Rapid and Reliable Peptide de Novo Sequencing Facilitated by Microfluidic Chip-Based Edman Degradation Wenzhang Chen,† Xuefeng Yin,*,† and Yan Yin‡ Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Hangzhou 310027, China, and Momenta Pharmaceuticals, Inc., Cambridge, Massachusetts 02142 Received July 25, 2007
Abstract: This paper expands the application of the newly developed highly sensitive microfluidic chip-based Edman degradation system. Comparison between the MS/MS spectra of a native peptide and its N-terminus truncated counterpart after carrying out one cycle of Edman degradation in a microfluidic chip can not only provide N-terminal residue information, but also facilitate the identification of different series of fragment ions. Manual peptide sequencing is more feasible and rapid using this method as demonstrated with three peptide examples including one neuropeptide. Furthermore, two cycles of Edman degradation allow the determination of the exact value of b2 ion of the intact peptide, which can serve as an internal calibrant to increase the mass accuracy of the MS/MS spectrum. Keywords: de Novo sequencing • Edman degradation • peptide • neuropeptide
Introduction Tandem mass spectrometry (MS/MS) combined with computer algorithms for database searching has become an essential tool for the identification of proteins. In cases when the protein of interest is not archived in the protein sequence database or when full characterization of a protein is desired, it usually calls for de novo sequencing.1 A major challenge in de novo sequencing analysis is the determination of the respective N-terminal and C-terminal fragment ion series2,3 and the identification of N-terminal residues in the MS/MS spectra.4 Cleavage of the peptide C-terminus with carboxypeptidase is an effective means of labeling the C-terminal fragments and distinguishing it from the N-terminal fragments.5,6 Limitations of this approach include the strong dependence of the enzyme cleavage kinetics on the amino acid sequence and the possible C-terminal inaccessibility of some proteins/peptides to the exopeptidase of choice.7 Many chemical derivatization schemes have also been developed to facilitate the de novo sequencing by introducing a mass shift at either the N-terminus or the C-terminus of a peptide, thus, enabling differentiation between b- and y-type fragment ion series in a complex MS/MS spectrum.8,13 How* To whom correspondence should be addressed: Prof. Xuefeng Yin. E-mail:
[email protected]. Tel: +86-571-87952070. † Zhejiang University. ‡ Momenta Pharmaceuticals, Inc.
766 Journal of Proteome Research 2008, 7, 766–770 Published on Web 11/30/2007
ever, these experiments may introduce nonspecific products or contaminants into the samples, further complicating spectral assignments. Also note that some chemical modifications at peptide termini may not be universally feasible, because the side chains of the terminal amino acids can be modified.14,15 For instance, the C-terminal methylation method would not be appropriate for peptides lacking free carboxyl termini, such as neuropeptides. In neuropeptides, C-terminal amidation is the most common post-translational modification. Therefore, the utility of this labeling approach for de novo sequencing native neuropeptides is somewhat problematic and requires further evaluation.2 High mass accuracy of the MS/MS data is crucially important as it allows for tighter constraints, leading to fewer errors.16 However, it is not practical to include calibrant masses in a MS/MS spectrum, as precursor ion isolation inevitably excludes other masses.15 There are two most popular choices for internal calibrant: immonium fragment ions or surviving precursor ions. But, either of these choices has its own limitation. Peaks of immonium ions reside in a densely populated part of spectra, which may overlap with other fragments. On the other hand, the surviving precursor ions may be polluted by impurities that accidentally coincide in m/z with the precursor ion of interest.17 Recently, Beardsley utilized both guanidination and amidination to assist peptide sequencing. Amidine groups promoted fragmentation of the N-terminal peptide bond to produce abundant b1 ion. When the b1 ion was used as the internal calibrant, it significantly reduced the mass errors in MS/MS spectra.15 Olsen demonstrated that the mass accuracy was generally within a 1 ppm absolute deviation from the calculated values using a lock mass strategy.16 However, the method was restricted to LTQ-Orbitrap mass spectrometer. Edman degradation is an accurate way for easy elucidation of long amino acid sequences. However, the application of this conventional method was limited by its sensitivity. In our previous paper,18 a sensitive microfluidic chip-based Edman degradation system was developed, in which Edman degradation of peptides at a subfemtomole level can be achieved in a microfluidic chip. However, it is a time-consuming procedure for complete peptide sequence interpretation solely by Edman degradataion. Here, we present a strategy that utilizes one cycle of Edman degradation in a microfluidic chip to assist peptide sequencing. Comparison between the MS/MS spectra of the native peptide and that of the N-terminal residue truncated peptide facilitates the identification of different series of fragment ions. Furthermore, the mass errors in MS/MS spectra 10.1021/pr070465p CCC: $40.75
2008 American Chemical Society
technical notes
Rapid and Reliable de Novo Sequencing
Figure 1. ESI-MS/MS mass spectra of (a) synthetic peptide [KLLGPHVLGV] and (b) of the N-terminal truncated peptide. The double charged parent ion is noted with a diamond. The experiment was performed using an ion trap mass spectrometry.
are significantly reduced using the b2 ion of the peptide as the internal calibrant, whose exact value can be determined by carrying out two consecutive cycles of Edman degradation.
Experimental Section Chemicals. Synthetic peptides were obtained from GL Biochem (Shanghai, China); trifluoroacetic acid (TFA) and phenyl isothiocyanate (PITC) were obtained from Sigma (St. Louis, MO). The stationary phase used to pack the microcolumn was Nucleosil ODS1 (Stacroma, Reinach, Switzerland), which is 3 µm porous modified C18-based particles. Methanol was from Merck (Darmstadt, Germany). Other reagents were of analytical grade and from local suppliers. Chemical Degradation. Edman degradation was carried out in a microfluidic chip following the gas phase sequencing procedure described previously with minor modification.18 Briefly, it consists of four main steps for one cycle of Edman degradation: (1) 50 pmol of synthetic peptides was loaded onto the reaction cartridge, and the volume of the reaction cartridge was about 250 nL; (2) the N-terminal amino was modified with PITC; (3) the N-terminal residue was cleaved off by acid
treatment; (4) the truncated peptides were eluted from the reaction cartridge with 10 µL of 80% methanol. To complete two cycles of Edman degradation for the determination of the exact value of b2 ion, step 2–3 were repeated. Mass Spectrometry. After chemical degradation, 10 µL of 50 pmol of the native peptides and the truncated peptides after Edman degradation dissolved in 80% methanol was diluted with 10 µL of 20% methanol, and then analyzed directly by ESIMS/MS. All ESI-MS and ESI-MS/MS experiments were performed using an ion-trap mass spectrometer (Esquire-3000, Bruker Daltonics, Germany) or a quadrupole time-of-flight (QTOF) mass spectrometer (API QStar Pulsar, Applied Biosystems, MA). The sample solution was introduced into the ion source at 4 µL/min using a syringe pump.
Results and Discussion Panels a and b of Figure 1 are the ESI- MS/MS spectra of synthetic peptide [KLLGPHVLGV] and its N-terminal truncated peptide after one cycle of Edman degradation, respectively. Comparison of the doubly charged parent ions, m/z of [M + 2H]2+ ) 517.0 for the native peptide and 452.9 for the truncated Journal of Proteome Research • Vol. 7, No. 2, 2008 767
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Figure 2. ESI-MS/MS mass spectra of (a) angiotensin II and (b) its N-terminal truncated peptide. The double charged parent ion is noted with a diamond. The experiment was performed using an ion trap mass spectrometry.
peptide fragment, revealed that the original N-terminal residue was lysine. Since both the native peptide and the truncated peptide share the same amino acid sequence toward the C-terminus, the common mass peaks in the collision-induced dissociation (CID) spectra of these two peptides would evidently be the C-terminal ions and common internal fragments ions. Comparison between panels a and b of Figure 1 gives the common mass peaks with m/z of 904.2, 791.2, 678.1, 621.1, 617.1, and 524.0. Because the mass difference between the peak at m/z 617.1 and the peak at m/z 621.1 or 678.1 did not match the molecular weight of any amino acid residue, the peak at m/z 617.1 in Figure 1a was not likely to be assigned as y ion. On the basis of the mass differences between the y ions among them, an amino acid sequence of K-L/I-L/I-G-P was deduced. It should be noted that the internal fragment (bm-yn–1) ion of the native peptide comprising n amino acid residues shares the same amino acid sequence with bm–1 ion of the truncated peptide. Therefore, the peak at m/z 617.1 in Figure 1a can be ascribed as the b7-y9 internal fragment ion, which has the same nominal mass (MH+ at m/z 617.1) as the b6 ion in the MS/MS spectrum of the truncated peptide (Figure 1b). Otherwise, the peak at m/z 617.1 in Figure 1a might be mistakenly assigned 768
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as the b6 ion of the native peptide because the mass difference between the b7 ion with m/z of 745.2 coincides with the mass of a lysine residue of 128.1, which could result in problematic peptide sequencing. Comparison between the CID spectra of the native peptide and that of the N-terminal residue truncated counterpart also allowed rapid assignment of all N-terminal fragment ions present, as the N-terminal ions showed a characteristic mass shift of 128 mass units (residue mass of a lysine). The relationship between the N-terminal fragment ions of the native peptide and that of the truncated peptide can be described as: mbn – mbtn–1 ) mr, where mbn and mbtn–1 represent the mass of the b-type ion of the native and the N-terminus truncated peptide, respectively and mr represents the N-terminal residue mass of the native peptide. With this distinct mass tag, a part of b-ion series (915.2/787.1, 858.2/730.1, 745.2/617.1, 646.1/518.0, and 355.0/226.9) was easily distinguished. The mass differences between these b-ions and the intact peptide mass 1032.6 indicated an amino acid sequence of H-V-L/I-G-V. On the basis of the knowledge of the N- and C-terminal fragment ions, the amino acid sequence of
Rapid and Reliable de Novo Sequencing the native peptide could be easily deduced as K-L/I-L/I-G-PH-V-L/I-G-V. The peptide angiotensin II [DRVYIHPF] with m/z of 1046.5 (Figure 2a) was chosen as another example to demonstrate the application of our method. After removal of the N-terminal residue by one cycle of Edman degradation, the comparison between the ESI-MS/MS spectra of the peptide with and without the N-terminal Asp residue is illustrated in Figure 2. The common mass peaks observed in the CID spectra of both intact and truncated angiotensin II peptide were m/z 775.3, 676.2, 513.1, 400.0, and 263.1. A part of the b-ion series differing by an Asp residue of 115.0 mass units (784.3/669.3, 756.3/641.2, 647.2/532.2, 619.2/504.2, and 534.1/419.1) could also be straightforwardly tracked as described above. The combination of these data revealed a partial sequence of D-R-V-Y-L/I-H. As observed in Figure 2, complementary b+/y+ ions, derived from the cleavage at the amide bond between HP, were dominant. It is in good agreement with previous reports that histidine is the amino acid residue most likely to show preferential cleavage at its C-terminal side in doubly protonated peptides.19,20 However, the corresponding b+/y+ product ions generated from the cleavage of amide bond between PF were absent in Figure 2. Therefore, like other de novo sequencing methods, fully sequencing of angiotensin II was not achieved due to the known fact that dissociation of the C-terminal peptide bond of proline is often suppressed in CID.20 The neuropeptide [KHKNYLRFamide] was chosen as the third application example. Panels a and b of Figure 3 are the ESI-MS/MS spectra of the native peptide and its N-terminal truncated peptide after one cycle of Edman degradation, respectively. As described above, the original N-terminal residue can be identified as lysine by comparing the doubly charged parent ions, m/z of [M + 2H]2+ ) 552.84 for the native peptide and 488.77 for the truncated peptide fragment. The common mass peaks observed in the CID spectra of both intact neuropeptide and the truncated peptide were m/z 839.51, 711.41, 597.37, 434.29, 321.21, and 266.16. However, the mass difference between the peak at m/z 321.21 and the peak at m/z 266.19 did not match the molecular weight of any amino acid residue. Therefore, we were not certain whether the peak at m/z 266.16 was a y-ion or not. On the basis of the already assigned y series ions (839.51, 711.41, 597.37, 434.29, and 321.21), an amino acid sequence of KNYI/L was easily deduced. The combination of lysine as the N-terminal residue and the mass difference between 1104.67 (calculated from doubly charged 552.84) and 839.51 revealed a partial N-terminal amino acid sequence of KHKNYI/L. This implied that the m/z value of b2 ion of both native and the truncated peptide was 266.16. There were six y ions and one b ion observed in the CID MS/MS spectrum of the native neuropeptide. However, some important ions were missing from the spectrum. The additional information provided in Figure 3b not only made the identification of fragment ions more reliable, but also made the complete peptide sequencing possible. In Figure 3b, in addition to these ions, three product ions at m/z 406.73 [M + 2H]2+, 380.20 [M + H]+, and 165.10 [M + H]+ were also detected. The peak at m/z 380.20 can be interpreted as the b3 ion based on the sequence deduced. For the doubly charged ion at m/z 406.726, the m/z value of its corresponding singly charged ion can be calculated as 812.45. Also note that the m/z value of singly charged parent ion of the truncated peptide is 976.53. According to the relationship21 mb + my ) mprecursor + 1.0078, where mb, my, and mprecursor represent the m/z values of singly
technical notes
Figure 3. ESI-QTOF MS/MS spectra of peptides after one and two cycles of Edman degradation: (a) native peptide, (b) one cycle degradation, and (c) two cycles degradation. The experiment was performed using QTOF mass spectrometry.
charged complementary b and y ions and the precursor ion, respectively, the peak at m/z 165.10 and the calculated singly charged ion at m/z 812.45 should be a complementary b and y ion pair. The deduced partial N-terminal amino acid sequence of KHKNYI/L revealed that the m/z value of b1, b2, and b3 ions of the truncated peptide was 138.06, 266.16, and 380.20, respectively. Therefore, the peak at m/z 165.10 was not likely a b ion. Because the mass difference between the peak at m/z 321.20, which had been assigned as a y ion, and the peak 165.10 was 156.10, which equals exactly to the mass of an arginine residue (156.10), the m/z 165.10 in the MS/MS spectra of truncated Journal of Proteome Research • Vol. 7, No. 2, 2008 769
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Table 1. Effect of Mass Correction When Using b2 Ion as Internal Calibrant with external calibration only calculated (Da)
measured (Da)
mass error (ppm)
measured (Da)
mass error (ppm)
y72+ 488.7779 y6 839.4890 y62+ 420.2480 y5 711.3941 y4 597.3512 y3 434.2879 y2 321.2038 b2 266.1617 [M + 2H]2+ 552.8254 Average Mass Error (ppm)
488.7888 839.5178 420.2614 711.4117 597.3716 434.2978 321.2135 266.1697 552.8392
22.3 34.3 31.9 24.8 34.2 22.8 30.2 30.0 25.0
488.7742 839.4926 420.2488 711.3903 597.3536 434.2847 321.2038 266.1617 552.8226
-7.5 4.3 1.8 -5.3 4.1 -7.2 0 0 -5.1
fragment I.D.
peptide should be assigned as a y ion and m/z 406.73 a b ion. The signal at m/z 165.10 also indicated the C-terminal as F-NH2. Therefore, the complete sequence was determined as K-H-K-N-Y-I/L-R-F-NH2. Internal Calibration Facilitated by Two Cycles of Edman Degradation. Figure 3a shows the ESI CID spectrum of the neuropeptide [KHKNYLRFamide]. The mass spectra obtained after one and two degradation cycles are illustrated in panels b and c, respectively, of Figure 3. Comparative analysis revealed that the N-terminal sequence was KH, which implied that the exact m/z value of b2 ion was 266.1617. With the use of the b2 ion, which is always present in a MS/MS spectrum,21,22 as the internal calibrant, the average mass error in the QTOF MS/MS spectrum was significantly reduced from 28.4 to 3.9 ppm, as summarized in Table 1.
Conclusions Comparative analysis using the CID spectra of intact and N-terminus truncated peptides provided a simple and straightforward method to distinguish the N- and C-terminal fragments. The fragment peaks demonstrating a characteristic mass shift of the N-terminal residue mass of the native peptide are N-terminal fragments, whereas the common peaks in both spectra are a mixture of C-terminal and internal fragments. The spectra comparison between intact and truncated peptides alleviates the complexity and ambiguity of mass data interpretation, which enables rapid and reliable assignment of peptide sequences. Compared to other tagging techniques, our method introduces mass tagging through one cycle of Edman degradation and works for C-terminal modified peptides, such as neuropeptides. This strategy exhibits the following important features. First, it can generate a single mass tag on any peptide derived from any protein sources, such as cell cultures, tissues, or biological fluids, as long as the peptide is not N-terminally blocked. Second, the strategy is compatible with MALDI and electrospray MS with no sensitivity reduction. Third, this taggenerating method is simple, inexpensive, and can be done with commercially available reagents. It has high reaction efficiency under mild reaction conditions. Fourth, with one more cycle of Edman degradation, an improved mass accuracy in the MS/MS spectra can be attained using the b2 ion as the internal calibrant. Last but not least, this approach has an advantage in de novo sequencing due to its ability to decipher
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28.4
3.9
the first two amino acids of a peptide at the subfentomole range, whose information is normally unavailable in a MS/MS spectrum.
Acknowledgment. This work was supported by the National Natural Science Foundation of China under project No. 20775072 and the National Key Basic Research and Development (973) Program (No2007CB714503) of China. References (1) Standing, K. G. Curr. Opin. Struct. Biol. 2003, 13, 595–601. (2) Fu, Q.; Li, L. Anal. Chem. 2005, 77, 7783–7795. (3) Johnson, R. S.; Davis, M. T.; Taylor, J. A.; Patterson, S. D. Methods 2005, 35, 223–236. (4) Boja, E. S.; Sokoloski, E. A.; Fales, H. M. Anal. Chem. 2004, 76, 3958–3970. (5) Hisada, M.; Konno, K.; Itagaki, Y.; Naoki, H.; Nakajima, T. Rapid Commun. Mass Spectrom. 2000, 14, 1828–1834. (6) Cotter, R. J. Anal. Chem. 1999, 445A–451A. (7) Sechi, S.; Chait, B. T. Anal. Chem. 2000, 72, 3374–3378. (8) Hunt, D. F.; Yates, J. R.; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Nati. Acad. Sci. U.S.A. 1986, 83, 6233–6237. (9) Münchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047–4057. (10) Ji, C.; Guo, N.; Li, L. J. Proteome Res. 2005, 4, 2099–2108. (11) Leitner, A.; Lindner, W. J. Chromatogr., B 2004, 813, 1–26. (12) Chen, P.; Nie, S.; Mi, W.; Wang, X. C.; Liang, S. P. Rapid Commun. Mass Spectrom. 2004, 18, 191>–198. (13) Goodlett, D. R.; Keller, A.; Watts, J. D.; Newitt, R.; Yi, E. C.; Purvine, S.; Eng, J. K.; von Haller, P.; Aebersold, R.; Kolker, E. Rapid Commun. Mass Spectrom. 2001, 15, 1214–1221. (14) Gu, S.; Pan, S.; Bradbury, E. M.; Chen, X. Anal. Chem. 2002, 74, 5774–5785. (15) Beardsley, R. L.; Sharon, L. A.; Reilly, J. P. Anal. Chem. 2005, 77, 6300–6309. (16) Olsen, J. V.; de Godoy, L. M. F.; Li, G.; Macek, B.; Mortensen, P.; Pesch, R.; Makarov, A.; Lange, O.; Horning, S.; Mann, M. Mol. Cell. Proteomics 2005, 4, 2010–2021. (17) Chernushevich, I. V.; Loboda, A. V.; Thomson, B. A. J. Mass Spectrom. 2001, 36, 849–865. (18) Chen, W. Z.; Yin, X. F.; Mu, J. X.; Yin, Y. Chem. Commun. 2007, 24, 2488–2490. (19) Tabb, D. L.; Smith, L, L.; Breci, L. A.; Wysocki, V. H.; Lin, D.; Yates, J. R. Anal. Chem. 2003, 75, 1155–1163. (20) Tsaprailis, G.; Nair, H.; Zhong, W.; Kuppannan, K.; Futrell, J. H.; Wysocki, V. H. Anal. Chem. 2004, 76, 2083–2094. (21) Kinter, M.; Sherman, N. E. Protein Sequencing and Identification Using Tandem Mass Spectrometry; Wiley-Interscience Series on Mass Spectrometry: New York, 2000; p 85. (22) Cittaro, D.; Borsotti, D.; Maiolica, A.; Argenzio, E.; Rappsilber, J. J. Proteome Res. 2005, 4, 1006–1011.
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