Collisionally Activated Dissociation and Electron Capture Dissociation

Nov 16, 2006 - One of the challenges in protein interaction studies with chemical cross-linking stems from the complexity of intra-, inter-, and dead-...
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Anal. Chem. 2006, 78, 8183-8193

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Collisionally Activated Dissociation and Electron Capture Dissociation of Several Mass Spectrometry-Identifiable Chemical Cross-Linkers Saiful M. Chowdhury, Gerhard R. Munske, Xiaoting Tang, and James E. Bruce*

Department of Chemistry, Washington State University, Pullman, Washington 99164-4630

One of the challenges in protein interaction studies with chemical cross-linking stems from the complexity of intra-, inter-, and dead-end cross-linked peptide mixtures. We have developed new cross-linkers to study proteinprotein interactions with mass spectrometry to improve the ability to deal with this complexity. Even the accurate mass capabilities of FTICR-MS alone cannot unambiguously identify cross-linked peptides from cell-labeling experiments due to the complexity of these mixtures resultant from the enormous number of possible crosslinked species. We have developed novel cross-linkers that have unique fragmentation features in the gas phase. The characteristics of these cross-linkers combined with the accurate mass capability of FTICR-MS can help distinguish cross-linking reaction products and assign protein identities. These cross-linkers that we call protein interaction reporters (PIRs) have been constructed with two reactive groups attached through two bonds that can be preferentially cleaved by low-energy CID of the respective protonated precursor ions. After cleavage of the labile bonds, the middle part of the linker serves as a reporter ion to aid identification of cross-linked peptides. This report highlights three new PIRs with new features that have been developed to improve the efficiency of release of reporter ions. The new cross-linkers reported here were tuned with the addition of an affinity tag, a hydrophilic group, a photocleavable group, and new low-energy MS/ MS cleavable bonds. This report presents our investigation of the MSMS fragmentation behavior of selected protonated ions of the new compounds. The comprehensive fragmentation of these PIRs and PIR-labeled crosslinked peptides with low-energy collisions and an example of electron capture dissociation in FTICR-MS is presented. These new cross-linkers will contribute to current systems biology research by allowing acquisition of global or largescale data on protein-protein interactions. Chemical tools to characterize protein expression and protein function are now becoming very popular in the field of mass * To whom correspondence should be addressed. Address: Department of Chemistry, P.O. Box 644630, Washington State University, Pullman, Washington 99164-4630. Phone: (509) 335-2116. Fax: (509) 335-8867. E-mail: james_bruce @wsu.edu. 10.1021/ac060789h CCC: $33.50 Published on Web 11/16/2006

© 2006 American Chemical Society

spectrometry-based proteomics. During the last 5 years, numerous analytical strategies have emerged in which chemistry played a major role to advance the field of proteomics.1-3 Most of these strategies utilized chemical probes that selectively label or enrich functional sites of proteins or protein complexes using the reactivity of certain functional groups in proteins. The advantage of a chemistry-based approach is that it is robust and can selectively label macromolecular complexes in their functional native settings. Thus, the advancement of proteomics research requires development and improvement of new chemical probes for global or large-scale identification of protein complexes. Chemical cross-linking combined with mass spectrometric analysis is a powerful technique for protein-protein interaction studies. Cross-linkers can covalently link two interacting proteins in complex systems. After enzymatic digestion, the cross-linked peptides can be subjected to MS/MS fragmentation to identify cross-linked species. A key aspect of the ability to study protein interactions with chemical cross-linking is the development of new cross-linking reagents. The cross-linker reagents currently available utilize the same architecture, two reactive groups connected through a linker. The reactive groups generally target primary amines or sulfhydryl groups in proteins. Because of the low abundance of cysteine in proteins, most of the cross-linkers are primary amine reactive homobifunctional cross-linkers. The most common primary amine reactive group utilized in current commercial cross-linkers is the N-hydroxysuccinimide (NHS) ester. NHS esters readily react with lysine side chains in proteins at solution conditions near physiological pH 7. One problem associated with NHS esters is that they can hydrolyze quickly in aqueous reaction buffer. This results in complex mixtures of dead-end, intra-, and inter-cross-linked peptides that are extremely difficult to distinguish when working with large-scale samples. To reduce the complexity, several researchers have contributed to the development and application of new cross-linkers for protein interaction studies. Rappsilber et al. analyzed the yeast (1) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (2) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Mol. Cell. Proteomics 2004, 3, 1154-1169. (3) Adam, G. C.; Sorensen, E. J.; Cravatt, B. F. Mol. Cell. Proteomics 2002, 1, 781-790.

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Figure 1. The chemical structures of three new PIRs. Low-energy MS/MS cleavable bonds are indicated by bold lines. In PIR-2, the photocleavable bond is indicated by a dashed line. After cleavage of photocleavable bond, the reactive part of the PIR-2 will be attached to the cross-linked peptide, and the remainder will be on the avidin beads.

nuclear pore complex utilizing commercial homo- and heterobifunctional cross-linkers.4 Trester-Zedlitz et al. introduced a modular solid-phase synthetic strategy for generating cross-linking reagents.5 This cross-linker has an affinity group and cleavable isotope tag to reduce the complexity of the sample. Muller et al. developed an isotope-tagged, cross-linking reagent in which a deuterium-labeled, cross-linking reagent was prepared together with its undeuterated counterpart.6 Efforts were also made by several other researchers to reduce the complexity by constructing cleavable and isotope-labeled cross-linkers.7-10 Petrotchenko et al. recently reported a new isotope-coded cross-linker that can be cleaved under base treatment.11 This hydrolytic cleavage can help to identify cross-linked species in complex mixtures by producing characteristic mass shift patterns of dead-end, intra-, and intercross-linked peptides. (4) Rappsilber, J.; Siniossoglou, S.; Hurt, E. C.; Mann, M. Anal. Chem. 2000, 72, 267-275. (5) Trester-Zedlitz, M.; Kamada, K.; Burley, S. K.; Fenyo, D.; Chait, B. T.; Muir, T. W. J. Am. Chem. Soc. 2003, 125, 2416-2425. (6) Muller, D. R.; Schindler, P.; Towbin, H.; Wirth, U.; Voshol, H.; Hoving, S.; Steinmetz, M. O. Anal. Chem. 2001, 73, 1927-1934. (7) Chen, X.; Chen, Y. H.; Anderson, V. E. Anal. Biochem. 1999, 273, 192203. (8) Pearson, K. M.; Pannell, L. K.; Fales, H. M. Rapid Commun. Mass Spectrom. 2002, 16, 149-159. (9) Bennett, K. L.; Kussmann, M.; Bjork, P.; Godzwon, M.; Mikkelsen, M.; Sorensen, P.; Roepstorff, P. Protein Sci. 2000, 9, 1503-1518. (10) Collins, C. J.; Schilling, B.; Young, M.; Dollinger, G.; Guy, R. K. Bioorg. Med. Chem. Lett. 2003, 13, 4023-4026. (11) Petrotchenko, E. V.; Olkhovik, V. K.; Borchers, C. H. Mol. Cell. Proteomics 2005, 4, 1167-1179.

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Sinz and coauthors utilized high-resolution FTICR-MS to identify cross-linked peptides.12,13 Among all mass spectrometers, FTICR-MS offers ultrahigh-resolution and high mass accuracy.14 For large-scale samples from complex organisms, accurate mass measurement alone is insufficient for unambiguous identification of cross-linked peptides, because the number of possible candidates may match hundreds of peptide combinations for a large protein complex, even at mass accuracy of better than 1 ppm.11 For proteome-wide interaction studies, the missed cleavage sites and broad chemical reactivity of cross-linking reagents must be taken into account, which further increases the number of possible candidates. In addition, MS/MS sequencing of cross-linked peptides is generally more difficult because of less predictable and convoluted MS/MS patterns of cross-linked peptides. Moreover, fragmentation of both the peptide backbone and cross-linker itself can make unambiguous assignment of cross-linked peptides difficult. To assign these cross-linked peptides from MS and MS/ MS studies, several software programs, such as ASAP (Automated Peptide Assignment Program) and MS2 assign were developed, but the problem still becomes overwhelming or intractable for complex samples15,16 (12) Kalkhof, S.; Ihling, C.; Mechtler, K.; Sinz, A. Anal. Chem. 2005, 77, 495503. (13) Schulz, D. M.; Ihling, C.; Clore, G. M.; Sinz, A. Biochemistry 2004, 43, 47034715. (14) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (15) Schilling, B.; Row, R. H.; Gibson, B. W.; Guo, X.; Young, M. M. J. Am. Soc. Mass Spectrom. 2003, 14, 834-850.

Figure 2. CID-FTICR mass spectra of PIR-1. The ion at m/z 1753.7185 corresponds to [M + H]+ of PIR-1 and was subjected to different collisional trap voltages. Mass spectra are shown for -3, -6, -9, and -21 V (A-D). The expected reporter ion is shown at m/z 1325.6001 and resulted from the cleavage of both low-energy mass spectrometry labile bonds (B-D). m/z 1539.6545 corresponds to the cleavage of only one of the two labile bonds.

Earlier, we introduced a new kind of cross-linker, which we call a protein interaction reporter (PIR).17 This cross-linker has specific fragmentation characteristics that can be exploited in lowenergy CID experiments and to generate reporter ions that can help identify dead-end, intra-, and inter-cross-linked species. The cross-linking of interacting species with this first-generation crosslinker was demonstrated with the RNase S protein complex. Those data illustrated successful mapping of the interaction between RNase S peptide and S protein. The advantage of the PIR approach is derived from the unique gas-phase fragmentation properties of the cross-linker species. In this Article, we highlight the gas-phase fragmentation behavior of three new cross-linkers utilizing specific chemical properties. These compounds were developed with either acidcleavable Rink or indole groups that have been found to fragment efficiently under low-energy CID. In our low-energy CID experiments with FTICR-MS, these compounds showed selective fragmentation to generate specific fragment masses, or reporter ions. We also incorporated several additional features to improve the efficiency of this reporter ion-based cross-linking strategy. For example, we incorporated an affinity tag, a hydrophilic group, a (16) Young, M. M.; Tang, N.; Hempel, J. C.; Oshiro, C. M.; Taylor, E. W.; Kuntz, I. D.; Gibson, B. W.; Dollinger, G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5802-5806. (17) Tang, X.; Munske, G. R.; Siems, W. F.; Bruce, J. E. Anal. Chem. 2005, 77, 311-318.

photocleavable group, and a new low-energy MS/MS-cleavable group to improve the utility and diversity of these new PIRs. The comprehensive fragmentation pattern of these new cross-linkers, including low-energy MS/MS and an example of ECD, is reported here for the first time. In addition, photocleavable properties were incorporated in the PIR and allow release of cross-linked peptides from the biotin-avidin complex by UV light cleavage instead of solvent-based elution. The specific chemical features of these cross-linkers, in combination with accurate mass capabilities and multiple fragmentation methods of FTICR-MS will be a powerful tool for large-scale identification of protein-protein interactions. Chemical cross-linking is also becoming more popular for elucidation of three-dimensional structures of proteins. We think the PIR strategy will be also a very useful tool for structural proteomics studies with chemical cross-linking.18 EXPERIMENTAL SECTION Chemicals were purchased from Sigma-Aldrich (St. Louis, MO), Novabiochem (San Diego, CA) unless otherwise stated. Substance P utilized for this study was [Met-OH11]-substance P. Cross-linking reactions of [Met-OH11]-substance P (RPKPQQFFGLM, MW 1347.7120 Da) (1 µL, 1 mM) with PIR-1, PIR-2, or PIR-3 (18) Novak, P.; Haskins, W. E.; Ayson, M. J.; Jacobsen, R. B.; Schoeniger, J. S.; Leavell, M. D.; Young, M. M.; Kruppa, G. H. Anal. Chem. 2005, 77, 51015106.

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Table 1. List of m/z Values and Calculated Neutral Masses Used to Assign Cross-Linked Reaction Productsa m/z monoisotopic mass of neutral compds (Da)

mass error, ppm

expected

obs

∆M

1752.7117 2032.8176 1039.4287 114.0191 214.0590 242.0903 100.0399 128.0712 1324.5937

1753.7190 (1+) 1017.4161 (2+) 1040.4360 (1+)

1753.7185 (1+) 1017.4169 (2+) 1040.4353 (1+)

5.0 × 10-4 8.0 × 10-4 7.0 × 10-4

0.3 0.8 0.7

1325.6010 (1+) 663.3041 (2+)

PIR-2, reporter, photofragment

709.2999

710.3072 (1+)

555.2482

556.2555 (1+)

substance P + PIR-1 (intra-link) substance P (Ox) + PIR-2 photo fragment (intra-link) substance P + PIR-3 (dead-end) substance P + two linker arms (PIR-1)

2870.3699* 2271.0710**

957.7973 (3+) 1136.5428 (2+)

9.0 × 10-4 1.0 × 10-4 5.0 × 10-4 4.0 × 10-4 2.5 × 10-3 8.0 × 10-4 1.7 × 10-3 2.7 × 10-3 2.4 × 10-3

0.7 0.2 0.8 0.6-3.5

PIR-3, reporter

1325.6001 (1+) 663.3042 (2+) 663.3046 (2+) 710.3076 (1+) 710.3097 (1+) 556.2563 (1+) 556.2572 (1+) 957.7946 (3+) 1136.5404 (2+)

2175.0974 1545.7762

1088.5560 (2+) 1546.7835 (1+)

1561.7711

1562.7784 (1+)

substance P + one linker arms PIR-3

1474.7754

1475.7827 (1+)

1.1 × 10-3 6.0 × 10-4 3.0 × 10-4 3.0 × 10-4 4.5 × 10-3 5.1 × 10-3 7.4 × 10-3

1.0 0.4-0.2

substance P (Ox) + two linker arms, PIR-2

1088.5549 (2+) 1546.7829 (1+) 1546.7838 (1+) 1562.7781 (1+) 1562.7829 (1+) 1475.7878 (1+) 1475.7901 (1+)

PIR-1 PIR-2 PIR-3 NHS mass (reactive group) NHS-SA linker mass, (PIR-1 and PIR-2) NHS-SA linker mass, (PIR-3) linker arm, PIR-1, PIR-2 linker arm, PIR-3 PIR-1, Reporter

1.4-3.0 2.8 2.1

0.2-2.9 3.5-5.0

a Calculation procedures for cross-linked reaction products are as follows: *1347.7120 (substance P mass) + 1752.7117 (cross-linker mass) 2(114.0191, NHS mass) - 2H (from amine groups) ) 2870.3699 Da; **1347.7120 (substance P mass) + 15.9949 (oxygen mass, methionine residue) + 1137.4179 (photocleavable part) - 2(114.0191, NHS mass) - 2H (from amine groups) ) 2271.0710 Da.

were performed in 100 µL of PBS buffer at pH 7.2. The molar ratio of substance P and cross-linker was maintained at 1:2. The reactions were conducted for 30 min at room temperature. The reactions were terminated by adding 50 µL of 10 mM Tris buffer. The salts were removed from the solution by SepPack C18 (Water, Milford, MA). The reaction solution of substance P and PIR-2 was captured with 50 µL of ultralink immobilized monomeric avidin beads (Pierce Biotech, Rockford, IL), and nonbinders were washed with PBS (pH 7.2) and 100 mM NH4HCO3 solutions. The avidin beads were resuspended in a 100-µL PBS solution, and photocleavage was performed using a UV lamp (model EN-160L, Spectronics Corp., Westbury, NY) of 360-nm wavelength for 4 h. After 4 h, the supernatant was collected for mass spectrometric analyses. All FTICR-MS spectra were obtained with a Bruker Daltonics, 7T APEX Q-FTICR mass spectrometer by direct infusion of samples with a nano ESI tip made with a fused-silica capillary (360µm o.d and 20-µm i.d), and the capillary tip was etched with 49% HF. The electrospray solution was acetonitrile/0.1% TFA or 50 mM ammonium acetate unless otherwise mentioned. Water was avoided in the spray solution used with pure PIR samples to prevent the hydrolysis of the reactive groups and to obtain mass spectra of the intact cross-linkers. Electron capture dissociation (ECD) was performed using a heated hollow cathode dispenser located outside the ICR cell to obtain the MS/MS data. The cathode dispenser was heated with 1.8-1.9 A. The sidekick19 trapping voltage was maintained between +6 and -6 V. Electrons (19) Caravatti, P.; Allemann, M. Org. Mass Spectrom. 1991, 26, 514-518.

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used for ECD were accelerated using 3 V. The electron injection time was 150-200 ms. The FTICR mass spectra were processed using the software ICR-2LS developed by Pacific Northwest National Laboratory.20 Time-domain signals were apodized (Welch) and zero-filled before Fourier transformation to the mass spectra. For calibration of the instrument, PIR-1 was mixed with two known standard peptides, substance P and bradykinin. The calibration was generated using a three-point calibration equation to obtain the accurate mass of PIR-1 and its reporter ion. The generated calibration constants were globally applied to process PIR-1 and PIR-2 and PIR-1-labeled substance P mass spectra. To calibrate PIR-3, one mass spectrum of PIR-3 was obtained with skimmer dissociation to generate fragment ions from cleavage of one or both mass spectrometry labile bonds. Calibration constants were generated from the accurate m/z of these ions using a two-point calibration equation. The generated calibration was applied globally to PIR-3 mass spectra to obtain accurate m/z values of the PIR-3 cross-linker ions. For spectra that show peptides coupled with photocleavable PIR-2 and PIR-3, one known peptide, CHWKQNDEQM, was used for calibration with a two-point calibration equation. Synthesis of PIRs. The cross-linkers were synthesized using a 431A peptide synthesizer (Applied Biosystem, Foster City, CA) with solid-phase peptide synthesis chemistry (Figure 1). Glycine was coupled to HMPB-MBHA (4-hydroxymethyl-3-methoxyphenoxybutyric acid) resin using the standard symmetric anhydride (20) Anderson, G. A., Bruce, J. E., Eds;, ICR-2LS1995; Pacific Northwest National Laboratory: Richland, WA, 1995.

Scheme 1. Schematic Representation of Cross-Linking Strategy with Photocleavable PIR-2a

a

The strategy is demonstrated with an intra-cross-linked peptide.

method. For PIR-1 and PIR-2, the biotin group and PEG linkers were added in the form of Fmoc-Glu(biotinyl-PEG)-OH using standard coupling reactions. For PIR-2, one photocleavable group, Fmoc-aminoethyl photolinker, was introduced between lysine and Glu(biotinyl-PEG)-OH using the same solid-phase chemistry. Lysine in the form of Fmoc-Lys--Fmoc was coupled to the FmocGlu(biotinyl-PEG)-OH that then formed the branch point for the cross-linkers. The Rink groups, succinic anhydride (SA), and N-hydroxysuccinimides (NHS) were coupled using the same standard activation, coupling, and deprotection procedures. For PIR-3, which does not contain a biotin group, one alanine residue coupled with Fmoc-Lys--Fmoc to form the branch point of the linkers. The two amino functional groups of the lysine residues were then coupled with (3-{[ethyl-Fmoc-amino]methyl}indol-1-yl)acetic acid. Succinic acids and N-hydroxysuccinimides were coupled as previously described. PIR-3 was synthesized with no affinity group but can be constructed with an affinity group using the same peptide synthesis procedures as used for PIR-1 and PIR2. It was found that reduced loading of the resin to about onethird is necessary to get efficient coupling of the last step, since the biotin group seems to limit the coupling of the Fmoc-LysFmoc. The final product was cleaved using either 0.5 or 1.0% TFA in chloroform and then neutralized with pyridine. The chloroform and TFA pyridine salts were removed under vacuum until constant weight was noted. RESULTS AND DISCUSSION New features of protein interaction reporter-1 (PIR-1) include an affinity group and a hydrophilic chain in the cross-linker (Figure 1, PIR-1). It is desirable to produce a variety of PIR compounds

with diverse physical properties. To vary the hydrophobic character, we incorporated a polyethylene glycol chain in the PIR-1 structure. In addition, a biotin moiety was introduced to allow enrichment of cross-linked peptides from complex mixtures. For studies of the gas-phase fragmentation behavior of the protein interaction reporter 1 (PIR-1), a 10 µM solution was directly infused into the FTICR-MS with a nanoelectrospray source using a flow rate of 20 µL/h. The ions at m/z 1753.7185 (expected m/z 1753.7190, mass accuracy, 0.3 ppm), corresponding to [M + H]+ of PIR-1, were isolated with a quadrupole and accumulated in a hexapole at -3 V collisional trap cell voltage (Figure 2A). To investigate the fragmentation of [M + H]+ of PIR-1, the collisional trap voltage was increased by 3-V increments. (Figure 2B-D). The characteristic cleavage of two labile bonds (NHS-SA linker loss, 2(214.0590)) results in the appearance of two fragment ions in the spectra at m/z )1539.6545 and 1325.6001. These correspond to the fragmentation of one and both MS/MS cleavable bonds, respectively. The ion at m/z ) 1325.6001 (1+), appeared in the MS spectra after fragmentation of both MS/MS cleavable bonds and is used as a reporter ion, since it should always appear in the mass spectra after low-energy CID (see Table 1 for measured and expected m/z values). The increase in collisional trap voltage (-6, -9, -21 V) increased the intensity of one MS/MS cleavable fragment and reporter fragment ion intensity (Figure 2B-D). These spectra showed the efficiency of reporter ion release from the cross-linker reagent alone and demonstrated the potential of this PIR-1 to release a specific reporter ion. Biotin is a commonly used affinity group in proteomics research. However, difficulties of eluting biotin-labeled peptides from the avidin beads have been reported.21 In addition, the biotin Analytical Chemistry, Vol. 78, No. 24, December 15, 2006

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Figure 3. CID-FTICR mass spectra of PIR-2. The ions at m/z 1017.4169 (2+ charge state) correspond to [M + 2H]2+ of PIR-2 and were subjected to different collisional trap voltages. Mass spectra shown were acquired for -6 -9, -12, and -18 V collisional cell voltages (A-D).

group and linker may also fragment during MS/MS experiments, which complicates the interpretation of mass spectra.22,23 To overcome this problem, we have introduced a photocleavable group in PIR-2 for more efficient sample recovery (Figure 1, PIR2). The cleavage of a photocleavable bond with UV light at wavelength 360 nm was reported to improve peptide recovery.24,25 With studies that use PIR-2, the digested reaction solution will be incubated with the avidin beads. After washing away the weak binders, the beads will be irradiated with a UV light to release the cross-linked peptides from the biotin-avidin complex. This cross-linker is larger in size than the previous PIR-1 due to the PEG and photocleavable groups, but it will be smaller after cleavage of the photocleavable bond. After UV light illumination, the photocleaved fragment will still contain the two low-energy MS/MS cleavable bonds that will be fragmented to identify the cross-linked product with same reporter ion strategy (Scheme 1). To confirm the incorporation of a photocleavable group in PIR-2, gas-phase fragmentation of intact PIR-2 was studied with FTICRMS. The cross-linker was directly infused in the nanoelectrospray source as previously described for PIR-1. In this case, the ion at (21) Steen, H.; Mann, M. J. Am. Soc. Mass Spectrom. 2002, 13, 996-1003. (22) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-382. (23) Brittain, S. M.; Ficarro, S. B.; Brock, A.; Peters, E. C. Nat. Biotechnol. 2005, 23, 463-468. (24) Zhou, H.; Ranish, J. A.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2002, 20, 512-515. (25) Qian, W. J.; Goshe, M. B.; Camp, D. G., 2nd; Yu, L. R.; Tang, K.; Smith, R. D. Anal. Chem. 2003, 75, 5441-5450.

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m/z 1017.4169 corresponds to a [M + 2H]2+ charge state of PIR-2 (Figure 3A). These ions were isolated in the quadrupole and fragmented in the collisional trap with different voltage settings. The fragmentation spectra shown were acquired with -6, -9, -12, and -18 V effective collision energies (Figure 3A-D). The fragmentation of both labile bonds was observed, resulting in loss of one or two N-hydroxysuccinyl monoamides (expected, 214.0590 Da) from PIR-2. The complete fragmentation of the molecular ion at m/z 1017.4169 to generate only the reporter ion for the intact PIR-2 (m/z 803.3575) in the mass spectrum was observed at -18 V (Figure 3D). The reporter ion for this PIR-2 will be smaller after photocleavage of the biotin group from the cross-linker. In the development of previous PIR structures, the Rink group was used to incorporate low-energy MS/MS cleavable bonds. The Rink group has acid-cleavable properties and was found to fragment under low-energy CID conditions in the gas phase 26,17 Here, we also investigated whether any other acid-cleavable groups can show similar low-energy fragmentation features in FTICR-MS. We designed and synthesized a new cross-linker using Indole acetic acid groups in the place of Rink groups (Figure 1, PIR-3). For PIR development, this seemed promising, since the acid-labile properties and synthesis of ethyl amide peptides with (3-{[ethyl-Fmoc-amino]methyl}indol-1-yl)acetic acid was reported.27 Although the Rink group acid cleavage was observed with 95% (26) Rink, H. Tetrahedron Lett. 1987, 28, 3787-3790. (27) Estep, K. G.; Neipp, C. E.; Stramiello, L. M. S.; Adam, M. D.; Allen, M.; Robinson S.; Roskamp E. J. J. Org. Chem. 1998, 63, 5300-5301.

Figure 4. CID-FTICR mass spectra of PIR-3. The ion at m/z 1040.4353 (1+ charge state) corresponds to [M + H]+ of PIR-3 and was subjected to different collisional trap voltages. Mass spectra shown were acquired for -6 -9, -12, and -18 V collisional cell voltages (A-D).

Figure 5. Schematic diagram of the relationship among fragment ions observed in the mass spectral analysis of intra-, inter-, and dead-end cross-linked peptides (A-C).

TFA, this group can be cleaved with much more mild acid conditions of 3-5% TFA. The nanoelectrospray conditions were maintained as previously described for PIR-1. As shown in Figure 4, the ion at m/z 1040.4353 corresponds to [M + H]+ of intact cross-linker, and this ion was isolated in the quadrupole. After isolation, we increased the voltage settings in the hexapole collisional trap, as previously described. The compound showed fragmentation as expected. Both MS/MS-labile bonds were cleaved, producing the 798.3476 and 556.2563 ions. PIR-3 showed efficient release of reporter ions, even at -9 V collisional trap voltage (Figure 4B). Complete fragmentation of the molecular ion to generate only the reporter ion (m/z 556.2568) in the mass spectrum was observed at -18 V (Figure 4D). This showed that the new acid-labile indole acetic acid groups efficiently fragmented under low-energy CID activation in FTICR-MS.

We have developed three new PIRs and confirmed their molecular weight with FTICR-MS with a mass accuracy of 0.30.8 ppm (Table 1). These data (Figures 2-4) demonstrated that the new features, including affinity group, hydrophilic chains, photocleavable group, and new low-energy labile groups, were successfully incorporated into these PIR scaffolds. The three new PIRs showed selective fragmentation in the gas phase to release the expected reporter ions. These data also verified the compatibility of these new features in the backbone of the PIRs without destroying their low-energy fragmentation properties. Next, we focus on the strategy to distinguish cross-linked peptides and mass spectrometry of peptides labeled with these new PIRs. The cross-linkers were designed with two primary amine reactive groups and two low-energy MS/MS cleavable bonds. Identification of cross-linked peptides is assisted by appearance Analytical Chemistry, Vol. 78, No. 24, December 15, 2006

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Figure 6. ESI-FTICR mass spectra of an intra-cross-linked peptide with PIR-1. (A) m/z 957.7946 (3+) corresponds to the [M + 3H]3+ ion of intralinked substance P with PIR-1 and was isolated in the quadrupole with -3 V trapping potential. (B) Fragmentation of the intra-cross-linked substance P at -9 V collisional trap voltage. (C) Fragmentation at -12 V collisional trap voltage settings.

of reporter ions in the fragmentation spectra. The residual linker masses that remain attached to the peptides from the two reactive ends of the cross-linker after low-energy MS/MS fragmentation are termed “linker arms”. The intralinked products fragment under low-energy CID conditions to release a reporter ion and a single modified peptide ion (peptide + two linker arms). The sum of the neutral masses of the reporter ion and modified peptide ion must match the observed cross-linked peptide neutral mass (Figure 5A). On the other hand, interlinked products will generate three fragment ions that include two modified peptide ions (peptide + one linker arm) and a reporter ion upon PIR activation. The sum of the modified peptide masses and reporter mass must match the interlinked precursor mass (Figure 5B). The dead-end reaction products can be distinguished by adding the modified peptide (peptide + one linker arm) and reporter ion masses generated under low-energy CID conditions. In this case, the sum does not match the mass of PIR-labeled dead-end precursor peptide because of the loss of one reactive group due to hydrolysis (Figure 5C). For dead-end products, two additional ions can appear in the spectrum. One marker ion results from the fragmentation of one labile bond from the coupled peptide end of the crosslinker, which is always constant for a particular hydrolyzed PIR. The other ion, which we call a diagnostic ion, can result from the fragmentation of one labile bond from the hydrolyzed end of the cross-linker. This mass depends on the cross-linked peptide mass and can be distinguished by subtracting this mass from the precursor dead-end cross-linked peptide mass. 8190 Analytical Chemistry, Vol. 78, No. 24, December 15, 2006

To demonstrate the cross-linking capabilities of these new reagents, we coupled [Met-OH11]-substance P with PIR-1, PIR-2, and PIR-3. The substance P sequence contains two primary amine functional groups: one is on the lysine side chain, and the other is on the N terminus. The reaction solution of substance P with PIR-1 was directly infused in FTICR-MS with the conditions previously described. The ions at m/z ) 957.7946 (3+), correspond to the [M + 3H]3+ charge state of an intra-cross-linked peptide of substance P, which was isolated in the quadrupole and then fragmented in the hexapole collisional trap (Figure 6A). The voltage in the hexapole collisional trap was first set to -9 V. Two distinctive fragment ions, m/z 1546.7829 (1+) and 663.3042 (2+) were observed in the mass spectrum (Figure 6B). As described above, the linker arms of the cross-linker will be retained by the cross-linked peptide after cleavage of two low-energy MS/MS bonds. For PIR-1 and PIR-2, the monoisotopic mass of each linker arm is 100.0390 Da (C4H6O2N1). For an intra-cross-linked peptide, after low-energy fragmentation, the mass of two linker arms is added to the cross-linked peptide after the loss of two hydrogen atoms from two amine groups of substance P. At -12 V, both the reporter ions and the modified peptide ions (substance P - 2H + two linker arms) were observed in the mass spectra with high intensity (Figure 6C). The ions at m/z 1546.7838 (1+) correspond to the expected molecular weight if substance P is intralinked with PIR-1 (expected m/z 1546.7835; mass accuracy, 0.2-0.4 ppm) and then fragmented at both of the low-energy MS/MS cleavable bonds (Table 1). The efficient release of reporter ions at m/z

Figure 7. ESI-FTICR mass spectra of an intra-cross-linked peptide with PIR-2. (A) The ion at m/z 1136.5404 corresponds to the [M + 2H]2+ ion of intralinked substance P with PIR-2 after photocleavage with UV light. (B) Fragmentation of these ions at -15 V collisional trap voltage. (C) Fragmentation at -18 V collisional trap voltage settings. The methionine residue in substance P was oxidized during UV light illumination. A asterisk (*) denotes noise peaks.

663.3046 (expected m/z 663.3041) was observed. The intensities of the reporter ions and the modified substance P ions were observed to increase with increased collisional trap voltages (Figure 6B-C). No significant fragmentation occurs in either the peptide or the cross-linker backbone beyond the expected lowenergy cleavable bonds. The precursor ion mass of the intralinked substance P (2870.3619 Da) matched the sum of the fragment ion masses of modified substance P and reporter ion (2870.3694 Da) with a mass accuracy of 2.6 ppm. To demonstrate the utility of introducing a photocleavable group in PIR-2, the release of a photocleavable fragment was optimized with a biotinylated peptide incorporated with a photocleavable group within the sequence [SSARL(photo)RK(biotin)G]. It was found that efficient release of the photofragment from the peptide captured on the avidin beads was observed with 4-5 h of UV-light illumination (Supporting Information available). We reacted substance P with PIR-2 at the conditions previously described. The solution was incubated with the avidin beads, and the peptide was eluted first with standard elution solvent utilized by Goshe et al.28 In MALDI-TOF mass spectra of these samples, we observed an ion of m/z ) 3151.80, which corresponds to the expected [M + H]+ of an intra-cross-linked substance P with the PIR-2. The reaction was conducted again to test the utility of photorelease instead of solvent elution. The supernatant from the photorelease experiment was collected, and MALDI-TOF mass spectra were obtained to confirm the release of photocleaved (28) Goshe, M. B.; Veenstra, T. D.; Panisko, E. A.; Conrads, T. P.; Angell, N. H.; Smith, R. D. Anal. Chem. 2002, 74, 607-616.

fragments. In the MALDI-TOF mass spectrum, we observed an ion at m/z 2272.10, which is 16 Da higher mass than the expected m/z (2256.0834 Da) of the cross-linked product. This is likely an oxidation product, since substance P contains a methionine residue that can be oxidized during UV light illumination.21 To confirm the cross-linking and release of reporter ions from this photocleaved fragment, 2+ ions at m/z 1136.5404 Da were isolated in the quadrupole (Figure 7A) in ESI-FTICR-MS (Table 1). These ions were then fragmented with different collisional voltage settings in the hexapole. Mass spectra were shown for -15 and -18 V (Figure 7B-C). The reporter ion and modified substance P for this photocleaved fragment were observed at m/z 710.3076 (1+) and m/z 1562.7781 (1+) respectively in the spectra. In addition, the sum of the fragment ion mass of the reporter ion and the modified peptide matched the experimental mass of precursor peptide with a mass accuracy of 2.1-5.1 ppm. These data demonstrated that substance P was intra-cross-linked with the PIR-2 and cleaved at the photocleavable bond during UV light illumination. In addition, these data showed that this photorelease technique can be an alternative elution of PIR-labeled peptides from the avidin beads. To further demonstrate the ability to distinguish between intra and dead-end products, we investigated the cross-linking reaction of substance P with PIR-3, which has indole groups substituted for Rink groups (Figure 8). The calculated product mass for two reactive groups coupled to substance P is 2157.0869 Da. We observed ions of m/z 2176.10 in MALDI-TOF mass spectra that correspond to a dead-end reaction product. The ions at m/z Analytical Chemistry, Vol. 78, No. 24, December 15, 2006

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Figure 8. An example of a dead-end cross-linked product from the reaction of PIR-3 with substance P. (A) The ions at m/z 1088.5549 correspond to a reaction product with 17-Da excess mass due to the hydrolysis of one of the reactive groups in PIR-3. (B) Fragmentation of m/z 1088.5549 at -18 V collisional trap voltage. (C) Fragmentation resulting from application of -27-V collisional trap voltage. Both the reporter ions and substance P ions added with one “linker arm” appeared in the spectra (B, C). The ion at m/z 1016.0129 corresponds to the loss of one MS/MS cleavable bond from the hydrolyzed end of the PIR-3 cross-linker.

Figure 9. Electron capture dissociation spectrum of an intra-cross-linked peptide of substance P and PIR-1 (see Figure 6 for CID spectra). All c ions (C10 - 1370.19, C9 - 1313.65, C8 - 1285.14, C7 - 1211.61, C6 - 1138.07) were observed with 2+ charge state. With ECD, fragmentation products resulting from the cleavage of labile bonds of PIR-labeled peptides were observed as minor products; however, peptide backbone cleavage products are observed with the PIR structures intact. An asterisk (*) denotes noise peaks.

1088.5549 (2+) (expected, m/z 1088.5560; mass accuracy, 1.0 ppm) were isolated in the quadrupole and fragmented in the hexapole with increasing collisional trap voltage (Figure 8A-C). At -18 V collisional trap voltage, both the reporter ion at m/z ) 556.2563 [M + H]+ and the modified substance P ion with one added linker arm at m/z ) 1475.7878 (1+) appeared in the 8192 Analytical Chemistry, Vol. 78, No. 24, December 15, 2006

spectrum (Figure 8B). One linker arm mass for PIR-3 was calculated to be 128.0712 Da (C6H10O2N1). The intensity of reporter ions (m/z 556.2572) and ions of modified peptides (m/z 1475.7901) were observed to increase with increased voltage (-27 V) in the collisional trap. These data show that PIR-3 was coupled at one end of substance P via a reactive group, and the other end was

hydrolyzed. For dead-end cross-linking, two additional ions may appear in the spectra. The diagnostic ion can result from the fragmentation of one labile bond from the hydrolyzed end of the cross-linker, which will be the loss of 145.07 Da from the deadend precursor peptide. The marker ion can result from the fragmentation of labile bond from the coupled peptide end of the cross-linker. This marker ion m/z will be constant, irrespective of dead-end precursor m/z. For this PIR-3, the dead-end hydrolyzed marker ion is 700.3220 Da. We did not observe any hydrolyzed marker ion at 700.3220 Da in the mass spectra, but we did observe the diagnostic ion m/z 1016.0129 (2+) that corresponds to the cleavage of one low-energy labile bond (145.0840 Da) from the hydrolyzed end of the PIR-3 (Figure 8BC). In addition, according to Figure 2C, the sum of the generated reporter and modified substance P (substance P + linker arm mass) fragment ion masses (2030.0285 Da) did not match the neutral molecular weight of the PIR-labeled precursor species (m/z 1088.5549 (2+), 2175.0942 Da). These spectra showed that with this new PIR-3, we can unambiguously distinguish dead-end crosslinking. To identify cross-linked peptides, accurate mass analysis and MS/MS can be performed on the released modified peptides. In Figure 9, we show an ECD spectrum of the intralinked substance P and PIR-1 that is distinctive from the observed CID behavior that can be used to identify the PIR-labeled cross-linked peptides. ECD generally results in cleavage of many more backbone bonds than collisional activation and allows preservation of labile side chain modifications.29 The ions at m/z ) 957.80, 3+ charge state, were isolated as previously described (Figure 6). The electron irradiation time was maintained at 150-200 ms. Several c-type fragment ions were generated from backbone cleavage of substance P. Both reporter ions and modified substance P ions were also observed in the spectrum at m/z ) 663.30, 1325.60, and 1546.78, respectively. All c ions were generated with added expected intact cross-linker mass. The sequence of substance P has two primary amine groups near the N-terminal end of the peptide, as mentioned before. From the c ions generated by ECD, we can conclude that this amine-reactive cross-linker was intralinked at the N-terminal end of substance P, where two possible primary amine functionalities are present. CONCLUSIONS We have investigated gas-phase fragmentation of three compounds that will have use as protein interaction reporters. (29) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563-573.

Distinctive fragmentation behavior of an intralinked peptide in lowenergy CID and ECD was also observed. The novelty of the PIR approach is a result of the engineered selective gas-phase fragmentation characteristics that generate reporter ions. These PIRs and PIR-labeled peptides showed the predicted fragmentation with low-energy CID conditions in the FTICR mass spectrometer. The essential feature as demonstrated in this paper is the capacity to distinguish dead-end and intra-cross-linked peptides and to produce sequence info on peptides by combination of ECD and CID. In addition, an alternative photo release strategy of PIRlabeled peptides from the avidin resin complex was demonstrated with a new PIR. A useful feature of the PIRs is that the mass and physical properties of the reporter ion can be tuned by substituting different amino acids in the linker. For this study, addition of an affinity group, a hydrophilic group, a photocleavable group, and a new low-energy labile group was accomplished while still preserving the low-energy fragmentation characteristics of these new PIRs. We feel these new cross-linkers and others of this class will be useful for large-scale identification of protein-protein interactions. ABBREVIATIONS CID, collision-induced dissociation; ECD, electron capture dissociation; FTICR-MS, Fourier transform ion cyclotron resonance mass apectrometry; NHS, N-hydroxysuccinimide; NHS-SA, N-hydroxysuccinimide reactive group/succinic acid linker; MALDITOF, matrix-assisted laser desorption ionization time of flight; MS/ MS, tandem mass spectrometry; PC, photocleavable group; PIR, protein interaction reporter; PEG, polyethylene glycol; RNase S, ribonuclease S; SA, succinic anhydride ACKNOWLEDGMENT This research was supported by the Office of Science (BER), U.S. Department of Energy, Grant No. DE-FG02-04ER63924 and The Murdock Charitable Trust. The authors thank Gordon A. Anderson and Nicola Tolic for the data analysis tools used in this work. SUPPORTING INFORMATION AVAILABLE MALDI-TOF mass spectra for the optimization of photocleavage time with UV light with the peptide [SARRL(photo)RK(Biotin)G]. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 27, 2006; Accepted September 22, 2006. AC060789H

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