Screening for Phosphorylated and Nonphosphorylated Peptides by

Article pubs.acs.org/ac

Screening for Phosphorylated and Nonphosphorylated Peptides by Infrared Photodissociation Spectroscopy Corey N. Stedwell, Amanda L. Patrick, Kerim Gulyuz, and Nicolas C. Polfer* Department of Chemistry, University of Florida, Post Office Box 117200, Gainesville, Florida 32611, United States S Supporting Information *

ABSTRACT: We present an infrared laser-based mass spectrometric strategy to differentiate peptides that are phosphorylated (i.e., containing pS, pT, or pY) from those that are nonphosphorylated (i.e., containing S, T, or Y), and those peptides that have none of these moieties (i.e., containing neither pS, pT, pY nor S, T, Y). This is demonstrated for a series of tripeptides and for two larger octapeptides, showing that the diagnostic phosphate OH stretch (indicative for pS, pT, or pY) can be distinguished from the alcohol OH stretch (indicative for S, T, or Y). In addition, the infrared multiple photon dissociation (IRMPD) spectra of multiple peptide analytes are recorded simultaneously in a multiplexed fashion. This is achieved by complexing each peptide precursor with a noncovalently bound 18-crown-6 ether, which is detached upon resonant infrared photon absorption.

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hosphoproteomics 1 is an important subbranch of proteomics, aimed at characterizing proteins that undergo the posttranslational modification (PTM) of phosphorylation. This reversible modification takes place on the alcohol groups of serine, threonine, or tyrosine side chains, and plays a key role in cell signaling.2,3 Mass spectrometric techniques are among the most powerful in identifying and localizing phosphorylations in peptides and proteins.4−7 These techniques involve dissociation of mass-selected peptides in a mass spectrometer in order to verify the sequence, and thus the location of the phosphate site. Chemically, phosphates are labile groups and are hence often lost in conventional heating techniques, such as collision-induced dissociation (CID), consequently complicating sequence analysis. In addition, it has been shown that the location of phosphate groups can be scrambled upon collisional activation, due to rearrangement reactions.8 Radical-mediated dissociation techniques, such as electron capture dissociation (ECD)9,10 and electron transfer dissociation (ETD),11,12 are much more amenable to phosphopeptide sequencing; ECD and ETD involve selective cleavage of backbone bonds while conserving labile PTMs, such as phosphorylations.11,13, As phosphorylations are acidic PTMs, phosphorylated peptides often ionize more efficiently in the negative ion mode.1 Equivalent negative-ion techniques to ECD and ETD, such as electron detachment dissociation (EDD),14 negative electron transfer dissociation (NETD)15−17 and negative electron capture dissociation (nECD),18 can increase the information in bottom-up phosphoproteomics. In the absence of chemical labeling strategies, the peptide mass does not identify which peptides are phosphorylated.19 For the sequencing approaches above, a screening technique would be useful to identify selectively those peptides that are or could be phosphorylated. Ion mobility has been applied to this © 2012 American Chemical Society

task with some success, as phosphorylated peptides are often more compact than their unphosphorylated counterparts, presumably due to the negatively charged phosphate groups.20,21 High-field asymmetric waveform ion mobility spectrometry (FAIMS) is also well suited to the separation of phosphopeptide isoforms.22,23 Nonetheless, while ion mobility often excels in separating mixtures of isomers, it is more difficult to establish a general feature in ion mobility data that unambiguously identifies phosphorylated peptides in unknown samples. An alternative approach involves detection of vibrational frequencies that are diagnostic for phosphorylated peptides. Infrared multiple photon dissociation (IRMPD) using fixedwavelength CO2 lasers at 10.6 μm (i.e., 943 cm−1) selectively photodissociate phosphorylated peptides while exhibiting little photodissociation of unphosphorylated peptides.24−28 Infrared spectroscopy studies on phosphorylated amino acids, aided by computational approaches, confirmed that the phosphate P−O stretching and POH bending have strong activity in this region.29 Similarly, an IRMPD spectrum on phosphotyrosine in the hydrogen stretching region has shown that the phosphate OH stretches are located at ∼3665 cm−1, which is higher in frequency than other vibrations in the molecule30 and could hence serve as a diagnostic region for identifying phosphopeptides. Here, we apply IRMPD spectroscopy to the task of identifying phosphorylated as well as non-phosphorylated peptides by virtue of their diagnostic vibrations in the OH Received: August 10, 2012 Accepted: October 18, 2012 Published: October 18, 2012 9907

dx.doi.org/10.1021/ac3023058 | Anal. Chem. 2012, 84, 9907−9912

Analytical Chemistry

Article

Scheme 1. Crown Ether 18-Crown-6 (18c6) Binding Partner Bound to Protonated Peptides Is Detached for the Example of Resonant IR Photon Absorption by the Serine OH Stretch Moiety

bursts shuttered at 10 Hz), in order to induce infrared multiple photon dissociation (IRMPD). At each wavelength step, the remaining precursor and photofragments were mass-analyzed in a time-of-flight (ToF) drift tube (Jordan TOF Products, Grass Valley, CA). The relative ion abundances were determined by integrating the ToF mass spectral peaks with in-house LabVIEW software. The IRMPD yield is defined here as the linear photodissociation yield = ∑(photofragments)/∑(photofragments + precursor), which was normalized with the relative OPO power at each wavelength step. The (wavelengthdependent) IRMPD spectrum was thus obtained by monitoring the IRMPD yield versus OPO wavenumber (cm−1).33,34 Control experiments have shown that the sensitivity of the ToF detector allows detection of 0.1 μM concentrations of these peptides, but 1 μM concentrations are required to allow reasonable signal-to-noise IRMPD spectra to be recorded. The aim of the experiment was to measure the IRMPD spectra of all ions simultaneously, in a multiplexed fashion (see the first section under Results and Discussion). The mass isolation capabilities of the QIT were somewhat limited. Lower m/z impurities were ejected by raising the rf amplitude temporarily (e.g., 2 ms); however, higher m/z impurities could not be ejected in these experiments. Nonetheless, only a few impurities were observed, and on the basis of control experiments it was confirmed that none of their mass channels overlapped with those of the analyte ions.

stretching region. It is shown that the phosphate OH stretches in phosphorylated peptides (i.e., containing pS, pT, or pY) can be clearly distinguished from the alcohol OH stretches in nonphosphorylated peptides (i.e., containing S, T, or Y), as opposed to the absence of either mode in peptides that lack any of these groups. In addition, the IRMPD spectra of these peptides are recorded simultaneously, in a multiplexed fashion, so that the multiple peptides can be screened for (putative) phosphorylation sites.

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EXPERIMENTAL SECTION Peptides. Phosphorylated amino acid residues were purchased from Novabiochem (Hohenbrunn, Germany), while all other amino acid residues were purchased from CreoSalus (Louisville, KY). The residues were used without further purification. Peptides were synthesized following conventional Fmoc solid-phase synthesis methods31 on an Applied Biosystems Synergy model 432A peptide synthesizer. The peptides were cleaved from the resin using a 3 mL solution of 95% trifluoroacetic acid (TFA), 2.5% HPLC-grade water, and 2.5% triisopropylsilane. The peptide was then precipitated from the cleavage solution with ice-cold diethyl ether, followed by three rounds of centrifugation and purification. After the last amount of ether was decanted, the product was air-dried overnight. Mass Spectrometry. All experiments were performed on a custom-built mass spectrometer, described in detail elsewhere.32 The peptide solutions were made up at 100 μM in water/methanol/formic acid (30:70:1). The crown ether 18crown-6 (18c6) (Sigma−Aldrich, St. Louis, MO) was added at a 2:1 stoichiometric ratio to the peptide solutions. The peptide−crown complexes were formed by electrospray ionization (ESI) in a modified commercial ion source (Analytica, Branford, CT), equipped with a laboratoryconstructed stainless steel inlet capillary and an ion funnel. The ESI-generated ions were trapped in a reduced-pressure (∼10−5 mbar) quadrupole ion trap (QIT) to allow lengthy (i.e., 1.3−1.8 s) irradiation with focused IR beams from a tunable benchtop optical parametric oscillator/amplifier (OPO/A) (Linos Photonics OS4000) and a continuous-wave CO2 laser (Apollo Lasers, Los Angeles, CA) operating at 0.45 W (50 ms

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RESULTS AND DISCUSSION Multiplexing the IRMPD Experiment. Multiplexed IRMPD Spectroscopy. A key limitation in IRMPD spectroscopy experiments is its extremely low duty cycle, as at any one time the photodissociation of one mass-selected ion packet is irradiated at one discrete wavelength. In order to overcome this constraint, we implemented a multiplexed approach to IRMPD spectroscopy. Simultaneously measuring the IRMPD spectra of multiple components in a mixture requires that the mass channels for the precursor and its corresponding photofragment are unambiguously assignable. One strategy to achieve this goal is to complex the precursor with a noncovalently bound molecule, which is then detached upon resonant infrared photon absorption. This approach is well-known from vibrational 9908



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Figure 1. Mass spectra of a mixture of 18c6-adducted unphosphorylated (left) and phosphorylated (right) tripeptides subjected to irradiation at different IR frequencies. Color-coding highlights unambiguous mass channels for precursors and corresponding photofragments. Unlabeled mass peaks arise from impurities and their photodissociation products.

crown ether had also been described in a recent threshold collision-induced dissociation (TCID) study.41 Figure 1 depicts the mass spectra of a mixture of 18c6adducted tripeptides with the sequence motif GXK [where X = D, E, S, T, Y (left) and X = pS, pT, pY (right)] at different OPO frequencies. The mass channels of the precursor and corresponding photofragment are shown by color-coding, supporting the view that IR activation leads to nearly exclusive loss of 18c6. The detailed assignment of this data is given in Table S1 (Supporting Information), showing that some subsequent dissociation takes place and that minor impurities are present. This observation is not unexpected, as the gasphase basicities of these lysine-containing peptides is higher than 18c6, thus leading to exclusive retention of the charge (i.e., proton) on the peptide. By virtue of this simple photodissociation pattern, the IRMPD spectra of a mixture of analytes can be conveniently recorded at the same time. Another striking observation in these data is that the photodissociation is wavelength-dependent and that some adducted peptides selectively photodissociate at discrete wavelengths, while others do not. It is evident that each ion photodissociates at 3578 cm−1, as each contains a carboxylic acid moiety; however, only ions with alcohol moieties (GYK, GTK, and GSK) photodissociate at 3657 cm−1, and only ions with phosphorylated residues (GpSK, GpTK, and GpYK) photodissociate at 3668 cm−1. This behavior is clearly related to the presence/absence of various moieties in the analyte ions. Detection of Alcohol OH and Phosphate OH Moieties. IRMPD Spectra. The (wavelength-dependent) IRMPD spectra for the five 18c6-complexed tripeptides discussed above, and for three phosphorylated analogues, are shown in Figure 2. These IR spectra encompass all of the modes in the hydrogenstretching region. Tentative band assignments are indicated by

predissociation spectroscopy, where ions are tagged with a weakly bound van der Waals atom or molecule.35,36 The latter method works for cold ions, cooled in a supersonic expansion or cryogenic trap. For room-temperature ions stored in conventional trapping mass spectrometers, more strongly bound binding partners need to be considered. Choice of Binding Partner. In terms of enhancing the efficiency of multiplexed IRMPD spectroscopy, the ideal binding partner (1) has a high binding affinity for the analyte ion (to maximize the complexation efficiency), (2) does not distort the diagnostic spectroscopic range of the analyte molecules in an unpredictable way, and (3) yields exclusive detachment of the binding partner upon IR photon absorption (thus producing a sole, unambiguous detection channel). Criteria 1 and 3 are somewhat contradictory, in that a higher binding affinity could lead to other dissociation channels being competitive with loss of the binding partner. Scheme 1 illustrates the complexation approach to multiplexed IRMPD spectroscopy for the case of the crown ether 18crown-6 (18c6) bound to a peptide. 18c6 has a high binding affinity for peptides and for protonated amine (i.e., NH3+) groups in particular, ensuring efficient adduct formation of the peptide in the electrospray ionization (ESI) process.37−39 The crown is then detached upon resonant photon absorption by an IR-active moiety. As shown in a recent IRMPD spectroscopy study on 18c6-adducted amino acids, the specific binding of 18c6 to protonated amine groups resulted in only minor blue shifts (10−15 cm−1) of local oscillator modes, such as the indole NH stretch, carboxylic acid OH stretch, and alcohol OH stretches.40 Nonetheless, in that study, the exclusive loss of the binding partner was not always observed, as the crown ether could also compete for the proton, notably for lower-basicity amino acids. Note that the role of the gas-phase basicity of the 9909



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Composition of IR Spectrum. A more detailed understanding of a particular IRMPD spectrum can be obtained by decomposing the spectrum into the individual components. For instance, as shown in Figure 3, the IRMPD spectrum for the

Figure 2. IRMPD spectra (linear yield) of 18c6-adducted tripeptides: GDK, GEK, GSK, GpSK, GTK, GpTK, GYK, and GpYK. Diagnostic bands (and associated amino acid residues) are color-coded: crown CH stretches (dark gray), NH3+ stretch modes (yellow), carboxylic acid OH stretches (blue), alcohol OH stretches (green), and phosphate OH stretches (purple).

Figure 3. IRMPD spectra (linear yield) of the crown-adducted, protonated tripeptide 18c6-GYK, and the crown-adducted, protonated amino acids tyrosine and lysine. Diagnostic bands are color-coded: crown CH stretches (dark gray), NH3+ stretch modes (yellow), carboxylic acid OH stretch (blue), and alcohol OH stretch (green).

color-coding and are based on quantum-chemical calculations from previous studies.30,40 Briefly, the crown ether CH stretches (dark gray), NH3+ stretch modes (yellow), carboxylic acid OH stretches (blue), alcohol OH stretches (green), and phosphate OH stretches (purple) can be distinguished, and these diagnostic regions are summarized in Table 1. The

tripeptide 18c6-GYK can be understood in terms of the contributions of the protonated amino acids Y (tyrosine) and K (lysine) (recorded previously).40 Notably, the phenol OH stretch is observed only for Y-containing species. While the crown CH stretches and carboxylic acid OH stretches are observed for each, the strength of the NH3+ vibrational modes observed for 18c6-GYK is attributed mainly to the presence of lysine. Due to the tight binding of the crown ether to the NH3+ group, the effect of crown ether complexation is most pronounced for the NH3+ stretches. In particular, the antisymmetric NH3+ stretch mode is noticeably red-shifted upon complexation, as discussed previously.40 On the other hand, the local oscillator modes are only weakly blue-shifted and give rise to discrete, resolvable bands. The present results support the thesis that crown ethers do not adversely affect the diagnostic region of local oscillator modes in peptides either. Larger Peptides. In order to test whether these diagnostic band positions for alcohol OH and phosphate OH stretches also apply to larger peptides, we have extended the study to the non-phosphorylated octapeptide GAAAAAYK and its phosphorylated counterpart GAAAAApYK. The IRMPD spectra are compared in Figure 4, clearly showing that the alcohol OH (green) and phosphate OH (purple) stretches can be distinguished. In addition, the diagnostic band positions remain identical to those reported for the tripeptides. In other words,

Table 1. Summary of Vibrational Ranges for 18c6-Adducted Peptides vibrational modes

range, cm−1

phosphate OH stretches phenol/alcohol OH stretches carboxylic acid OH stretches NH3+ stretches crown CH stretches

3665−3685 3640−3670 3550−3590 3000−3280 2850−2980

presence and absence of various bands are consistent with expectations. GDK and GEK are representatives of peptides that lack both phosphate and alcohol OH stretch bands. GSK, GTK, and GYK are representatives for non-phosphorylated peptides; they exhibit prominent alcohol OH stretch bands but lack a definite phosphate OH stretch. Finally, GpSK, GpTK, and GpYK are representatives for phosphorylated peptides, exhibiting discrete phosphate OH stretches but lacking an alcohol OH stretch. It should be noted that the alcohol OH stretch displays some background photodissociation that is overlapping with the phosphate OH stretches. Nonetheless, this is produced more as a shoulder on the high-frequency side of the IR band, rather than as a discrete IR feature. 9910



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Article

ASSOCIATED CONTENT

S Supporting Information *

One table giving an assignment of the peaks in Figure 1. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone 352-392-0492; fax 352-392-0872; e-mail polfer@chem. ufl.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.C.P. thanks the University of Florida for generous start-up funds. This research is financially supported by the National Science Foundation under Grant CHE-084545. Professor John R. Eyler is thanked for providing access to his OPO, which was funded from an In-House Research Program (IHRP) grant from the National High Magnetic Field Laboratory (NHMFL). The authors’ collaborators from Ardara Technologies and, particularly, Randall E. Pedder and Christopher Taormina are thanked for their help in designing and setting up the custombuilt mass spectrometer described here. Damon T. Allen is acknowledged for developing the LabVIEW software applied in the mass spectral analysis. Finally, we thank our colleagues in the mechanical and electronic workshops in the Department of Chemistry for all their help and, in particular, Todd Prox, Brian Smith, Joe Shalosky, and Steven Miles.

Figure 4. IRMPD spectra of the 18c6-adducted, protonated octapeptides GA5YK and GA5pYK. Diagnostic bands are colorcoded: carboxylic acid OH stretch (blue), alcohol OH stretch (green), and phosphate OH stretches (purple).

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the diagnostic band positions are seemingly not affected by the size of the molecule, and discrete bands remain resolvable.

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SUMMARY AND CONCLUSIONS IRMPD spectroscopy was utilized to differentiate between peptides that contain alcohol and phosphate groups and those that do not. For a series of nonphosphorylated/phosphorylated tripeptide pairs, GSK/GpSK, GTK/GpTK, and GYK/GpYK, the alcohol OH stretching modes (3640−3670 cm−1) and the phosphate OH stretching modes (3665−3685 cm−1) are clearly resolvable and distinguishable, despite some overlap. This approach is also extendable to larger peptides, such as octapeptides (e.g., GA5YK/GA5pYK). The IRMPD spectra presented here were recorded by complexing the peptides with noncovalently bound 18-crown-6 ether during the electrospray process and subsequently detaching the crown via resonant absorption of infrared photons. This allows an unambiguous assignment of the precursor and corresponding photofragment mass channels, and hence permits multiplexing of the IRMPD spectroscopy experiment to record the IR spectra of multiple analytes simultaneously. These proof-of-principle experiments on a mixture of tryptic digest-type peptides demonstrate that phosphorylated peptides (i.e., containing pS, pT, or pY) can be readily distinguished from those peptides that could be phosphorylated but are not (i.e., peptides containing S, T, or Y), and those peptides that could not be phosphorylated (i.e., containing neither pS, pT, pY nor S, T, Y). These results underline the promise of this methodology in screening for phosphorylated peptides in enzymatic digests in phosphoproteomics. 9911



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Screening for Phosphorylated and Nonphosphorylated Peptides by

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