Charge Switch Derivatization of Phosphopeptides for Enhanced Surface-Enhanced Raman Spectroscopy and Mass Spectrometry Detection Handong Li and Narayan Sundararajan* Biomedical and Life Sciences, Digital Health Group, Intel Corporation, Santa Clara, California 95054 Received November 22, 2006
We report an aqueous one-pot reaction chemistry to derivatize phosphopeptides by switching the negatively charged phosphate group to a positively charged phosphonium or ammonium moiety. The phosphonium or ammonium tagged peptides then serve as peptide or protein phosphorylation signatures allowing extended and more sensitive analyses using surface-enhanced Raman spectroscopy (SERS) and mass spectrometry. Keywords: phosphopeptides • charge switching • β-elimination • Michael addition • phosphonium or ammonium tagged peptides • surface-enhanced Raman spectroscopy (SERS) • mass spectrometry
Introduction Reversible protein phosphorylation, principally on serine, threonine, or tyrosine residues, is one of the most important and well-studied post-translational modifications.1,2 Phosphorylation3-6 plays a critical role in the regulation of many cellular processes including cell cycle, growth, apoptosis, and signal transduction pathways due to its reversible nature. To understand the molecular basis of these regulatory mechanisms, it is necessary to identify and characterize these phosphorylation sites in proteins. However, there are many challenges in phosphopeptide analysis because of the following intrinsic problems: (1) phosphate groups attached to serine and threonine are labile due to β-elimination and have low ionization efficiency due to suppression by more abundant non-phosphorylated species during mass spectrometry analysis; (2) phosphorylated species have low stoichiometry due to dynamic kinase and phosphatase activity, (3) phospho-groups make peptides more hydrophilic, hence, making reverse phase HPLC separation and purification difficult, adversely affecting subsequent MS or other detection methods; (4) phospho-groups have affinity to metals like aluminum and iron causing sample loss; and (5) finally, phospho-groups are negatively charged and hence are electrostatically unfavorable to detection using negatively charged nanoparticles (silver or gold) typically used in surface-enhanced Raman spectroscopy (SERS) measurements.7 Recently, we reported the use of SERS for the ultra-sensitive detection and characterization of post-translational modifications (PTMs).8 It was demonstrated that SERS can detect as little as zeptomoles (10-18 mol) of peptides with a variety of protein PTMs, including trimethylation. However, SERS phosphorylation signatures were found to be subtle, and detection required sophisticated data analysis techniques. * To whom correspondence should be addressed. Tel: 408-765-8929. Fax: 408-765-2393. E-mail:
[email protected]. 10.1021/pr0606225 CCC: $37.00
2007 American Chemical Society
Scheme 1 . Schematic of a One-Pot Aqueous β-Elimination and Michael Addition Reactiona
a The Michael addition generated diasteromers. The number of total isomers is 2n where n equals the number of phosphogroups present in peptide sequence.
To address the analysis challenges toward phosphorylation and to meet the demand for ultra-sensitive detection and characterization of phosphopeptides, we have designed a chemical derivatization strategy to switch the negatively charged phosphate to positively charged phosphonium or ammonium that is compatible with standard SERS and mass spectroscopy measurements (Scheme 1).
Methods Reagents and Chemicals. The monophosphopeptide and tetraphosphopeptide kit purified from β-casein digest, trypsin, angiotensin I, and bovine β-casein, and all other chemicals were purchased from Sigma-Aldrich. 2-Mercaptoethyltrimethylammonium chloride was custom-synthesized at the Ryss lab (Union City, CA). All peptides were purchased from CPC Scientific, Inc. (San Jose, CA). HPLC grade water and acetonitrile were purchased from VWR. A 1 M LiCl stock solution was prepared from LiCl and HPLC grade water. Colloidal silver suspensions used in the SERS experiments were prepared by Journal of Proteome Research 2007, 6, 2973-2977
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Figure 1. SERS spectra of phosphopeptide control (FQpSEEQQQTEDELQDK), derivatized phosphopeptide, and tag molecule (thiocholine). The peaks at 780 and 908 cm-1 from the derivatized phosphopeptide spectra can be assigned to CH3 rocking and C-S stretching vibrational modes, respectively, indicating that the phospho-group has been converted to the thioethyltrimethylammonium tag.
a modified method adapted from the citrate reduction of silver nitrate as described by Lee and Meisel.7 The suspension had a final silver concentration of 1.00 mM. Its zeta potential, after diluting 20 times with d,i, water was measured to be -62 ( 3 mV (Zetasizer 3000HS, Malvern). Tryptic Digestion. Bovine β-casein (4 mg) was dissolved in freshly prepared 8 M urea (40 µL) and incubated in a 37 °C water bath for 20 min to denature. Ammonium bicarbonate (50 mM, pH 8.0, 80 µL) and trypsin (1 µg/µl stock in ammonium bicarbonate buffer, 40 µL) were added. The final urea concentration was 2 M. The weight ratio of trypsin to β-casein was 1:100. The final reaction mixture was incubated overnight in a 37 °C water bath (14 h). The reaction was quenched by adding formic acid to a final pH of 3.0 and desalted with a C18 column. The desalted β-casein digest sample was vacuum-dried and stored at -20 °C. β-Elimination and Michael Addition (BEMA). The β-casein digest sample (500 µg) was reconstituted in 250 µL water and a freshly prepared solution of saturated barium hydroxide (250 µL) was added under nitrogen. The solution was then mixed well and incubated at room temperature for 6 h. Cysteamine hydrochloride solution (2 M), 2-mercaptoethyltrimethylammonium solution (2 M), or phosphines (2 M) in water were freshly made and stored under nitrogen. In the case of non-water soluble phosphines, 50% methanol was added to improve solubility. This freshly made nucleophile solution (100 µL) was added to the β-elimination reaction mixture under nitrogen. The final solution (pH ∼ 8.0) was incubated in the dark for 14 h and quenched by adding formic acid and bringing the solution to a final pH of 3.0. The final solution was then desalted with a C18 column. The C18 column (1.0 mL bed volume) was washed with 3 mL of methanol and equilibrated with 4 mL of water containing 0.1% formic acid (buffer A). The column was washed with 2 mL of buffer A after the sample was loaded. The sample was eluted with 1 mL of acetonitrile containing 0.1% formic acid (buffer B), vacuum-dried, and 2974
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reconstituted in water for HPLC fractionation, SERS measurement, and LC/MS analysis. HPLC Fractionation of BEMA Derivatized β-Casein Digest. HPLC separation of the peptides from the derivatized β-casein digest was performed using an Agilent Zorbax C18 column (150 mm × 0.3 mm) with the mobile phase consisting of 0.1% formic acid in water (v/v) (buffer A) and 0.1% formic acid in acetonitrile (v/v) (buffer B) at a flow rate of 30 µL/min. The elution gradient started with 0% B for 20 min, increased from 0 to 60% B over 60 min, from 60% to 90% B for 5 min, 90-0% B for 5 min, and equilibrated with 0% B for 10 min. Fractions were collected every 2 min using an automated fraction collector and directly used for subsequent SERS measurement and LC/MS analysis. The 0% acetonitrile gradient was chosen to ensure complete separation of the free Raman tag small molecule and the derivatized peptide. It did not seem to cause phase collapse with the HPLC column used. Raman Spectrometer Setup. The Raman spectrometer setup used for the SERS experiments consisted of a titanium:sapphire laser operating at 785 nm with power levels of ∼750 mW, and a 20× microscope objective to focus the laser beam onto the sample plane. The Raman-scattered light was back-collected using a combination of optical components, including a dichroic filter and a holographic notch filter, and imaged onto the slit of the spectrophotometer connected to a thermoelectrically cooled charge-coupled device (CCD) detector. SERS Measurements. SERS measurements were performed directly on the HPLC fractions. The stock solution of the synthesized colloidal silver, with a final silver concentration of 1.0 mM, was diluted 1 to 2 parts in volume with d.i. water. Typically, 10 µL of the HPLC fraction was incubated at room temperature with 80 µL of the diluted silver solution for 0-15 min. A volume of 20 µL of 1.0 M LiCl solution was added after the incubation, and the solution was mixed thoroughly with a pipet tip and dropped onto an aluminum plate (cleaned with pure water, ethanol, and water) for immediate SERS measure-
Charge Switch Derivatization of Phosphopeptides
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Figure 2. (Continued) Journal of Proteome Research • Vol. 6, No. 8, 2007 2975
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Figure 2. (a) TIC (total ion current) of thiocholine derivatized β-casein digest. The highlighted areas are identified as thiocholine derivatized monophosphopeptide (empty arrow) and tetraphosphopeptide (filled arrow), respectively. (b) Identified at an elution time of 22.42422.858 min, thiocholine derivatized monophospeptide has a molecular weight of 2083. M/(Z+2H) at 1042.8 (1042.5), M/(Z+3H) at 696.0 (695.4), and M/(Z+4H) at 523.3 (521.8) were observed and are indicated by asterisks. (c) Identified at an elution time of 29.041-29.241 min, thiocholine derivatized tetraphosphopeptide has a molecular weight of 3207. M/(Z+4H) at 803.7 (theoretical value: 802.8), M/(Z+5H) at 643.1 (642.4), and M/(Z+6H) at 536.2 (535.5) were observed and are indicated by asterisks.
ments. SERS spectra were collected for each fraction by focusing the laser inside the sample droplet. A typical spectrum was acquired at 1 s acquisition time per frame for 30 frames. LC-ESI-MS Analysis. An Agilent model 1100 (binary) highperformance liquid chromatograph coupled with a hybrid triple quadrupole/linear ion trap mass spectrometer, model 4000 Q TRAP LC/MS/MS system (Applied Biosystems MDS SCIEX) was used in all analyses. The analytical column was Agilent Zorbax SB-C18, 5 µm, 150 mm × 0.5 mm. The injection volume was 5 µL. The mobile phase consisted of (A) 0.1% formic acid in water (v/v); (B) 0.1% formic acid in acetonitrile (v/v) at a flow rate of 30 µL/min under a linear gradient of 0% B to 80% B over 30 min. MS data were acquired in the positive ion electrospray ionization (ESI) mode, using the following TurboIonSpray source conditions: temperature ) 500 °C, curtain gas ) 40 (arbitrary units), GS1 ) 70, GS2 ) 60, CAD gas pressure high, ion spray voltage ) 5500. MALDI-MS Analysis. MALDI mass spectrometry was carried out on a 4800 TOF/TOF mass spectrometer (Applied Biosystems, Boston, MA). A total of 1.0 µL of sample solution was mixed with 2 µL of R-cyano-4-hydroxycinnamic acid solution (saturated in 0.1% TFA, 50% water, and 50% acetonitrile). The sample matrix mixture (0.5 µL) was spotted onto a MALDI plate. Mass spectrometric spectra were obtained at a laser power of 2976
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2700 kW cm-2. All MALDI spectra were calibrated with standards of a known peptide mixture.
Results and Discussion The derivatization was carried out by performing β-elimination followed by Michael addition as a one-pot reaction. A variety of nucleophiles such as thiols, phosphines, hydrazides, amines, and azides were tested for the Michael addition to introduce the desired structures. Thiols and phosphines were found to be the most effective nucleophiles (please see Supporting Information). Addition of phosphines resulted in a phosphonium moiety, while cysteamine or thiocholine was used to introduce an amine or ammonium moiety through a thioether linkage. The positively charged moieties were then the so-called SERS or mass tags. It is important to note that phosphotheronine has a much slower β-elimination rate (20 times less) than phosphoserine, and phosphotyrosine is amenable to β-elimination reaction. Typically, phosphopeptide signature differences are found to be subtle by SERS due to the repulsive forces between the phosphate groups and citrate coated metal particle surfaces.5 Figure 1 shows a model phosphopeptide (FQpSEEQQQTEDELQDK) becoming easily detectable by SERS even at 10fold lower concentration by switching the charge on the
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phosphorylation site. It is also clear from the Figure 1 that the spectrum of the derivatized phosphopeptide is unique due to the methyl terminal rocking mode (∼780 cm-1). This signature is from the trimethyl ammonium moiety in the tag that helps in identification of phosphorylation modification (please see Supporting Information). The derivatized peptide was HPLCpurified, and the derivatization was re-confirmed by LC/MS as well. To optimize the working protocol for derivatization, the following model peptides were tested: FQpSEEQQQTEDELQDK (monophosphopeptide from β-casein), RELEELNVPGEIVEpSLpSpSpSEESITR (tetraphosphopeptide from β-casein), DLDVPIPGRFDRRVpSVAAE (calcineurin substrate, synthetic), KMpSTLSYR, KMSpTLSYR, YPpSTSPSK,, and KpSTGGKAPR (synthetic peptides). The β-elimination was completed in 6 h in 90 mM barium hydroxide aqueous solution at room temperature. Neither stronger base sodium hydroxide nor an organic solvent was used.9,10 It was also found that if the phosphorylation site was on the second residue to the N-terminus of the peptide, a cyclization reaction occurs at the β-elimination step because the N-terminal amine group could attack the newly formed double bond undergoing an intramolecular Michael addition to form a six-member ring product. This problem was solved by acetylation of the amine groups before β-elimination (please see Supporting Information). The N-terminal amine and the amine residues from all lysine residues were acetylated.11 An optimized working protocol was then applied to β-casein digest to demonstrate its applicability to a more complex peptide mixture sample. Since the cyclization reaction was concentration-dependent and the rate was not significant under the conditions used and also the sequences of the phosphopeptides in the β-casein tryptic digest did not pose the intramolecular cyclization problem, the acetylation step was not used with the β-casein digest. A typical protocol used for bovine β-casein was as follows: The β-casein tryptic digest sample was dissolved in deionized water and an equal volume of freshly prepared saturated barium hydroxide was added under nitrogen. The β-elimination reaction was typically completed in 6 h at room temperature. Freshly made nucleophiles (2 M) were added to the β-elimination reaction mixture under nitrogen with the final pH adjusted to 8.0 and incubated overnight under dark. The reaction was then quenched by adding formic acid and bringing the reaction mixture to a final pH of 3.0, after which it was desalted with a C18 column. The desalted sample was lyophilized, resuspended, and then fractionated using a reversephase HPLC column. Different fractions from the HPLC run were then subjected to SERS measurement to identify the derivatized phosphopeptides. The identified fractions were then characterized using LC/MS to reconfirm the SERS identification. MALDI-MS analysis was performed in parallel for further identification. Mass spectrometry detection in a complex peptide mixture (e.g., a protein digest) is especially challenging. After derivatization of the negatively charged phosphogroup of a model monophosphopeptide to a positively charged trimethylammonium moiety, however, the MALDI mass detectability increased 100 (see Supporting Information). A 10-fold greater sensitivity
in detection of phosphopeptides in the model β-casein digest was also achieved after the phosphogroups were permanently converted into positively charged and more stable ammonium or phosphonium moieties. As indicated in Figure 2b and c (see also Supporting Information), the conversion of phosphogroups was quantitative. In the case of the tetraphosphopeptide, four adjacent phosphogroups were all converted, and no partial conversion was observed as confirmed by the MS characterization results (Figure 2c).
Conclusions This novel application of charge switch chemical derivatization strategy enabled sensitive phosphorylation detection and identification by SERS and mass spectrometry in a model complex peptide mixture. It remains to be seen whether this strategy permits detection of phosphorylation in complex tissue samples. The combination of this derivatization strategy along with SERS and MS detection schemes can be extended to detect other post-translational modifications of interest such as glycosylation, thereby allowing sensitive PTM detection toward biomarker discovery applications.
Acknowledgment. The authors thank Drs. Jingwu Zhang and Sarah Ngola for preparing silver nanoparticles, Dr. Anil Patwardhan for SERS spectrum data processing, and Dr. Bree Mitchell for helping with MALDI mass analysis. Supporting Information Available: Nucleophiles for β-elimination and Michael addition, acetylation of N-terminal amine to block intramolecular Michael addition, Raman scattering shift assignment and identification of SERS tagged phosphopeptides, mass detection sensitivity increased after charge switch derivatization This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Arrigoni, G.; Resjo, S.; Levander, F.; Nilsson, R.; Degerman, E.; Quadroni, M.; Pinna, L. A.;James, P. Proteomics 2006, 6 (3), 757766. (2) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101 (2), 269296. (3) Kruger, R.; Kubler, D.; Pallisse, R.; Burkovski, A.; Lehmann, and W. D. Anal. Chem. 2006, 78 (6), 1987-1994. (4) Jin, M.; Bateup, H.; Padovan, J. C.; Greengard, P.; Nairn, A. C.; Chait, B. T. Anal. Chem. 2005, 77 (24), 7845-7851 (5) Corso, T. N.; Van Pelt, C. K.; Li, J.; Ptak, C.; Huang, X. Anal. Chem. 2006; 78 (7), 2209-2219. (6) King, J. B.; Gross, J.; Lovly, C. M.; Rohrs, H.; Piwnica-Worms, H.; Townsend, R. R. Anal. Chem. 2006, 78 (7), 2171-2181. (7) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391. (8) Sundararajan, N.; Mao, D.; Chan, S.; Koo, T. W.; Su, X.; Sun, L.; Zhang, J.; Sung, K. B.;Yamakawa, M.; Gafken, P. R.; Randolph, T.; McLerran, D.; Feng, Z. D.; Berlin, A. A.; Roth, M. B. Anal. Chem. 2006, 78 (11), 3543-3550. (9) Knight, Z. K.; Schilling, B.;Row, R. H.; Kenski, D. M.; Gibson, B. W.; Shokat, K. M. Nature Biotech. 2003, 21 (9), 1047-1054. (10) Steen H.; Mann M. J. Am. Soc. Mass Spectrom. 2002, 13, 9961003. (11) Suckau, D.; Mak, M.; Przybylski, M. Proc. Natl. Acad. Sci. U.S.A. 1992, 89 (12), 5630-5634.
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