Anal. Chem. 2005, 77, 4673-4676
Deamidation as a Consequence of β-Elimination of Phosphopeptides Jonathan A. Karty and James P. Reilly*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
β-Elimination procedures often precede mass spectrometric analyses of phosphorylated peptides. Unfortunately, the commonly employed reaction conditions facilitate the deamidation of amide-containing residues. In addition to being 1 Da heavier than their amide counterparts, the newly created acidic residues greatly influence peptide tandem mass spectra. The effects of deamidation are investigated for five different amide-containing synthetic peptides exposed to β-elimination conditions. MALDI-generated ions are analyzed with a tandem TOF mass analyzer. Methodologies for estimating the degree of deamidation from peptide mass spectra are presented, the influence that adjacent residues exert on the rate of deamidation is catalogued, and the impact that deamidation can have on peptide tandem mass spectra is demonstrated. The complications this side reaction can cause for automated data interpretation are also noted. Phosphopeptides and phosphoproteins often require chemical derivatization to make them more amenable to mass spectrometry. One of the most commonly employed protocols is β-elimination of the phosphate moiety followed by Michael addition of a thiolcontaining reagent to the resulting unsaturated amino acid.1-4 This is not a perfect solution, however, as β-elimination can introduce complexity into phosphopeptide analyses. The dehydroalanine formed by elimination of the phosphate can cross-link to active residues such as cysteine, histidine, and lysine.5 Heterogeneity is introduced into the sample when β-elimination/Michael addition does not go to completion (especially problematic with phosphoThr residues2). Furthermore, the conditions required for complete β-elimination of phosphate from phosphoserine and phosphothreonine residues (e.g., 3 h @ pH 12, 45 °C)2 favor deamidation of asparagine residues, especially if they are followed by small polar amino acids (e.g., Ser or Gly).6-12 (1) Meyer, H. E.; Hoffman-Posorske, E.; Korte, H.; Heimeyer, L. M. G., Jr. FEBS Lett. 1986, 204, 61-66. (2) Molloy, M. P.; Andrews, P. C. Anal. Chem. 2001, 73, 5387-5394. (3) Knight, Z. A.; Schilling, B.; Row: R. H.; Kenski, D. M.; Gibson, B. W.; Shokat, K. M. Nat. Biotechnol. 2003, 21, 1047-1054. (4) Thompson, A. J.; Hart, S. R.; Clemens, F.; Barnouin, K.; Ridley, A.; Cramer, R. Anal. Chem. 2003, 75, 3232-3243. (5) Linetsky, M.; Hill, J. M. W.; LeGrand, R. D.; Hu, F. Exp. Eye Res. 2004, 79, 499-512. (6) Bornstein, P. Biochemistry 1970, 9, 2408-2421. (7) Patel, K.; Borchardt, R. T. Pharm. Res. 1990, 7, 703-711. (8) Patel, K.; Borchardt, R. T. Pharm. Res. 1990, 7, 787-793. (9) Wright, H. T. Crit. Rev. Biochem. Mol. Biol. 1991, 26, 1-52. 10.1021/ac050294c CCC: $30.25 Published on Web 05/26/2005
© 2005 American Chemical Society
Deamidation is the conversion of amino acids with amide side chains (N and Q) to the corresponding acidic residues (D and E). This side reaction can further frustrate phosphopeptide analyses. Deamidation creates both R- and β-forms of aspartic acid.6 Peptides containing these isomers often separate chromatographically,7,8 decreasing sensitivity. Deamidation during β-elimination directly affects peptide mass spectra in two important ways. First, each new acidic site increases a peptide’s molecular weight by 0.984 Da. Second, replacement of amide groups with acidic ones has a dramatic impact on peptide tandem mass spectra.13-15 In particular, fragments resulting from cleavage of the peptide backbone immediately C-terminal to aspartate residues often dominate the CID mass spectra of singly charged ions.16 Deamidation can seriously hinder the ability to analyze β-eliminated phosphopeptides by CID when MALDI is employed. A brief series of experiments characterized the severity of these effects. EXPERIMENTAL SECTION In the first set of experiments, four synthetic peptides with the sequences AAAANXAAAR (where X was Gly, Ala, Ser, or Asn) and AAAAQGAAAR (Thermo Electron Corp., Milford, MA) were subjected to the β-elimination conditions reported by Thompson and co-workers4 for up to 4 h. In another experiment, a tryptic digest of Arabidopsis phosphoprotein PBS117 was reacted for 2 h under the same conditions. MALDI-TOF and MALDI-CID-TOFTOF mass spectra of the peptides were recorded with an Applied Biosystems 4700 Proteomics Analyzer tandem TOF mass spectrometer (Applied Biosystems Inc., Foster City, CA). Samples were prepared for MALDI by mixing one part peptide mixture with nine parts 7 g/L R-cyano-4-hydroxycinnamic acid matrix (Waters Corp., Milford, MA) in 50% v/v acetonitrile, 0.1% v/v trifluoracetic acid. (10) Clarke, S.; Stephenson, R. C.; Lowenson, J. D. In Stability of Protein Pharmaceuticals, Part A; Ahern, T. J., Manning, M. C., Eds.; Plenum Press: New York, 1992; pp 1-29. (11) Karty, J. A.; Ireland, M. M. E.; Brun, Y.; Reilly, J. P. J. Chromatogr. B 2002, 782, 363-383. (12) Robinson, N. E.; Robinson, A. B. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 944-949. (13) Yu, W.; Vath, J. E.; Huberty, M. C.; Martin, S. A. Anal. Chem. 1993, 65, 3015-3023. (14) Tsaprailis, G.; Nair, H.; Somogyi, A.; Wysocki, V. H.; Zhong, W.; Futrell, J. H.; Summerfield, S. G.; Gaskell, S. J. J. Am. Chem. Soc. 1999, 121, 51425154. (15) Gu, C.; Tsaprailis, G.; Breci, L.; Wysocki, V. H. Anal. Chem. 2000, 72, 5804-5813. (16) Lee, S.-W.; Hyun, S. K.; Beauchamp, J. L. J. Am. Chem. Soc. 1998, 120, 3188-3195. (17) Shao, F.; Golstein, C.; Ade, J.; Stoutemyer, M.; Dixon, J. E.; Innes, R. W. Science 2003, 301, 1230-1233.
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All mass spectra were calibrated externally and peak areas were automatically computed by the Data Explorer software package supplied with the mass spectrometer. Estimates of the degree of deamidation were computed using a method similar to that described by Robinson and Robinson.12 Briefly, isotopic envelopes from the mass spectra of the peptides after incubation were compared to those observed prior to any derivatization. If XNX, XDX, and XDD represent the mole fractions of non-deamidated, singly deamidated, and doubly deamidated forms of a peptide, respectively, then the normalized intensities (In) of the first three peaks in the isotopic envelope of a partially deamidated peptide sample are given by
In ) An/(A1 + A2 + A3...+ An)
(1)
I1 ) XNX‚R0NX
(2)
I2 ) (XNX‚R1NX) + (XDX‚R0DX)
(3)
I3 ) (XNX‚R2NX) + (XDX‚R1DX) + (XDD‚R0DD)
(4)
Here, An is the area of the nth peak in an experimentally observed isotope distribution (all peaks in the isotopic envelopes were considered in the normalization). The R coefficients are normalized relative peak intensities that are derived from experimentally measured isotope distributions from unreacted samples of the peptides. R0NX is associated with the first isotope peak in the mass spectrum of an unreacted peptide and corresponds to the fraction of non-deamidated molecules that contain only the lightest isotope of each element. R1NX is the relative intensity of the second peak in the unreacted peptide mass spectrum and corresponds to the fraction of non-deamidated molecules that contain 1 amu of heavier isotope atoms (e.g., 13C). R2NX is the relative intensity of the third isotope peak and corresponds to the fraction of non-deamidated molecules containing 2 amu of heavier isotope atoms. R0DX is associated with the first isotope peak of the singly deamidated sample mass spectrum. R1DX is the relative intensity of the second isotope peak in the singly deamidated sample mass spectrum. R0DD is associated with the first isotope peak in the mass spectrum of the doubly deamidated peptide mass spectrum. (R0DD is zero for all peptides other than AAAANNAAAR in this analysis.) All deviations in the isotopic envelopes were attributed to deamidation of the asparagine or glutamine residues in the peptides. The ionization and detection efficiencies for the peptides before and after deamidation were assumed to be equal, and each deamidation estimate is the average of three replicates. RESULTS Table 1 lists the deamidated mole fractions observed in the synthetic peptide time-course experiments. The pattern of deamidation rates for the asparagine-containing peptides (NG > NS > NA) were in agreement with those reported by Robinson and Robinson.12 These results suggest that tryptic peptides containing the sequence features NG, NS, NN, and NH are especially susceptible to deamidation during β-elimination procedures. Both asparagines in AAAANNAAAR were susceptible to deamidation. Double deamidation progressed at a markedly slow rate however, suggesting that an appreciable population of singly deamidated molecules must accumulate before double deamidation becomes 4674 Analytical Chemistry, Vol. 77, No. 14, July 15, 2005
Table 1. Observed Deamidated Mole Fractions for Samples of the Synthetic Peptides subjected to β-elimination Reaction Conditions peptide sequence AAAAXZAAAR XZ ) incubation time 5 min 15 min 30 min 60 min 90 min 120 min 240 min
NG
NA
NS
0.424 -0.013 -0.027 0.907 -0.023 0.003 0.989 0.081 0.108 1.005 0.183 0.245 1.002 0.253 0.379 1.008 0.335 0.475 0.993 0.662 0.871
NN NN singly doubly deamidated deamidated -0.004 0.042 0.187 0.311 0.413 0.477 0.447
0.006 0.002 -0.033 0.031 0.091 0.143 0.487
QG -0.023 -0.013 0.025 0.063 0.045 0.058 0.130
Figure 1. MALDI-CID-TOF-TOF spectra of synthetic peptide AAAANSAAAR after (A) 0 min, (B) 60 min, and (C) 120 min incubation in the β-elimination solution. The + refers to y6 ions whose masses are 1 Da heavier due to deamidation (see inset).
detectable. A previous study reported that a peptide with two NG elements demonstrated complete double deamidation after a 20 min incubation in 3.2 M NH4OH at 65 °C.18 In contrast, glutamine was much more stable; AAAAQGAAAR had a deamidated mole fraction of 0.130 after 4 h of incubation, indicating that deamidation of glutamine is a relatively slow process. This is also in agreement with previously published results.9 The CID results have serious implications for moderate to high throughput mass spectrometric analyses of phosphorylated tryptic peptides. Figure 1 contains representative MALDI-CID-TOF-TOF mass spectra from AAAANSAAAR after three different incubation times. The 0 min sample was not deamidated at all; but the 60 and 120 min samples had deamidated mole fractions of 0.245 and 0.475, respectively. The y5 ion was the second most intense feature in Figure 1B even though, based on the data in Table 1, only 24% of the molecules had the sequence AAAADSAAAR. This is due to the fragmentation-directing ability of the newly created aspartate residue. Unexpectedly, the y6 ion resulting from fragmentation of AAAADSAAAR is observed to be much more intense than the y6 ion from AAAANSAAAR (see inset of Figure 1B). The nondeamidated y6 ion is nearly invisible in Figure 1C. The results from the β-elimination/Michael addition experiment performed on the PBS1 tryptic peptide were even more (18) Karty, J. A.; Ireland, M. M. E.; Brun, Y. V.; Reilly, J. P. J. Proteome Res. 2002, 1, 325-335.
Figure 2. MALDI-CID-TOF-TOF mass spectra of phosphopeptide RQS*EQGTSESNSTGEFLEPG isolated from the tryptic digest of Arabidopsis protein PBS1 (A) before and (B) after β-elimination/ Michael addition of cysteamine. The sample responsible for Figure 2B had an estimated deamidated mole fraction of 0.465.
striking. Figure 2 demonstrates the effect of deamidation on the MALDI-CID-TOF-TOF mass spectrum of a phosphorylated tryptic peptide RQS*EQGTSESNSTGEFLEPG (S* ) phosphoserine) isolated and modified according to the procedure recommended by Thompson et al.4 Having arginine at the peptide’s N-terminus favored the observation of b-type ions over y-type ions. Fragment ions labeled with * were observed at masses consistent with peptide ions containing either phosphoserine (Figure 2A) or S-(2aminoethyl)cysteine (Figure 2B). The b3 ions in both samples and the b4 ion in the unreacted sample were observed at masses 98 Da less than predicted, implying that their serines had been replaced by dehydroalanines (denoted by b, in Figure 2). These ions may arise due to loss of phosphoric acid from phosphoserine, incomplete Michael addition of cysteamine, or creation of dehydroalanine during post-source decay. The most intense feature in Figure 2B appears at a mass 1 Da heavier than expected for b11*. Aspartate facilitates cleavage of the peptide backbone immediately C-terminal to itself,14 and the resulting b-type ion retains the aspartate. Thus, the deamidated fragment is observed to be 1 Da heavier than predicted for the asparagine-containing fragment. The other significant effect on the CID mass spectrum was the reduced intensity of the other fragment ions. Figure 2A has several more intense b-type ions than Figure 2B. The enhanced fragmentation C-terminal to the deamidated asparagine reduced the extent of cleavage at other sites. DISCUSSION The synthetic peptide experiments demonstrated unequivocably that the chemical conditions required for β-elimination facilitate deamidation of asparagine residues. Deamidation radically alters the isotopic envelopes in the peptide mass spectra. This was especially true for the CID mass spectra of the peptides. The fragment ion formed by cleavage of the peptide backbone immediately C-terminal to the deamidated asparagines dominated the CID mass spectra after approximately 40% of the asparagines
in the sample had been modified. The rate of deamidation varied as a function of the amino acid immediately C-terminal to the asparagine. The peptide with NG deamidated very quickly, followed by the NS-containing peptide, then the NN-containing peptide, and finally the peptide AAAANAAAAR. The peptide AAAAQGAAAR was mostly unmodified even after 4 h of incubation. As might be expected, the peptides AAAANSAAAR and RQS*EQGTSESNSTGEFLEPG had nearly identical deamidated mole fractions after 2 h in the β-elimination solution. β-Elimination is often required to facilitate mass spectrometric analyses of phosphorylated samples. It was needed to localize the phosphorylation site in the PBS1 peptide as it had six serines and threonines. The propensity of phosphorylated peptide ions to lose HPO3 and H3PO4 upon fragmentation19 made precise localization of the phosphorylation impossible using data from the non-βeliminated sample. This tendency is quite apparent in Figure 2A; fragment ions b3, b4, b5, b9, and b13 are seen with one of those two losses. Only the b3 fragment is observed with any side chain fragmentation in Figure 2B. Deamidation is problematic when mass analyzers with moderate resolution are used to separate ions. A resolution20 in excess of 45 000 is needed to separate the second isotope of AAAANSAAAR from the first isotope of AAAADSAAAR. At present, this resolution is available only with magnetic sector and Fourier transform ion cyclotron resonance instruments. The majority of biological mass spectrometry experiments are performed with time-of-flight mass analyzers, quadrupoles, or a hybrid of the two, implying that the changes induced by deamidation will be convolved into the isotopic envelope of the unmodified sample. CONCLUSION The reaction conditions required for efficient β-elimination of phosphate from phosphoserine and phosphothreonine favor the deamidation of asparagines immediately followed by small polar residues (e.g., Gly, Ser, Ala, His, Asn). The altered isotopic distributions created by partial deamidation could confound efforts to identify peptide/fragment masses. Shorter reaction times can limit the deamidation of most asparagines, but NG is extremely labile. The synthetic peptide AAAANGAAAR was completely deamidated after 15 min of incubation in the β-elimination solution, implying that peptides containing NG will almost always deamidate during β-elimination. The impact of deamidation on CID tandem mass spectra is more worrisome. The synthetic peptide data demonstrated that fragmentation C-terminal to a deamidated asparagine can dominate CID mass spectra when less than half of the asparagines have been modified. This enhanced fragmentation has two important effects. The first is that other fragment ions diminish in intensity as the favored fragment increases, reducing the amount of information contained in the tandem mass spectrum. The second is that all fragment ions that contain the deamidated asparagine will have altered isotopic envelopes that could confuse data analysis algorithms. To account for the effects of deamidation, mass spectrometry data interpretation software packages (e.g., Sequest,21 Mascot,22 and Protein Prospector23) must be directed to account for the 1 Da increase in the masses (19) Annan, R. S.; Carr, S. A. Anal. Chem. 1996, 68, 3413-3421. (20) Ashcroft, A. E. Ionization Methods in Organic Mass Spectrometry; The Royal Society of Chemistry: Cambridge, UK, 1997.
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of both the parent and fragment ions induced by deamidation when β-elimination is employed. Also, when de novo sequencing is performed using data from β-eliminated samples, sequence features such as DG, DS, and DH could actually be deamidated versions of NG, NS, or NH. (21) Eng, J. K.; McCormack, A. L.; Yates, J. R., III. J. Am. Soc. Mass Spectrom. 1994, 5, 976-989. (22) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-3567. (23) Clauser, K. R.; Baker, P. R.; Burlingame, A. L. Anal. Chem. 1999, 71, 28712882.
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ACKNOWLEDGMENT This work was supported by the National Institutes of Health Grant GM 61336 and the National Science Foundation Grant CHE0994579. The authors thank the laboratory of Dr. Roger Innes for providing the Arabidopsis PBS1 tryptic digest.
Received for review February 17, 2005. Accepted April 27, 2005. AC050294C
