Electron Transfer Dissociation in Conjunction with Collision Activation

May 12, 2009 - Molecular Life Sciences, 8057 Zurich, Switzerland, ThermoFisher ... for Systems Biology, Seattle, Washington 98103, Faculty of Science,...
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Electron Transfer Dissociation in Conjunction with Collision Activation To Investigate the Drosophila melanogaster Phosphoproteome Bruno Domon,*,†,# Bernd Bodenmiller,†,‡,# Christine Carapito,†,# Zhiqi Hao,§ Andreas Huehmer,§ and Ruedi Aebersold†,|,⊥,∇ Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland, Zurich Ph.D. Program in Molecular Life Sciences, 8057 Zurich, Switzerland, ThermoFisher Scientific, San Jose, California 95134, Institute for Systems Biology, Seattle, Washington 98103, Faculty of Science, University of Zurich, 8057 Zurich, Switzerland, and Competence Center for Systems Physiology and Metabolic Disease, ETH Zurich, 8093 Zurich, Switzerland Received May 21, 2008

Better understanding how cells are regulated and adapt to their environment based on the reversible phosphorylation of proteins is a key question of current molecular and systems biology research. In this study, an advanced mass spectrometry based approach leveraging the electron transfer dissociation (ETD) technique in combination with CID using a linear ion trap mass spectrometer is described. The technique was applied, for the first time, to the identification of phosphorylated peptides isolated from the Drosophila melanogaster Kc167 cell line. We demonstrate that the method is particularly useful for the characterization of large phosphopeptides, including those with multiple phosphorylation sites, as extensive series of c′ and z• fragment-ions were observed. Finally, we have applied a directed tandem mass spectrometric workflow using inclusion lists to increase the number of identified peptides. Keywords: ETD • fragmentation mechanism • phosphopeptide • supplemental activation • directed proteomics

Introduction Sequencing peptides by tandem mass spectrometry is a cornerstone of proteomics and has become central to many biological studies. Collision induced dissociation (CID) is the most widely used technique to fragment peptide ions in a mass spectrometer and has proven to be extremely useful for amino acid sequence assignment.1-3 Nevertheless, several limitations justify the need for alternative fragmentation techniques. First, the preferential cleavage of the frequently labile post-translational modification (PTM) bonds leading to fragment ion spectra dominated by the signal of the bare peptide after loss of the modification, with little sequence information. This problem is pronounced for peptides with labile modifications such as phosphorylations and O-glycosylations. Second, the well-known favored cleavage of fragile peptidic bonds such as Xxx-Pro or Asp-Xxx and GluXxx results in incomplete fragment ion series and thus only partial sequence information.4 Third, large peptides with * To whom correspondence should be addressed. Tel: +41 44 633 20 88. E-mail: [email protected]. † Institute of Molecular Systems Biology, ETH Zurich. ‡ Zurich Ph.D. Program in Molecular Life Sciences. # These authors contributed equally to this work. § ThermoFisher Scientific. | Institute for Systems Biology. ⊥ Faculty of Science, University of Zurich. ∇ Competence Center for Systems Physiology and Metabolic Disease, ETH Zurich. 10.1021/pr800834e CCC: $40.75

 2009 American Chemical Society

molecular masses exceeding 2500 Da are difficult to fragment by CID because the intramolecular vibrational energy redistributes over the large number of degrees of freedom, and is no longer sufficient to cleave peptidic bonds. Alternative fragmentation principles such as electron capture dissociation (ECD)5 and more recently electron transfer dissociation (ETD)6,7 have opened new avenues for the structural analysis of peptides (and proteins). While ECD has been primarily implemented in ion cyclotron resonance cells, ETD is typically performed in linear ion traps.6,7 Both techniques rely on the charge reduction of multiply charged peptide ions through electron transfer and the subsequent unimolecular dissociation of the resulting activated species to yield fragments of the peptidic backbone. The electron transfer produces an odd-electron molecular species (e.g., [M + 2H]+•), which undergoes subsequent cleavage of the N-CR bond to generate fragment ions of the c′ and z• types.6,8,9 ETD was first applied to analyze peptides with labile PTMs such as phosphorylated, sulfated or O-GlcNAc-ylated peptides.6,8,10-12 In contrast to CID, the modification remains attached to the backbone under ETD conditions and the resulting spectra typically exhibit informative ion series indicating both the amino acid sequence and the modification site(s). Furthermore, ETD (as ECD) is fairly insensitive to the size of the peptides and full sequence coverage can be obtained for larger peptides (20-75 residues) when the number of charges is sufficient to observe them at m/z values below 850.7 Journal of Proteome Research 2009, 8, 2633–2639 2633 Published on Web 05/12/2009

research articles Nevertheless, N-terminal cleavage to proline residues is not observed on ETD fragmentation spectra as it would require the dissociation of two bonds. Even though they are very informative in regards of peptide sequence information, the interpretation of ETD fragmentation spectra can be tedious due to the presence of c - 1 and z + 1 ion types introducing an additional complexity level in the spectra.13 Mass analyzers with high resolution and mass accuracy in both MS and MS/MS modes separating these ion species and determining the charge states of the peptide fragment ions will facilitate the data analysis.14 Accurate mass measurement is of paramount importance for the validation of modified peptide identifications and the reduction of false positive identifications has already been addressed in previous studies for CID data.15,16 In spite of the just mentioned advantages of ETD over CID, it has a low efficiency in fragmenting doubly charged peptides. This can be overcome by combining ETD with supplemental collision activation, as the energy transferred by ETD alone may not be sufficient to induce spontaneous dissociation of the radical-ion species.17,18 In this study, we applied a directed LC-MS/MS workflow using CID, ETD and electron transfer (ET) in combination with supplemental collisional activation (ET/CID) to analyze complex mixtures of phosphorylated peptides isolated from Drosophila melanogaster Kc167 cells. The analysis using CID and ETD in alternating scan mode leverages the complementarity of the two techniques to increase the coverage of the phosphoproteome. The ET/CID technique was applied to improve the characterization of large, multiply charged and highly hydrophilic peptides (rich in aspartic and glutamic acids) comprising multiple phosphorylation sites, which are difficult to be analyzed by CID.

Results Analysis of the Fly Phosphoproteome Using ETD in Combination with CID in a Directed LC-MS/MS Workflow. To map phosphorylation sites from the fly, we first performed a phosphopeptide isolation from Kc167 whole cell proteome digests using IMAC and then we performed LC-MS/MS analyses using CID and/or ETD on these isolates. As a result of these measurements, 304 and 153 phosphorylation sites were identified by CID and ETD, respectively. In addition, we found that the overlap between the two data sets was about 26% (Supporting Information Table 1), illustrating the complementarity of both approaches. We further wanted to test whether a directed proteomics approach based on inclusion lists could be implemented on a LIT instrument. The classical shotgun approach discriminates in favor of the most abundant components present in the sample as the intense ions observed on the MS spectra are preferentially selected for sequencing by CID and ETD (undersampling effect). Additionally, multiply phosphorylated peptides can display poor ionization efficiency, therefore, exhibiting weak signals and are consequently easily omitted. To test if these limitations of the DDA acquisition mode can be overcome, a directed LC-MS/MS workflow was tested.19,20 In this workflow, first, the sample was analyzed on a high mass accuracy and high resolution instrument (e.g., LTQ-FTICR) in three replicate runs. Second, an offline bioinformatics analysis of the data using the software tool SuperHirn21 allowed establishing a list of all ions reproducibly present in all three replicates with their cor2634

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Domon et al. responding attributes (m/z value, retention time and peak intensity).22 Third, a directed LC-MS/MS experiment was performed on an LTQ-ETD instrument in which all peptides of this list (inclusion list) are specifically fragmented by CID and ETD. Each directed LC-MS/MS run containing a part of the inclusion list yielded a unique set of identified (phospho)peptides. In the first run, 98 unique (phospho)peptides were identified. Continuing the analysis using two additional inclusion lists increased the overall number of identified (phospho)peptides to 297. These findings are in accordance with the findings described previously19 in which the directed LC-MS/MS sequencing was applied to samples produced from serum and yielded better results than repeated DDA LC-MS/MS analyses of the same sample.20 Thus, besides the obvious complementarity obtained between CID and ETD data, these results also demonstrate that a directed MS/MS sequencing strategy can be implemented in a beneficial way on a linear ion trap operated in CID/ ETD mode. Electron Transfer Followed by Collision Induced Dissociation, An Efficient Technique for Sequencing Large and Multiply Phosphorylated Peptides. The ion trap instrument offers the unique capability of multiple stages of mass selection and activation. In the case of poor spontaneous fragmentation of the partially charge reduced peptide ions after electron transfer, one specific charge state can be isolated and activated to induce dissociation as indicated in Figure 1. The additional collision activation applied to the most intense charge reduced species uses a low collision energy combined with a lower RF amplitude to preferentially fragment only the odd electron species (or the resulting complex), thus, producing an extensive series of c′ and z• ions. We will call this MS3 level experiment ET/CID. Figure 1 illustrates an example of ET/CID fragmentation on the peptide KKESNS*EDELEYDPSLYPQR. The electron transfer generated the charge reduced species [M + 3H]2+• and [M + 3H]+•• at m/z 1254.3 and at m/z 2510.3, respectively (Figure 1). The doubly charged reduced species was then selected and isolated (MS3 experiment) and gently collisionally activated to cleave the peptide backbone to generate c′ and z• ions. The extended series of ions allowed immediate assignment of the amino acid sequence of the peptide derived from the protein CG11844 (Figure 1 and Supporting Information Figure 3). As expected, the gentle collisional activation also resulted in a neutral loss peak, even though this was of low intensity. The ET/CID fragmentation technique was especially efficient for large and very acidic peptides. This is illustrated in Figure 2 using the quadruply charged 4896.4 Da peptide AAWSGTPLPQLAGGKPTVAAAAKPAADDDDDVDLFGS*DDEEDEEAER (ET precursor ion [M + 4H]4+ at m/z 1225.1, CID precursor ion [M + 4H]3+• at m/z 1632.7) as an example. The extensive series of fragment ions present in the spectrum allowed the identification of the peptide by database search, thus, demonstrating that large, multiply charged peptides can readily be sequenced when dissociated via supplemental collision energy. It is of note that a vast majority of the fragment ions predicted were detected either as singly or doubly charged species. The c37′2+ and z14+• fragment ions at m/z 1838 and m/z 1704.6 bearing the phosphate residue unambiguously localize the modification site (Supporting Information Figure 3). ET/CID also showed to be the technique of choice for fragmenting peptides bearing multiple phosphate residues.

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Figure 1. Fragmentation of a peptide by concomitant use of electron transfer and collision activated dissociation. (A) Charge reduction and formation the c′/z• complex upon ET of the precursor ion [M + 3H]3+ at m/z 837.0. (B) Fragmentation of the peptide KKESNS*EDELEYDPSLYPQR upon collision activation of the c′/z• complex [M + 3H]2+• at m/z 1254.3 from the protein CG11844 is shown.

Figure 3 illustrates this with 3 peptides bearing 2 and even 3 phosphorylation sites. Extensive series of c′ and z• ions allowed assigning the amino acid sequences with high confidence (Supporting Information Figure 3). The sites of phosphorylation were precisely determined by the presence of fragment ions exhibiting a +80 Da mass shift. The peptide KPLAPKPISEPIDIS*S*GDENEDDSNTK (Figure 3a, ET precursor ion [M + 3H]3+ at m/z 1020, CID precursor ion [M + 3H]2+• at m/z 1529.5) exhibits the dominant c′ ions at m/z 1683.9 (c15′+), 1850.9 (c16′+) as well as z• ions at m/z 1374.4 (z12+•) and 1541.4 (z13+•), unambiguously assigning the sites of phosphorylation. The peptide KPEANDDDDDVDLFGS*DS*EEEDGEAAR (Figure 3b, ET precursor ion [M + 3H]3+ at m/z 1034.6, CID precursor ion [M + 3H]2+• at m/z 1551.4) exhibits dominant c′ ions at m/z 1830.7 (c16′+) and m/z 2112.7 (c18′+) as well as a z• ion at m/z 1438.4 (z12+•) indicative of the modification sites. Finally, for the peptide SLRT*PT*PPGS*PTPSTSTAAQNLR (Figure 3c, ET precursor ion [M + 3H]3+ at m/z 860.2, CID precursor ion [M + 3H]2+• at m/z 1289.8), modified z• fragment ions at m/z 1494.7 (z14+•) and at m/z 2204.9 (z20+•) and the unmodified fragment ion at m/z 1230.6 (z12+•) pointed to phosphorylation of the residues in position 4, 6 and 10.

These results demonstrate that ET/CID allows effective sequencing of large peptides, with one or multiple phosphorylation sites, and that their identification and characterization is often straightforward. Also, peptides with a charge state higher than 2 can effectively be sequenced by the ET/CID technique. Of note, even though observable, the neutral loss ions visible in most tandem mass spectra generated using ET/ CID are of low intensity and do not disturb the unambiguous phosphorylation site assignment. When the list of peptides identified with ET/CID was compared with that of peptides identified using CID and ETD, it was found that (as expected) CID mostly identified doubly and triply charged peptides, whereas ETD and ET/CID have a bias toward charge states equal or higher than 3 and therefore peptides of higher mass (Supporting Information Figure 1). Finally, when the number of phosphorylation sites per phosphopeptide was analyzed, a bias of ETD toward singly phosphorylated phosphopeptides became apparent (Supporting Information Figure 2). As the peptides identified via ET/CID are derived from ET charge reduced precursors which did not dissociate, the pool of peptides identified via the ET/CID technique and pure ETD are complementary. Journal of Proteome Research • Vol. 8, No. 6, 2009 2635

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Figure 2. Electron transfer and collision activated dissociation mass spectrum of a 4896.4 Da phosphorylated peptide. Electron transfer and collision activation dissociation mass spectrum of the singly phosphorylated peptide AAWSGTPLPQLAGGKPTVAAAAKPAADDDDDVDLFGS*DDEEDEEAER with the ET precursor ion [M + 4H]4+ at m/z 1225.1 and the CID precursor ion [M + 4H]3+• at m/z 1632.7 from the protein EF1 beta is shown.

Conclusion This study has focused on identifying phosphorylated peptides in the fly phosphoproteome and on precisely localizing the modification sites on the peptide backbone by using ETD in combination with CID. A useful complementarity was observed between the two fragmentation techniques, as had already been illustrated in previous work.7 Additionally, the combination of electron transfer with supplemental collision activation enabled identification of high mass peptides with one or several phosphorylated residues. Many of the peptides identified throughout this study exhibit long amino acid sequences rich in acidic residues, and multiple phosphorylated residues for which the use of ETD with supplemental activation was essential for their successful identification. This technique opens new avenues in proteomics, as larger, multiply charged peptides can be readily sequenced. Further developments of the technique such as its implementation on hybrid instruments (e.g., orbitrap or quadrupole time-of-flight instruments) with high resolution and mass accuracy capabilities will drastically simplify the data analysis as the charge state of the fragment ions can be readily determined.

Material and Methods Materials. All chemicals, if not otherwise mentioned, were bought with the highest available purity from Sigma-Aldrich, Buchs, Switzerland. Sample Preparation. D. melanogaster Kc167 cells were grown as described previously.23 Starved cells were exposed to 100 nM insulin for 30 min. After this treatment, the samples were shock frozen in liquid nitrogen to preserve their phosphorylation state. Cell lysis, protein extraction, digestion and phosphopeptide isolation using IMAC was performed as described previously.22-24 In short, the isolation of phosphopeptides using IMAC was done as follows: 1.5 mg of peptide derived from the whole proteome digest was reconstituted in 30% acetonitrile (ACN) and 250 mM acetic acid at pH 2.7 and mixed with 30 µL of equilibrated PHOS-Select gel (Sigma-Aldrich) in a blocked mobicol spin column (MoBiTec, Go¨ttingen, Ger2636

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many). The mixture was incubated for 2 h with end-over-end rotation. Then, the PHOS-Select gel was washed two times with 250 mM acetic acid, 30% ACN at pH 2.7 and once with ultra pure water. Finally, phosphopeptides were eluted once with 50 mM and once with 100 mM phosphate buffer, pH 8.9. The pH of the eluate was adjusted to 2.75 for the subsequent reverse phase cleanup of the phosphopeptides. LC/MS/MS. The phosphopeptide isolates were analyzed by LC-MS/MS using a reverse phase HPLC coupled with a linear ion trap mass spectrometer Finnigan LTQXL (Thermo Scientific, San Jose, CA) equipped with ETD. Approximately, 1 µg of the sample was directly injected onto a Picofrit bioBasic C18 column, 10 cm × 75 µm (New Objective) and separated with a 80 min 0-40% linear gradient (A, 0.1% formic acid; B, 100% ACN/0.1% formic acid) at a flow rate of 0.3 µL/min using a Finnigan Surveyor HPLC equipped with a Micro AS and nanospray source (Thermo Fisher Scientific, San Jose, CA). The analyses were performed in the data-dependent acquisition mode (DDA). Briefly, a full MS spectrum was first acquired, and depending on the experimental setup, then, most intense peaks were selected for subsequent MS2 experiments. To achieve a resolution allowing determining a higher mass accuracy for the precursor masses and an efficient exclusion of singly charged precursors, the enhanced scan rate was used for the full MS spectrum. For the precursor charge states, the highly abundant charge reduced species present in the generated ETD spectra are reliable indicators for a proper charge state determination during the MS/MS data processing step.25 For each selected precursor, one CID and an ETD spectrum were acquired. In some specific experiments, a third scan was added where the charge reduced peptide ion generated by ET is selected for a subsequent experiment (MS3 without isolation) in which the ion is further activated by gentle collision activation. For the measurement in which only ETD was performed, for each MS1 scan, three MS2 ETD scans were triggered. For measurements in which ETD and CID were alternated, for each MS1 scan, subsequently three CID and three ETD scans were performed. For the ET/CID measure-

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Figure 3. Electron transfer and collision activated dissociation mass spectra of peptides with multiple phosphorylation sites. (a) Electron transfer and collision activated dissociation mass spectrum of the diphosphorylated peptide KPLAPKPISEPIDIS*S*GDENEDDSNTK with ET precursor ion [M + 3H]3+ at m/z 1020 and the CID precursor ion [M + 3H]2+• at m/z 1529.5 from the protein smid is shown. (b) Electron transfer and collision activation dissociation mass spectrum of the diphosphorylated peptide KPEANDDDDDVDLFGS*DS*EEEDGEAAR with the ET precursor ion [M + 3H]3+ at m/z 1034.6 and the CID precursor ion [M + 3H]2+• at m/z 1551.4 from the protein EG1 delta is shown. (c) Electron transfer and collision activation dissociation mass spectrum of the triphosphorylated peptide SLRT*PT*PPGS*PTPSTSTAAQNLR of the protein CG32425 with the ET precursor ion [M + 3H]2+• at m/z 860.2 and the CID precursor ion [M + 3H]2+• at m/z 1289.8.

ments, the most intense ion detected in the MS1 scan was selected for a subsequent MS2 experiment. Finally, the most intense ion present in the MS2 spectrum was further activated via CA (MS3 experiment). For the full MS scan, the AGC target value was set to 30 000.

CID was performed using an isolation width of 3 m/z and relative collision energy of 35% and with an AGC target value set to 10 000. ETD was performed using an isolation width of 3 m/z and an activation time of 100 ms. The precursor cation AGC target value was set at 80 000, whereas a value of 100 000 Journal of Proteome Research • Vol. 8, No. 6, 2009 2637

research articles was used for the fluoranthene anion population. For ET/CID of the charge reduced precursor ions, the isolation width was 50 m/z and the relative collision energy was 10% along with a low activation Q of 0.15. Data Analysis. All MS2 and MS3 spectra obtained were searched against a combined forward + reverse Drosophila FlyBase protein database (Version 4.3, www.flybase.com) containing the protein sequences of trypsin and human keratins (total of 38 930 entries) with the Sequest search engine (Bioworks, Version 3.3.1, Thermo Electron, San Jose, CA). The search criteria were as follows: full tryptic specificity was required (cleavage after K, R but not if followed by a proline); 2 missed cleavages were allowed; carbamidomethylation (C) was set as fixed modification and phosphorylations (S, T, Y) were applied as variable modifications; mass tolerance of the precursor ion and the fragment ions was 3 and 1.0 Da, respectively. The decoy database search was performed as described previously to assess the number of false positive peptide identifications.26 On the basis of this approach, the error rate was estimated to less than 5% (error rate ) 2 × percentage of decoy hits). For the analysis of the ETD and ET/CID data, the same settings as described for CID were used with the modifications that c and z ions were defined for the peptide fragment ions. Finally, phosphopeptides identified using CID with an associated dCn > 0.1 were considered to have a localized phosphorylation site, while for phosphopeptides with a dCn < 0.1, the site of phosphorylation is not accurately localized.16 Abbreviations: ETD, electron transfer dissociation; ECD, electron capture dissociation; CID, collision induced dissociation; NCI, negative chemical ionization; ET/CID, electron transfer in combination with supplemental collisional activation; IMAC, immobilized metal affinity chromatography.

Acknowledgment. We thank Paola Picotti and Martin Sadilek for fruitful discussions. This project has been funded in part by ETH Zurich, the Swiss National Science Foundation under Grant No. 31000-10767. R.A. was supported in part by a grant from F Hoffmann-La Roche, Ltd. (Basel, Switzerland) provided to the Competence Center for Systems Physiology and Metabolic Disease. B.B. was the recipient of a fellowship by the Boehringer Ingelheim Fonds. Nomenclature appendix The ion nomenclature proposed by Zubarev27 is applied. In essence, fragments resulting from the cleavage of the N-CR bonds, yielding an N-terminal even electron ion and its complementary C-terminal radical species were labeled c′ and z•, respectively. The odd electron species (i.e., that had gained one electron) is represented with a dot (z•), while the hydrogen atom transferred to the peptide backbone will be indicated by a prime (c′). The charge carried by either the c′ or z• ions result from additional protons attached to the fragment. Each proton added accounts for one charge; for instance, z•, z+• and z2+• denote a neutral fragment (formed in case of dissociation of a singly charged precursor), singly protonated and doubly protonated species, respectively.

Supporting Information Available: Peptides identified using ETD; peptides identified using CID; peptides identified using ET/CID; charge state distribution of identified phosphopeptides using CID, ETD or ET/CID for peptide fragmentation; 2638

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Domon et al. phosphorylation site distribution of identified phosphopeptides using CID, ETD or ET/CID for peptide fragmentation; detailed information on the ETD fragmentation spectra of the peptides presented in Figures 1-3. This material is available free of charge via the Internet at http://pubs.acs.org.

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