After the digestion was complete, phenylmethylsulfonylfluoride was added to produce a final concentration of 1 mM. A packed avidin column (2 mL bed volume) was prepared and equilibrated with PBS. The column was blocked with 2 mM biotin in PBS (pH 7.2), and reversible binding sites were stripped from the column using 0.1 M glycine (pH 2.8). The column was reequilibrated with 2× PBS (pH 7.2). After the ICAT-d0/d8-labeled peptide mixture was heated at 100 °C for 5 min, it was loaded onto the avidin column. The loaded avidin column was incubated at room temperature for 30 min. After washing the column with 5 bed volumes of 2× PBS (pH 7.2) followed by 5 bed volumes of 50 mM NH4HCO3 (pH 8.4), the bound Cys-peptides were eluted using 30% acetonitrile with 0.4% TFA. Capillary HPLC Methods. LC-MS analysis of the RNase A sample was performed using an Agilent 1100 Series capillary LC system (Agilent Technologies, Inc., Palo Alto, CA) coupled directly on-line with an LCQ ion-trap mass spectrometer using an in-housemanufactured ESI interface. The reversed-phase capillary HPLC column contained 5-µm Jupiter C18 stationary phase (Phenomenex, Torrence, CA) packed into a 360 µm o.d. × 150 µm i.d. × 30 cm length capillary (Polymicro Technologies Inc., Phoenix, AZ). The mobile phase consisted of (A) 0.05% acetic acid and 0.1% TFA with 5% acetonitrile in water and (B) 0.05% acetic acid and 0.1% TFA in acetonitrile. After injecting a sample volume of 8 µL onto the reversed-phase capillary HPLC column, the mobile phase was held at 95% A for 10 min, followed by a linear gradient to 80% B over 90 min at a flow rate of 1.5 µL/min. The peptide mixture from the ICAT-labeled D. radiodurans sample isolated using immobilized avidin chromatography was separated by capillary LC using a system consisting of two HPLC syringe pumps (Isco, Inc. Lincoln, NE) for gradient elution of the peptides at a flow rate of ∼1 µL/min, as previously described.18 A sample volume of 10 µL was loaded onto a 360 µm o.d. × 150 µm i.d. × 60 cm length capillary (Polymicro Technologies, Phoenix, AZ) packed with 5-µm Jupiter C18 stationary phase. The mobile phase consisted of (A) 0.2% acetic acid and 0.05% TFA in water and (B) 0.1% TFA with 90% acetonitrile in water. MS Protocols. To study the effects of labeling on peptide stability during CID, an LCQ ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA) was used. The peptides were dissolved in 50% methanol and 1% acetic acid in H2O to achieve a concentration of ∼50 µg/mL and directly infused into the LCQ at a flow rate of 1 µL/min using an in-house-manufactured ESI interface. For MS/MS CID experiments, the LCQ software supplied with the instrument provides a user-controlled parameter called “normalized collision energy.” It was used in our MS/MS experiments as a measure of collisional excitation in CID. It should be noted that the “normalized collision energy” setting does not directly correspond to the actual collision energy (center-of-mass, or lab(18) Shen, Y. F.; Zhao, R.; Belov, M. E.; Conrads, T. P.; Anderson, G. A.; Tang, K. Q.; Pasˇa-Tolic´, L.; Veenstra, T. D.; Lipton, M. S.; Udseth, H. R.; Smith, R. D. Anal. Chem 2001, 73, 1766-1775.

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frame), the peak-to-peak voltage applied to the end caps of the trap, or the internal energy gained by ions from collisions with neutral gas. Thus, we will refer to this software parameter as “CID amplitude”. On the basis of this CID amplitude setting, the software calculates the peak-to-peak voltage of the end caps (Vp-p) according to the relationship

Vp-p )

NCE × [a(m/z) + b] 30

(1)

where NCE is the “normalized collision energy” setting and m/z is the mass-to-charge ratio. The values of a and b (slope and intercept, respectively) are set during factory calibration and are accessible in the operating software. The data acquisition sequence used for all LC-MS/MS analyses involved a full MS scan followed by three MS/MS scans, where the three most intense ions are dynamically selected for CID from the precursor MS scan. In a standard MS/MS experiment, the CID amplitude was set to 28, excitation time was 30 ms, and the q value was kept at 0.250. Peptides were identified by searching the MS/MS spectra against the appropriate databases using the computer program SEQUEST.17 RESULTS AND DISCUSSION The Effect of the Acetyl-PEO-biotin Label on Peptide Fragmentation. Unmodified and acetyl-PEO-biotin-labeled laminin (CDPGYIGSR) were analyzed by ESI-MS using direct infusion. CID of unmodified and labeled [M + H]+ precursor ions (m/z 967.3 and 1381.5, respectively) results in selective cleavage across the Asp-Pro amide bond for both peptides as shown in Figure 1A,B. The predominance of the y7+ ion agrees with the known fragility of this peptide bond.19-21 In the modified peptide, an additional product ion at m/z 1007 was observed and corresponds to the neutral loss of 374 u from the singly charged parent ion, a result of the partial fragmentation of the PEO-biotin label (discussed below). The addition of a second proton has a dramatic effect on the fragmentation of unmodified and acetyl-PEO-biotin labeled-laminin and can be explained in terms of a “mobile proton” model.22,23 Although cleavage along the Asp-Pro amide bond is still preferred during CID of the [M + 2H]2+ species, a greater number of product ions are observed for each peptide, as shown in Figure 1C,D. Cleavage along this bond in unmodified laminin produces predominantly the y72+ ion at m/z 375. In contrast, the Cys-labeled peptide produces complimentary b2+ and y7+ ions at m/z 633 and 749, respectively, with the intact acetyl-PEO-biotin moiety covalently bound to the b2+ ion. For both [M + 2H]2+ species, one of the charge-carrying protons is retained by the Arg residue; thus, the location of the other proton will determine the formation of (19) Yu, W.; Vath, J. E.; Huberty, M. C.; Martin, S. A. Anal. Chem. 1993, 65, 3015-3023. (20) Gu, C. G.; Tsaprailis, G.; Breci, L.; Wysocki, V. H. Anal. Chem. 2000, 72, 5804-5813. (21) Tsaprailis, G.; Nair, H.; Somogyi, A.; Wysocki, V. H.; Zhong, W. Q.; Futrell, J. H.; Summerfield, S. G.; Gaskell, S. J. J. Am. Chem. Soc. 1999, 121, 51425154. (22) Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. J. Mass Spectrom. 2000, 35, 1399-1406. (23) Reid, G. E.; Wu, J.; Chrisman, P. A.; Wells, J. M.; McLuckey, S. A. Anal. Chem. 2001, 73, 3274-3281.

Figure 1. MS/MS spectra of labeled and unlabeled laminin. Collision-induced dissociation (CID) spectra of singly charged (A) unmodified laminin (CDPGYIGSR) and (B) acetyl-PEO-biotin-labeled laminin (C*DPGYIGSR) and those of doubly charged (C) unmodified and (D) acetylPEO-biotin-labeled laminin peptides are shown.

the y72+ ion or the b2+ and y7+ ion pair. For unmodified laminin, the second proton is localized on the carboxy-terminal side of the cleaved Asp-Pro amide bond, favoring y72+ ion formation. For the labeled peptide, the second proton is positioned on the aminoterminal side, promoting b2+ ion formation. It is likely that the presence of the label on the Cys residue contributes to proton delocalization. The similarity in the summed intensities of the product ions resulting from the Asp-Pro amide bond cleavage (i.e., y72+ vs b2+ and y7+) suggests that the presence of the acetyl-PEObiotin group on the peptide does not affect the overall yield of product ions produced by this bond cleavage relative to other fragmentation channels. The ability of the acetyl-PEO-biotin tag to delocalize protons was also observed for other Cys-labeled peptides. The MS/MS spectra of both unmodified and labeled peptides, VTCG and GRGDSC, are presented in Figure 2. For the [M + H]+ ion of the unmodified VTCG peptide (Figure 2A), the proton is localized at the amino terminus, as evident by the preferential cleavage of the Thr-Cys amide bond to form the b2+ product ion. Although the labeled peptide cleaves along the same amide bond, the y2+ ion is preferentially produced (Figure 2B). These data suggest that the acetyl-PEO-biotin label out-competes the amino terminus for the proton and promotes the formation of the y2+ ion. In Figure 2C, the MS/MS spectrum for the GRGDSC peptide is dominated by b ions, consistent with the Arg residue as the probable site of protonation. However, for the labeled version, y fragments are more abundant, indicating partial localization of a proton on the label. It is likely that the lone electron pairs present on the nitrogen and oxygen atoms of the acetyl-PEO-biotin label facilitate its protonation.

Identification of Characteristic Acetyl-PEO-biotin Fragments. For all MS/MS spectra produced by CID of the labeled and unlabeled Cys-peptides studied, a series of peaks were consistently observed for only the modified versions of each peptide. For example, along with the formation of peptide-specific fragments, peaks corresponding to the neutral losses of 269, 331, and 374 u and product ions at m/z 227, 270, 332, 375, and 449 were observed during CID of acetyl-PEO-labeled peptides (Figures 1D, 2B,D). The formation of these ions is attributed to the partial fragmentation of the acetyl-PEO-biotin label, as shown in Figure 3. Although fragmentation of the label occurs to some extent, the majority of the Cys-labeled residue remains intact to permit identification. The Effect of CID Amplitude on Peptide Fragmentation. To investigate the effect of ion-trap CID amplitude on fragmentation efficiencies for unmodified and acetyl-PEO-labeled peptides, MS/MS experiments were conducted at various CID amplitude settings. Figure 4A shows the intensity of the y7+ ion of the [M + H]+ precursor of unmodified and acetyl-PEO-biotin-labeled laminin as a function of the CID amplitude. For [M + H]+ precursor ions, the formation of the y7+ ion, a result of Asp-Pro amide bond cleavage, is not significantly affected by the presence of the label. The partial fragmentation of the acetyl-PEO-biotin tag (loss of 374 u), on the other hand, occurs at lower CID amplitudes. At higher CID amplitudes, this dissociation channel becomes less productive as other channels become accessible. In contrast, unmodified [M + 2H]2+ laminin is easier to fragment than its labeled analogue, as demonstrated by the production of b5+ and y4+ ions in Figure 4B,C. Analytical Chemistry, Vol. 74, No. 10, May 15, 2002

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Figure 2. MS/MS spectra of labeled and unlabeled Cys-peptides. The CID spectra of singly charged unmodified (A) VTCG and (C) GRGDSC and acetyl-PEO-biotin Cys-labeled (B) VTC*G and (D) GRGDSC* are shown. The acetyl-PEO-biotin-specific fragments are indicated in the CID spectra of the modified peptides.

The data presented in Figure 4C illustrate several important aspects of Cys-peptide labeling. First, in the presence of a second proton, the onset of the ion signals associated with the fragmentation of the acetyl-PEO-biotin label occurs at the same CID amplitude settings as those of laminin-specific fragments. Second, the label remains intact on the b5 fragment, as evidenced by its m/z shift from 536 for unmodified to 950 for labeled laminin. Although formation of the product ion at m/z 270 (as well as other acetyl-PEO-biotin-specific fragments listed in Figure 3, except for the ion at m/z 375) occurs at higher CID amplitude settings, in terms of the resonant CID protocol, it is unlikely that this ion originated from the product ion at m/z 375. For the Cys-labeled peptides studied, the fragmentation efficiency curves of labelspecific fragments and sequence-specific fragments indicate that the production of these ions proceeds through different dissociation pathways; thus, they are independent of one another. For example, the relative abundance of the y2+ ion (containing the intact label) of labeled VTCG did not change significantly when the intensity of the acetyl-PEO-biotin fragment at m/z 375 increased with increasing CID amplitude (data not shown). In fact, the carboxy- and amino-terminal ions with intact acetyl-PEO-biotin tags did not fragment further to lose part of the label. Under the CID conditions used in this study, it follows that the formation of acetyl-PEO-biotin-specific fragments occurs in parallel to the cleavage along the peptide backbone. For unmodified peptides, the value of the CID amplitude (as defined by the LCQ software and according to eq 1) corresponding to ∼50% precursor fragmentation requires the application of a 2288

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resonance voltage to the trap end caps at a level that depends linearly on m/z, as shown in Figure 5. This linear dependence was found to have a slope of 1.45 × 10-3 V/(m/z) and an intercept of -4.0 × 10-2 V, with a correlation coefficient of 0.92. The negative intercept is insignificant on the basis of the scale of the sampled range of CID peak-to-peak voltages (Vp-p). Cys-peptides labeled with acetyl-PEO-biotin underwent ∼50% fragmentation along the same line: 1.43 × 10-3 V/(m/z) and -2.5 × 10-2 V for the slope and intercept, respectively, with a correlation coefficient of 0.93. In terms of CID dynamics, this suggests that acetyl-PEObiotin labeling is equivalent to the addition of a hypothetical 414 Da peptide segment to the Cys-peptide. Microscopically, this result is not unreasonable, since the structure of the acetyl-PEO-biotin tag closely resembles a peptide as a result of the presence of several amide bonds. Together, these results suggest that the acetyl-PEO-biotin modification “creates” a complex similar to a longer peptide in terms of backbone strength and does not affect peptide CID stability under the conditions used in typical LCQMS/MS experiments. Acetyl-PEO-biotin and ICAT-Labeled Peptides Analyzed by LC-MS/MS. After characterizing the CID of acetyl-PEObiotin-labeled peptides using ESI and direct infusion, experiments were conducted to examine the effects of CID during LC-MS/ MS analysis of acetyl-PEO-biotin- and ICAT-labeled laminin. A sample containing a mixture of laminin modified with either iodoacetyl-PEO-biotin, or heavy (ICAT-d8), or light (ICAT-d0) ICAT reagents was analyzed by LC-MS/MS. As shown in Figure 6A, acetyl-PEO-biotin labeled laminin elutes ∼2-3 min before the

Figure 3. Characteristic fragment ions observed during CID of Cys-labeled peptides. The addition of acetyl-PEO-biotin, ICAT-d0, or ICAT-d8 to a Cys residue of a peptide increases the mass of the Cys residue by 414.2, 442.2, and 450.3 Da, respectively. The putative structures and m/z values of the label-specific fragment ions observed during the CID of modified peptides are shown.

ICAT-labeled peptides, consistent with the greater hydrophobicity of the ICAT tag. The differential elution observed between ICATd0- and ICAT-d8-labeled laminin has been observed repeatedly in our laboratory for other ICAT-labeled peptides, as well. This partial resolution of isotopically labeled peptides is a result of an isotope effect due to the presence of eight alkyl C-D bonds on the ICAT-d8 tag and will have a profound effect on peptide quantitation but not on peptide identification. The CID characteristics of the three modified versions of laminin were also compared. As shown in Figure 3, the structural similarities of the acetyl-PEO-biotin and ICAT tags are reflected in the fragmentation ions produced from CID of the labels. Figure 6B compares the relative intensities of label-specific and peptidespecific fragments of modified laminin obtained during LC-MS/

MS. Examination of the label-specific fragments indicates that the same bonds within each label are cleaved: S-CH2, HN-CO, and O-CL2 where L is H or D for acetyl-PEO-biotin and ICAT-d0 or ICAT-d8, respectively. The Effect of Cys-Label Fragmentation on Peptide Identification. Proteome studies based on Cys-labeling approaches use automated database searching algorithms such as SEQUEST for peptide identification. It is imperative that any peptide modifications not impede product ion assignment. Partial or complete loss of a label will complicate or prevent correct sequence identification based on MS/MS data. To study the effect of Cys-labeling on peptide identification using SEQUEST, both unmodified and acetyl-PEO-biotin-labeled laminin were subjected to various CID amplitude settings, and the resulting MS/MS data Analytical Chemistry, Vol. 74, No. 10, May 15, 2002

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Figure 5. The dependence of CID resonance peak-to-peak end cap rf voltage on peptide m/z ratios. The CID resonance peak-topeak end cap rf voltages (Vp-p) were calculated using eq 1 in the Experimental Procedures and corresponded to ∼50% fragmentation of the various acetyl-PEO-biotin Cys-labeled and unmodified peptides used in this study. Linear regression based on the combined data for the labeled and unlabeled Cys-peptides produced a slope of 1.44 × 10-3 V/(m/z) and an intercept of -3.2 × 10-2 V with a correlation coefficient of 0.94.

Figure 4. The fragmentation efficiency curves for CID of unmodified and labeled laminin. (A) The intensities of y7+ and [M + H - 374]+ product ions formed from singly charged acetyl-PEO-biotin-labeled laminin (C*DPGYIGSR) and that of y7+ from unmodified laminin (CDPGYIGSR) are plotted as a function of the CID amplitude setting. The intensities of y4+, b5+, and the acetyl-PEO-biotin-specific fragments are plotted as a function of the CID amplitude setting applied to the [M + 2H]2+ form of (B) unmodified and (C) acetyl-PEO-biotinlabeled laminin.

was analyzed using SEQUEST. The results shown in Figure 7 indicate that the CID amplitude required to elicit a correct SEQUEST identification (a cross-correlation, Xcorr, score g 2) for labeled laminin is greater than that required for unmodified laminin. As illustrated in Figure 4B,C, the addition of the label requires a higher CID amplitude to effectively fragment the peptide, which as shown in Figure 7, is also required for correct identification. This is consistent with the shift of the fragmentation efficiency curves of labeled peptides to higher CID amplitude settings. Together, these data indicate that ∼50% of the precursor ions must be fragmented to achieve an Xcorr of 2. The incorrect identifications for the modified peptide at lower CID amplitude 2290 Analytical Chemistry, Vol. 74, No. 10, May 15, 2002

Figure 6. The effect of Cys labeling on peptide elution. (A) The extracted ion chromatograms of laminin labeled with either acetylPEO-biotin, ICAT-d0, or ICAT-d8 produced by capillary reversed-phase HPLC. (B) The relative intensities of label-specific and laminin-specific fragments observed in the MS/MS spectra generated during LCMS/MS using a CID amplitude setting of 35.

settings may be related to the low abundance of sequence-specific fragments with a concurrent presence of acetyl-PEO-biotin-specific fragments. At higher CID amplitudes, acetyl-PEO-biotin-specific fragments still appear in the spectrum, but the number of informative fragments increases, leading to correct identification.

Figure 7. The dependence of the SEQUEST cross-correlation score on the CID amplitude settings for unmodified and Cys-labeled laminin. The cross-correlation (Xcorr) score was obtained for MS/MS spectra generated at various CID amplitude settings applied to the [M + 2H]2+ ions of acetyl-PEO-biotin-labeled and unmodified laminin. As shown in the plot, an acceptable Xcorr score of ∼2 for the labeled peptide requires a higher CID amplitude setting (∼10 units) than its unlabeled counterpart.

Identification of Cys-Labeled Peptides from RNase A. To further characterize acetyl-PEO-biotin labeling and identification of Cys-modified peptides using LC-MS/MS, experiments were conducted using RNase A. Tryptic peptides obtained from the derivatization of RNase A with iodoacetyl-PEO-biotin were analyzed by LC-MS/MS and identified by database searching the product ions using SEQUEST. Twelve unique peptides from this sample were identified with a significant score (Xcorr score g 2). Digestion with trypsin produced peptides predominantly without missed cleavages; however, several peptides were identified as having no more than one missed-cleavage site. Out of the 12 tryptic peptides, all 6 Cys-peptides were identified as labeled with one or two acetyl-PEO-biotin tags corresponding to the number of Cys residues within the peptide. To determine labeling efficiency, the SEQUEST database search was performed using a “differential modification mode” where a Cys residue was considered as labeled or unmodified. It was determined that all of the Cys residues were effectively labeled during the derivatization procedure used, including those peptides that contained two Cys residues. The MS/MS spectrum of the RNase A peptide C*KPVNTFV HESLADVKAVC*SQK containing two Cys labels and producing an Xcorr score of 5.38 is shown in Figure 8A. Inspection of this MS/MS spectrum and those of the other five Cys-labeled peptides originating from RNase A revealed that the same acetyl-PEObiotin-specific fragments shown in Figure 3 were present for these Cys-labeled peptides, as well. The only Cys-labeled peptide that produced a Xcorr score less than 2 (Xcorr score of 1.03) was NVAC*K, whose MS/MS spectrum is shown in Figure 8B. This result is not surprising, since SEQUEST typically fails to produce high Xcorr scores for peptides with a mass below 1000 Da. However, as shown in Figure 8B, the presence of the characteristic acetyl-PEO-biotin-specific fragments indicates this peptide as being labeled. ICAT-Labeled D. radiodurans Proteome Sample. The utility of identifying the presence of label-specific fragment ions to assist in the identification of ICAT-d0- and ICAT-d8-modified peptides was demonstrated using Cys-labeled peptides extracted from D. radiodurans. A representative MS/MS spectrum for an

Figure 8. MS/MS spectra of Cys-labeled peptides from RNase A. The MS/MS spectra produced by (A) the [M + 3H]3+ form of the Cyslabeled peptide C*KPVNTFVHESLADVKAVC*SQK containing two acetyl-PEO-biotin labels and (B) the [M + H]+ form of the Cys-labeled peptide NVAC*K containing one acetyl-PEO-biotin label. The peptides were identified from the LC-MS/MS analysis of peptides generated from acetyl-PEO-biotin-labeled RNase A. The m/z values of the characteristic acetyl-PEO-biotin label fragments are indicated.

ICAT-d0/d8 Cys-labeled peptide pair is shown in Figure 9. SEQUEST identified the peptide as C*TFTLGAVWGALEAITGQTFLGEHTESVLR with a charge state of 3+ and Xcorr scores of 4.22 and 5.71 for the ICAT-d0 (Figure 9A) and the ICAT-d8 (Figure 9B) version, respectively. As shown for each spectrum, the presence of ICAT-fragment ions confirms the SEQUEST assignments. Differences in the m/z values of the ICAT-related fragments are due to the isotopically labeled sites within the reagent. Peaks separated by 8 Da (m/z 403.3/411.3 and 477.1/485.1) include all of the isotopically labeled sites within the reagent, whereas those that differ by 4 Da (m/z 345.9 and 349.9), are indicative of fragmentation between isotopically labeled positions. These peaks indicate the presence of the label on the peptide and allow the use of a Cys constraint to increase the confidence in the identification of the peptide. The presence of these reagentspecific product ions was used to distinguish ICAT-labeled peptides from the unmodified peptides that were nonspecifically isolated during avidin affinity chromatography. CONCLUSIONS The CID fragmentation efficiencies of peptides are dictated by their amino acid composition, which includes the presence of acidic and basic residues, amino acid modifications, the charge state of the parent ion, and size.21,24-26 From this perspective, it is important to understand the effect of Cys labeling on peptide CID fragmentation efficiencies, especially when these peptides are identified by automated database searching against their fragment (24) Nair, H.; Wysocki, V. H. Int. J. Mass Spectrom. Ion Proc. 1998, 174, 95100. (25) Tsaprailis, G.; Somogyi, A.; Nikolaev, E. N.; Wysocki, V. H. Int. J. Mass Spectrom. 2000, 196, 467-479. (26) Summerfield, S. G.; Cox, K. A.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 1997, 8, 25-31.

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Figure 9. MS/MS spectra of ICAT-d0- and ICAT-d8-labeled peptides from D. radiodurans. The peptides were identified from the LC-MS/MS analysis of a proteome sample extracted from D. radiodurans. The ICAT reagent-specific fragments observed in the product ion spectra for the (A) ICAT-d0- and (B) ICAT-d8-labeled peptide C*TFTLGAVWGALEAITGQTFLGEHTESVLR are shown.

ions. Acetyl-PEO-biotin and ICAT Cys-labeled peptides produce fragmentation patterns similar to their unmodified analogues but possess different CID stabilities, thus requiring higher CID amplitudes to achieve effective fragmentation. In most cases, the CID amplitudes required to fragment the label are equal to or greater than those required to fragment the peptide backbone and are sequence dependent. The unique product ions originating from the labels themselves provide useful indicators that validate the presence of the Cys modification. In a manner analogous to using the detection of ions formed from the loss of H3PO4 or HPO3 to indicate the presence of a phosphorylated peptide by MS/MS, these reagent-specific product ions can be used to distinguish between labeled and unmodified peptides and enhance automated database peptide identification used in proteome-wide analysis involving Cys-capture reagents. Moreover, the isotope-specific

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fragment ions of ICAT-labeled peptides can be used to differentiate identical Cys-peptides labeled with either ICAT-d0 or ICAT-d8. ACKNOWLEDGMENT We thank the National Cancer Institute (under Grants CA86340 and CA93306) and the United States Department of Energy Office of Biological and Environmental Research for the support of portions of this research. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for the U.S. Department of Energy under contract DE-AC06-76RLO 1830.

Received for review February 19, 2002. AC010974P

September

5,

2001.

Accepted