Letter pubs.acs.org/ac

Gas-Phase Rearrangement in Lysine Phosphorylated Peptides During Electron-Transfer Dissociation Tandem Mass Spectrometry Jordi Bertran-Vicente,*,†,§ Michael Schümann,† Christian P. R. Hackenberger,†,‡ and Eberhard Krause*,† †

Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle Str. 10, 13125 Berlin, Germany Department Chemie, Humboldt Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany § Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany ‡

S Supporting Information *

ABSTRACT: Tandem mass spectrometry (MS/MS) strategies coupled with collision-induced dissociation (CID) or radical-driven fragmentation techniques such as electron-capture dissociation (ECD) or electron-transfer dissociation (ETD) have been successfully used for comprehensive phosphoproteome analysis. However, the unambiguous characterization of the phosphorylation site remains a significant challenge due to phosphate-related neutral losses and gasphase rearrangements, which have been observed during CID. In particular, for the analysis of labile N-phosphorylated peptides, ECD and ETD are emerging as a complementary method. In contrast to CID, the phosphorylation site of histidine, arginine, and lysine phosphorylated peptides can be characterized by ETD. Here, we present a study on the application of ETD for analysis of phospholysine (pLys) peptides. We show that, depending on the charge state of the precursor ion as well as the presence of basic amino acid side chains, phosphate transfer reactions during the ETD process can be observed leading to ambiguous fragment ion spectra. Basically, pLys is stable under ETD conditions allowing an unambiguous assignment of the site of phosphorylation, but some factors/parameters have to be considered to avoid gas-phase rearrangement which would lead to false positive results in phosphoproteomic studies.

P

labeled by the following c and z nomenclature.11 The most important advantage of ETD/ECD is that CID-labile protein modifications such as phosphorylation on Ser or Thr remain unaffected during this fragmentation, which allows their unambiguous localization from the resulting MS/MS spectrum. Electron-driven fragmentation techniques have also been used to determine acid-labile N-phosphorylation of arginine (Arg), histidine (His), and lysine (Lys). Thus, for arginine and histidine phosphorylation, the phosphate is stable upon electron-transfer dissociation while MS/MS fragmentation using collisional activation leads to extensive elimination of phosphoric acid and increases the numbers of false localizations.12,13 Within this acid-labile group, phospholysine (pLys) is by far the least studied. Biochemical studies point toward a role of lysine phosphorylation in phosphoryl transfer reactions of nuclear proteins.14 Kinases and phosphatases with specificity toward Lys and pLys residues have been identified suggesting a role in regulation of Lys phosphorylation in histone H1.15 However, to date, no phosphorylation sites have been found due to the limitations of the synthetic and analytical tools available. Recently, the ECD method was shown to be capable of detecting pLys residues from synthetic peptides. However, significant phosphate-related losses were observed.16

hosphorylation of the hydroxyl functions of amino acids is a ubiquitous modification of proteins, involved in signal transduction processes such as cell differentiation, proliferation, energy storage, and apoptosis.1 Mass spectrometry (MS) has become the technique of choice to identify phosphorylated residues within proteins, whereas fragmentation analysis by tandem mass spectrometry (MS/MS) is used to confirm the peptide sequence and to assign the site of modification.2−4 However, the reliable localization of phosphorylation sites in proteins represents one of the most challenging tasks in phosphoproteomic studies even though a number of techniques have been developed for specific enrichment, chromatographic separation, and subsequent MS/MS analysis of phosphopeptides. Using collision-induced dissociation (CID), the conventional tandem MS method for analysis of phosphoserine (pSer), and phosphothreonine (pThr) peptides, a loss of phosphoric acid (H3PO4) and gas-phase rearrangements of the phosphate moiety onto an unmodified serine (Ser) or threonine (Thr) side chain have been reported.5−7 Recently, radical-driven fragmentation techniques, such as electron-transfer dissociation (ETD) and electron-capture dissociation (ECD), have gained attraction for labile PTMs assignment like phosphorylation and glycosylation.8−10 ETD provides peptide fragmentation by transferring an electron from a radical anion to a positively charged peptide ion. The resulting ETD spectrum displays generally Cα-N cleavage fragments, which are commonly © XXXX American Chemical Society

Received: April 14, 2015 Accepted: June 25, 2015

A

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Analytical Chemistry Table 1. Phosphorylated Peptides Analyzed by nLC-MS-ETDa peptide 1 2 3 4 5 6 7 8 9 10 a

peptide sequence

precursor ion charge state (z)

RLYKLpKASKLAR12 1 RLYKLKApSKLAR12 1 RLYpKLKASKLAR12 1 RLYKLKASpKLAR12 1 RLpYKLKASKLAR12 1 RLYKLpKASKLAK12 1 KLYKLpKASKLAR12 1 KLYKLpKASKLAK12 1 ALYKLpKASKLAR12 1 ALYKLpKASKLAA12

4 3 4 4 3 4 4 4 4 3

1

observed fragment ions c1, c1, c1, c1, c1, c1, c2, c2, c2, c2,

c2, c2, c2, c2, c2, c2, c3, c3, c3, c3,

c3, c4, c5, c6, c7, c8, c9, c10, c11 z1, z2, z3, z4, z5, z6, z7, z8, z9, z10, z11 c3, c4, c4, c5, c6, c7, c8, c9, c10 z1, z2, z4, z5, z6, z7, z8, z9, z10, z11 c3, c4, c4, c5, c6, c7, c8, c9, c10, c11 z1, z2, z3, z4, z5, z6, z7, z8, z9, z10, z11 c3, c4, c4, c5, c6, c7, c8, c9, c10, c11 z1, z2, z3, z4, z5, z6, z7, z8, z9, z10, z11 c3, c4, c5, c6, c7, c8, c9, c10, c11 z1, z2, z3, z4, z5, z6, z7, z8, z9, z10, z11 c3, c4, c5, c6, c7, c8, c9, c10, c11 z2, z3, z4, z5, z6, z7, z8, z9, z10, z11 c4, c5, c6, c7, c8, c9, c10, c11 z2, z3, z4, z5, z6, z7, z8, z9, z10, z11 c4, c5, c6, c7, c8, c9, c10, c11 z2, z3, z4, z5, z6, z7, z8, z9, z10, z11 c4, c5, c6, c7, c8, c9, c10, c11 z2, z3, z4, z5, z6, z7, z8, z9, z10, z11 c4, c5, c6, c7, c8, c9, c10, c11 z3, z4, z5, z6, z7, z8, z9, z10, z11

Fragments ions containing a phosphate group are indicated in bold.

Figure 1. (A) ETD MS/MS of the triply charged precursor ion ([M + 3H]3+) of peptide 1 in which the expected fragment ions are labeled. Phospho-site assignment at position Lys6 (B), (C) at position Lys4, and (D) at position Ser8 indicating gas-phase rearrangement reactions in peptide 1. Fragments ions with an asterisk indicate the presence of nonphosphorylated fragments.

water and 0.1% formic acid in acetonitrile (ACN) were purchased from Biosolve (Valkenswaard, The Netherlands). Synthesis of Phosphorylated Peptides. pLys lysine peptides were synthesized following our recently reported synthetic protocol.17 In brief, azido lysine peptides were synthesized by SPPS and reacted with phosphite esters. Final phosphoramidate ester deprotection was performed under basic conditions to deliver pLys peptides. pSer and pTyr peptides were synthesized following standard Fmoc-SPPS protocols and using the corresponding commercially available Fmoc-protected phosphorylated building blocks. ESI Tandem MS Using ETD Source. For LC-MS analysis, peptides were dissolved in 6 μL of water (7 pmol/μL) and analyzed by a reversed-phase capillary liquid chromatography system (Dionex Ultimate 3000 NCS-3500RS Nano, Thermo Scientific) connected to an LTQ Orbitrap XL mass spectrometer (Thermo Scientific) equipped with an ETD module (Thermo Scientific). LC separations were performed

We recently reported the unambiguous characterization of pLys peptides using ETD fragmentation by using a novel approach for site-specific incorporation of the N-phosphorylated lysine residues into peptides.17,18 In the present work, we report a first study about migration processes during the analysis of pLys peptides by ETD tandem mass spectrometry. By using an artificial peptide sequence based on the amino acid composition of Histone H1, we show the influence of the charge state selection with the phosphorylation site assignment process as well as the influence of basic amino acid side chains during ETD fragmentation.

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EXPERIMENTAL SECTION Materials. Fmoc-protected amino acids and TG HMBA resin were purchased from Novabiochem (Darmstadt, Germany). Fmoc-Lys(N3)-OH was purchased from Iris Biotech (Marktredwitz, Germany). LC/MS grade 0.1% formic acid in B

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Analytical Chemistry on a capillary column (Acclaim PepMap C18, 2 μm 100 Å, 250 mm × 75 μm i.d., Dionex) with an eluent flow rate of 300 nL/ min using a gradient of 2−23% B in 20 min. Mobile phase A contained 0.1% formic acid in water, and mobile phase B contained 0.1% formic acid in acetonitrile. Mass spectra were acquired in a data-dependent mode with one MS survey scan (with a resolution of 30 000) in the Orbitrap and ETD scans of the phosphorylated peptide precursor ions in the Orbitrap mass analyzer at a resolution of 7500. Peptide ions (z = 3 and 4) were fragmented using an include list. Activation time for ETD was 70 ms for all measurements. Before ETD experiments, the ETD source was tuned to the optimal signal of fluoranthene anions. ETD spectra were acquired without using supplemental activation. MS spectra and ETD fragment ion spectra of phosphopeptides were manually verified and compared with the theoretical fragment ions phosphorylated-lysine peptides considering all possible phosphorylation sites.

mentioned before, this observation has been previously reported for Ser and Thr-phosphorylated peptides during CID fragmentation,6,7 leading to an ongoing discussion about the influence of CID mediated gas-phase rearrangement on the reliability of site localization using classical phosphoproteomics approaches.6,19−21 Gas-phase rearrangements during CID, which are highly dependent on charge state and amino acid composition of the precursor ion, might be connected to an elimination of phosphoric acid and water during the fragmentation process. However, the detailed mechanism of gas-phase phosphate group rearrangement remains unclear.6 Our data clearly show that phosphate migration does occur not only in CID but also in electron-based fragmentation. We hypothesize that the mechanism might be the same as in CID leading to a relatively stable rearrangement product, which can be observed by ETD fragmentation. To the best of our knowledge, a phosphate rearrangement in ETD has not been reported before. To evaluate the influence of the charge state, we performed ETD using the precursor ion with charge 4+ (Figure S3, Supporting Information). Interestingly, this spectra did not show fragment ions indicating rearrangement reactions as observed when fragmenting the precursor ion with charge 3+ (Figure 2). Specifically, the fragment ions (z5# and c4#)

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RESULTS AND DISCUSSION In order to study the ETD fragmentation behavior of lysinephosphorylated peptides in greater detail, model peptides were synthesized as recently described.17 The peptides sequences (1−10) were based on the amino acid composition of the Nterminal region of Histone H1 which is highly enriched with basic amino acid side chains such as Arg and Lys. Thereby, we chose the following artificial peptide sequence X1LYKLKASKLAX12, where the charge and hydrophobicity of the peptides (1, 6−10) with phosphorylation at position 6 were varied by Arg/Lys and Arg/Ala replacements (Table 1). All phosphorylated peptides were analyzed by nanoLC-ESI-MS/ MS using ETD, and the fragment ions were measured in the Orbitrap mass analyzer. In the outset of our studies, when analyzing peptide 1 containing Arg in positions 1 and 12 and phosphorylated at Lys6, we observed that the ETD MS/MS spectra of the triply charged (z = 3) precursor ion clearly confirm the phosphorylation in position Lys6 in contrast to CID where complete neutral loss of phosphate is observed (Figures 1A,B and S2, Supporting Information). However, some ambiguous fragment ions with respect to the phosphorylation site assignment analysis were found in ETD. A detailed analysis of the MS/MS spectra revealed c and z fragment ions indicating phosphorylation of other phosphoacceptor residues. Specifically, ion fragments indicating phosphorylation at Lys4 and Ser8/Lys9 were found (Figure 1C,D). Additionally, we observed the presence of nonphosphorylated z7*, c6*, c4*, z5*, and c8* fragments for peptide 1, indicating elimination of the phosphate moiety for each phosphorylated assignment (Figure 1B−D). As a consequence of this result, we decided to synthesize all the peptides with all potential phosphorylation sites in order to rule out that this observation is due to the synthetic approach and not a merely gas-phase effect. As mentioned before, we recently reported that the synthetic approach used here is site-specific, targeting only the azide moiety and no other nucleophilic side chains.17 Analysis by liquid chromatography (LC) of all potential phosphopeptides showed that peptides 1, 2, 3, 4, and 5 have different retention times (Figure S1, Supporting Information) which excludes that a phosphate transfer during the synthesis of peptide 1 or during sample preparation (pH = 7.1 in water) is responsible for the presence of fragment ions indicating phosphorylation at Lys4 and Ser8/Lys9. We therefore considered a phosphate transfer in the gas-phase as the main hypothesis for an such effect. As

Figure 2. ETD MS/MS of the (A) triply charged precursor ion of peptide 1 showing the z5# and c4# fragments of phosphorylation at Lys4 and Ser8 or Lys9 and (B) quadruply charged precursor ion of peptide 1 containing only the expected ion fragments of phosphorylation at Lys6. Red lines showed the disappearance of z5# and c4#.

indicating phosphorylation at Lys4 and Ser8 or Lys9 did not show up, and only fragments confirming phosphorylation at Lys6 were observed (Figures 2, S4, and S5, Supporting Information, and Table 1). Furthermore, the stability of the phosphate-modified lysine side chain during ETD MS/MS of the 4+ charge state (Figure S3, Supporting Information) is demonstrated by the absence of nonphosphorylated z7* and c6* fragments. A similar effect was reported during CID-MS/ MS analysis of synthetic pSer, pThr, and pTyr peptides.6 The C

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Analytical Chemistry

Figure 3. ETD MS/MS of the triply charged precursor ion ([M + 3H]3+) of peptide 10 indicating the phosphorylation assignment at position Lys6. No c6* and z7* nonphosphorylated fragment ions were found.

observed transfer of phosphate group from the phosphorylated residue to an unmodified hydroxyl-containing amino acid was highly dependent on the precursor charge state and proton mobility, being observed predominantly for peptides under “nonmobile” or “partially mobile” protonation conditions.22 Interestingly, fragmentation of the phosphoester peptides 2 (pSer) and 5 (pTyr) by ETD selecting the 3+ charge state precursor ion led to unambiguous phosphorylation site assignment, without indication of gas-phase phosphate group rearrangement (Figures S6 and S11, Supporting Information). Since the basic amino acid side chains of Arg and Lys play an important role on the proton mobility in the gas phase, we decided to synthesize peptide sequences, which contain amino acid substitutions at the very terminal positions (Table 1). First, we checked whether exchanging Arg to Lys residues on both Nand C-terminal sides have an influence on the fragmentation process with the 3+ charge state. Thereby, we synthesized peptides 6, 7, and 8 having one or both lysine exchanged. As expected, the presence of nonphosphorylated c6* and z7* fragments ions was observed when analyzing the 3+ charge state for peptides 6, 7, and 8 (Figures S12, S14, and S16, Supporting Information). In addition, fragment ions indicating phosphorylation at positions Lys4, Ser8, and Lys9 were found. In contrast, the ETD MS/MS spectra of the 4+ charge precursor ion led to an unambiguous phosphorylation site assignment to Lys6 without the presence of the nonphosphorylated c6* and z7* fragments ions (Figures S13, S15, and S17, Supporting Information). With this information in hand, we decided to check whether or not exchanging Arg to a more hydrophobic amino acid like alanine (Ala) in both the N- and C-terminal domains has an influence. Peptides 9 and 10 were synthesized accordingly (Table 1). In peptide 9, replacing Arg with Ala at the very Nterminus did not have any influence, and as before, fragment ions indicating gas-phase rearrangement were observed when fragmenting the 3+ charge state precursor (Figure S18, Supporting Information). In contrast, fragmentation of the 3+

charge state precursor ion of peptide 10, having both N- and Cterminal basic amino acids exchanged to alanine, showed neither nonphosphorylated (z7* and c6*) nor fragment ions indicating a phosphate transfer reaction. Exclusively, the expected fragment ions for phosphorylation at position Lys6 were observed (Table 1, Figures 3 and S20, Supporting Information).

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SUMMARY AND CONCLUSIONS Taken together, pLys is sufficiently stable under ETD conditions. However, fragmentation of phospholysine peptides displayed secondary fragment ions, which indicate phosphate transfer to other phosphoacceptor amino acids. Interestingly, gas-phase rearrangements of the phosphate moiety were observed only when fragmenting the 3+ charge state precursor ion but not when selecting the 4+ charge state precursor ion. Furthermore, the presence of basic Arg and Lys residues has an impact on the formation of gas-phase rearrangement products by reducing the proton mobility. As described for CID fragmentation of O-phosphorylated peptides,6,23 in ETD of N-phosphorylated peptides, neutral loss of phosphoric acid and gas-phase rearrangement seems to be related to “nonmobile” or “partially mobile” protonation conditions. Our data shows that ETD MS/MS is generally useful for identification of lysine-phosphorylated peptides in phosphoproteomics approaches. However, as described for CID analysis of O-phosphorylated peptides, gas-phase phosphate group rearrangement reactions (“phosphate scrambling”) cannot be excluded. To avoid that the phosphate group rearrangement affects the identification and the reliability of phosphorylation site localization in pLys-phosphoproteomics, proper experimental conditions (proton mobility conditions) and careful data analysis should be applied. Taking into consideration that phospholysine has been found in lysine-rich histone H1 protein,14c this work might be a valuable contribution when fragmenting by ETD MS lysine-rich peptides containing phospholysine residues. D

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(21) Aguiar, M.; Haas, W.; Beausoleil, S. A.; Rush, J.; Gygi, S. P. J. Proteome Res. 2010, 9, 3103−3107. (22) Kapp, E. A.; Schutz, F.; Reid, G. E.; Eddes, J. S.; Moritz, R. L.; O’Hair, R. A. J.; Speed, T. P.; Simpson, R. J. Anal. Chem. 2003, 75, 6251−6264. (23) Palumbo, A. M.; Tepe, J. J.; Reid, G. E. J. Proteome Res. 2008, 7, 771−779.

ASSOCIATED CONTENT

S Supporting Information *

LC chromatograms and MS spectra of peptides as noted in the text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01389.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge support from the DFG (SFB 765 and SPP 1623), the Fonds der Chemischen Industrie, the Einstein Foundation, and the Boehringer-Ingelheim Foundation (Plus 3 award).

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