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Nano-electrospray mass spectrometry and edman sequencing of peptides and proteins collected from capillary electrophoresis
Nano-electrospray Mass Spectrometry and Edman Sequencing of Peptides and Proteins Collected from Capillary Electrophoresis Mark D. Bauer, Yiping Sun and Feng Wang The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH 45253-8707
I.
Introduction
Capillary electrophoresis (CE) is rapidly becoming an important complementary method to high performance liquid chromatography (HPLC). It provides several advantages, including high separation efficiency, small sample consumption and short analysis time. Because of the small sample volumes required, CE is becoming the analytical tool of choice for biological applications which are sample limited. Electrospray mass spectrometry (ES/MS) is a powerful technique for the structural characterization of biomolecules. Although on-line CE-MS has been demonstrated to be useful for the analysis of biomolecules, the interfacing of CE and ES/MS is nontrivial and much more complicated than the LC-ES/MS interfacing (1-4). Some limitations for on-line CE-MS are: the capillary has to be long enough for the coupling of the CE to the MS; MS/MS sensitivity is relatively low due to the limited sample size loaded onto the capillary; and CE buffers used have to be compatible with MS analysis. Off-line approaches, which combined CE fraction collection and desorption mass spectrometry (plasma desorption and matrix-assisted laser desorption ionization) for peptide and protein analyses, have been demonstrated (5-8). The advantage of off-line approaches is the possibility of independently optimizing both CE separation and mass spectral analysis. Nanoelectrospray (nES) is a new technique for characterizing biomolecules in small volumes (0.5-2 |il) at low picomole levels (9-11). In nES, signals from a single sample loading typically last more than 30 minutes, which permits optimization of instrument parameters and MS/MS sequencing with high sensitivity. Both nES/MS and nES/MS/MS data can be obtained from a single sample loading. These features make nES an attractive off-line technique for sequencing peptides collected from CE. In this paper, an off-line approach, which combines nES/MS analysis and Edman sequencing of peptide/protein fractions collected from CE, is presented. Automatic peak collection was accomplished using a computer-controlled Beckman P/ACE 5000 instrument (12). Several different samples containing 5lOpicomoles of material, including a peptide mixture (angiotensin-I, methionine enkephalin and substance-P), a tryptic digest of cytochrome-C and proteins like myoglobin, insulin and lysozyme, were used to demonstrate this method. TECHNIQUES IN PROTEIN CHEMISTRY VIII Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.
37
38
Mark D. Bauer et al
II. Materials and Methods A.
Peptides and Proteins
Angiotensin-I, methionine enkephalin, substance-P, bovine trypsin, horse cytochrome-C, horse myoglobin, bovine insulin and egg-white lysozyme were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. B.
Enzymatic
Digestion
Both trypsin and cytochrome-C were dissolved separately to 1 mg/ml in 100 mM NH4AC, pH 8.1. 1 |Lil of the trypsin solution was added to 100 |Lil of the cytochrome-C solution. The reaction mixture was incubated at 37°C for 2 hours. The reaction was stopped by freezing. C.
Capillary
Electrophoresis
A Beckman P/ACE 5000 (Schaumberg, IL) was used for all CE separations. The background electrolyte (BGE) was CH3CN/H2O/HCOOH (50:45:5). Washing solution was 1% NH4OH. All untreated fused-silica capillaries were obtained from Polymicro Technologies (Phoenix, AZ). Column dimensions were 97 cm x 75 |LiM. Samples ranging from 0.3 mg/ml to 2 mg/ml (each analyte) were pressure injected for 10 to 30 sec with the goal to load 5 - 1 2 picomoles of each analyte. During separation, the voltage was maintained at 30kV except during fraction collection when the voltage was reduced to 7.5 kV. To collect a CE fraction, the following stepwise procedures which were used on the Beckman P/ACE 5000 instrument: (1) tum off the voltage just before the peak entered the UV window, (2) switch to the collection vial, (3) tum on the voltage to 25% of the maximum until the peak eluted from the capillary, and (4) turn off the voltage and switch back to the original outlet vial and resume the run under initial voltage conditions. The distance from the flow cell to the capillary outlet end is 7 cm. The fraction collection window is 5-10 minutes, depending on the peak width of interest. Fraction collection was accomplished by using the outlet carousel on the P/ACE 5000 to switch to a collection vial which contained 100 |il BGE. Each fraction was dried in-vacuo. Samples were redissolved in 4 |LI1 of CH3OH/H2O/HCOOH (50:50:0.1) and subjected to nES/MS and nES/MS/MS analysis and Edman sequencing. Protein samples collected from CE were subjected to Edman sequencing without drying the fractions. D.
Nano-electrospray
Mass
Spectrometry
All nES/MS and nES/MS/MS measurements were made on a PerkinElmer Sciex API-in triple quadrupole mass spectrometer (Thomhill, Canada) equipped with the nanoelectrospray source designed by Matthias Wilm and Matthias Mann at the European Molecular Biology Laboratory, Germany (9). The long signal duration allowed the instrumental parameters to be optimized for each peptide interrogated by nES/MS/MS. Argon gas was used as the collision gas. The colHsion gas thickness was 100-200x10 atoms/cm .
Nano-electrospray MS and Edman Sequencing Using CE
E.
39
Edman Sequencing
A PE-ABD 494 Precise protein sequencer was used for Edman sequence analysis of the collected CE fractions. Sample was transferred into a ProSpin (PE-ABD) pre-wetted by 20 L| L1 of methanol. A gentle nitrogen stream was applied to reduce the concentration of acetonitrile. The volume was then brought up to 400 |il with water. The ProSpin unit was centrifuged to dryness at 5,600g. The PVDF disc was cut out and washed three times with water before being placed in a sequencing cartridge. A modified sequencing program based on NORMAL-BLOT was used.
III. Results and Discussion Capillary electrophoresis is a rapid, reliable method for separating complex mixtures according to differences in the charge-to-mass ratio of each component. Fraction collection of the components of interest is necessary for further structural characterization by other analytical methods, e.g. mass spectrometry and Edman sequencing for peptides and proteins. Because of the need to maintain a high voltage across the capillary, fraction collection of analytes is relatively difficult by CE. One approach is to electrokinetically collect fractions into separate tubes by taking advantage of the automated outiet carousel available on the Beckman P/ACE 5000 CE system (12). First, it is necessary to establish reproducible electropherograms using solvent systems that are compatible with the desired subsequent MS analysis. Buffers such as Tris, phosphate or borate, though common for CE separations, are less compatible with electrospray MS. In addition, NaOH washes of the capillary introduce high levels of sodium into the system. Therefore, only volatile solvents and buffers were used in our initial experiments. The background electrolyte (BGE) contained only acetonitrile, water and formic acid, while 1% NH4OH was used as the capillary wash solution. These restrictions on the BGE composition limited the CE separation efficiency. Nonetheless, peptides and proteins could still be separated from one another under these conditions. The collected fraction was dried in-vacuo in preparation for nES/MS analysis and Edman sequencing. Figure 1 depicts the nano-electrospray source. nES/MS offers several features that recommend it for use with fractions collected by CE. First, nES requires small loading volumes at low picomole levels. This minimizes the amount of dilution which the sample must undergo to be effectively introduced into the nES source. Second, the signal obtained by nES lasts for 30 minutes or longer. This feature allows for the extensive interrogation of the sample by MS/MS on appropriately equipped instruments. As a result, all of the data can be generated from one CE run and one nES experiment in which both MS and MS/MS data are obtained. The nES needles, which are gold-coated, pulled glass capillaries, are generally treated as disposable so once loaded all of the data is acquired at one time. Sometimes a needle is defective or is damaged early in the nES experiment. This can result in the loss of the collected CE fraction.
Mark D. Bauer et al
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A peptide mixture (angiotensin-I, substance-P and met-enkephalin) was used in our initial experiment. After reproducible CE runs were obtained, each peptide in the nfiixture was collected individually. Figure 2 shows the electropherogram of CE-UV of the peptide mixture and the nES mass spectrum of peak "a" collected from a single CE run. The peaks at m/z 649.2 and 433.2 correspond to the doubly- and triply-charged ions of angiotensin-I, respectively. A fairly high background in the low-mass region was observed in every sample eluted from CE. To reduce the background in nES/MS analysis, the base wash by 1% NH4OH can be eliminated between CE runs. Figure 3 depicts the MS/MS of the doubly- and triply-charged precursor ions of angiotensin-I (DRVYIHPFHL). These spectra were all obtained from a single sample loading. Although the doubly- and triply-charged ions of angiotensin-I showed relatively weak peaks in Figure 2, their corresponding MS/MS spectra showed good signalto-noise ratios. Because the arginine residue was located near the N-terminus, the a- and b-series ions were predominent in both spectra (for nomenclature see ref 13). Two other peptides, substance-P and met-enkephalin, were also analyzed by nES/MS. Figure 4 shows the nES/MS and nES/MS/MS results for metenkephalin (peak c) collected from the CE (see inset Figure 2). Note that the Cterminal metiiionine was oxidized to the sulfoxide (m/z 590.2) during CE sample preparation. The methionine oxidation to the sulfoxide was also observed in substance-P CE fraction. The oxidation reaction of the methionine-containing peptides could occur during the sample drying process. Figure 5 shows that the signal from met-enkephalin lasted for at least 26 minutes with no loss in signal intensity. All three peptides were also successfully sequenced by Edman degradation using half of the material from the same collected CE fractions.
Nano-electrospray MS and Edman Sequencing Using CE
41
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Off-line CE-nES/MS Angiotensin-I
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^ -UV Peptide Angiotensin-I Substance-P Met-enkephalinJ
Figure 2. nES mass spectrum of the peak collected at 11.2 minutes. D-R-V-Y-l-H-P-F-H-L
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Figure 3. Off-line CE-nES/MS/MS of both the doubly and triply-charged precursor ions of angiotensin-1.
Mark D. Bauer et al
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Nano-electrospray MS and Edman Sequencing Using CE
43
Once the standard peptide mixture was successfully analyzed by off-line CE-nES/MS, the technique was attempted on a more complicated peptide mixture. More than 15 peptides were presented in the 2-hour tryptic digest of cytochromeC. CE separation of the peptides was achieved witiiin 30 minutes. Peaks of interest were collected in 100 |Lil BGE, dried and redissolved in 4 |i,l of the nES solvent. Figure 6 shows the nES mass spectrum of the CE peak at 21.9 minutes (inset) from the tryptic digest. Only one peptide was detected in the CE fraction. Both the singly- and doubly-charged ions of the peptide (m/z 964.5) corresponding to residues 92-99 (EDLIAYLK) of cytochrome-C were observed. Figure 7 is the nES/MS/MS of the doubly-charged precursor ion at m/z 483.0 of the peptide (residues 92-99), yielding a complete series of "y" ions. Fragmentation of the doubly-charged ion was much easier than the singly-charged ion. Both nES/MS and nES/MS/MS spectra of the peptide were obtained from a single loading of a 2-jil sample solution. Edman sequencing of the peak at 21.9 minutes, collected from a separate run, further verified the peptide sequence. Based on both MS and Edman sequencing data, there was no carryover between closely separated peaks.
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92 99 E-D-L-l-A-Y-L-K +2 483.0
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Figure 6. nES/MS of the peak (21.9 min) collected from the CE separation of a tryptic cytochrome-C.
Mark D. Bauer et al.
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Figure 8a. nES spectrum of myoglobin collected from CE. 12 pmoles of protein were loaded onto the CE.
Proteins like insulin, myoglobin and lysozyme were also loaded onto the non-coated CE capillary and collected for nES/MS analysis and Edman sequencing. Because the peak width of proteins is larger than that of the peptides on the non-coated column, the window is relatively wide (about 10 minutes) under 7.5 kV for fraction collection. Figure 8a is the nES/MS spectrum of
Nano-electrospray MS and Edman Sequencing Using CE
45
myoglobin collected from CE using about 6 picomoles of material. It was noticed that the multiply-charged state produced in nES/MS for myoglobin was shifted to a higher values, compared to the normal electrospray MS. Again, the measured mass was 16 Da higher than the calculated mass (16951.5 Da), corresponding to one oxygen added to the protein. Figure 8b is the Edman sequencing data from the myoglobin CE fraction using about 6 picomoles of protein. The samples collected from a CE run were usually not suitable for direct Edman sequencing. High background interference was often observed which may result in a wrong sequence call. ProSpin can effectively eliminate small molecule contamination. Since the collected CE fraction contained 50% acetonitrile, the sample was partially pre-dried using a nitrogen stream before the centrifugation process. As Figure 8b shows, twelve N-terminal cycles of myoglobin were obtained from a single CE fraction with a quite clean background.
Figure 8b. Edman sequencing data of the CE fraction of myoglobin showing the N-terminal 12 cycles.
IV.
Conclusion
An off-line approach that is simple and useful for peptide/protein sequencing using 5-10 picomoles of material has been demonstrated. Peptide and protein samples were first separated by capillary electrophoresis. Selected peaks were fraction collected and analyzed by both nano-electrospray mass spectrometry and Edman sequencing. A standard peptide mixture, a tryptic-digested protein and intact proteins were used to illustrate this method. Successful fraction collection of each component required reproducible electropherograms, the ability to automatically switch the outlet buffer vessel and the ability to maintain electrophoretic integrity while eluting a peak of interest into a small outlet buffer
46
Mark D. Bauer et al
volume. Successful MS and MS/MS required the use of electrospray-compatible buffers in the initial CE separation along with the nES source which provided signals of sufficient duration to fully interrogate the ions of interest. Recently, Matthias Wilm et al. (10) reported a simple technique for peptide analysis isolated from polyacrylamide gel electrophoresis, using perfusion sorbent for sample clean-up before nano-electrospray MS analysis. This approach might be very useful for sample clean-up of CE fractions, allowing the use of different CE buffers and different types of capillary columns. Work is under way using coated amine capillaries for better CE separation of proteins.
Acknowledgments The authors gratefully acknowledge Dr. Thomas W. Keough and Dr. Kenny Morand for their help with the nano-electrospray source installation.
References 1. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12. 13.
Cai, J. and Henion, J. (1995), J. Chromatography, 703, 667. Pleasance, S., Thibault, P. and Kelly, J. (1992), J. Chromatography, 591, 325. Locke, S.J. and Thibault, P. (1994), Anal Chem., 66, 3436. Sun, Y., Bauer, M.D. and Wang, P., "Analysis of Peptides and Proteins by On-line CE-ES/MS and Off-line CE-nES/MS", Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR (1996). Herold, M. and Wu, S. (1994), LC-GC, 12, No. 7, 531. Takigiku, R., Keough, T., Lacey, M. P., Schneider, R. E. (1990), Rapid Commun. Mass Spectrom., 4(1), 24. Keough, T., Takigiku, R., Lacey, M. P., and Purdon, M. (1992), Anal. Chem., 64, 1594. Licklider L., Kuhr, W. G., Lacey, M. P., Keough, T. Purdon, M. P., and Takigiku, R., (1995), Anal Chem., 67, 4170. Wilm, M.S. and Mann, M. (1994), International J. Mass Spectrom. and Ion Processes, 136, 167. Wilm, M.S., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T. and Mann, M. (1996), Nature, 379, 466. Shevchenko, A., Wilm, M.S., Vorm, O. and Mann, M. (1996), Anal Chem., 68, 850. Biehler, R. and Schwartz, H.E., Beckman Instruments technical bulletin, TIBC-105. Roepstorff, P. and Fohlman, J., 1984, 11, 601.
I.
Introduction
Capillary electrophoresis (CE) is rapidly becoming an important complementary method to high performance liquid chromatography (HPLC). It provides several advantages, including high separation efficiency, small sample consumption and short analysis time. Because of the small sample volumes required, CE is becoming the analytical tool of choice for biological applications which are sample limited. Electrospray mass spectrometry (ES/MS) is a powerful technique for the structural characterization of biomolecules. Although on-line CE-MS has been demonstrated to be useful for the analysis of biomolecules, the interfacing of CE and ES/MS is nontrivial and much more complicated than the LC-ES/MS interfacing (1-4). Some limitations for on-line CE-MS are: the capillary has to be long enough for the coupling of the CE to the MS; MS/MS sensitivity is relatively low due to the limited sample size loaded onto the capillary; and CE buffers used have to be compatible with MS analysis. Off-line approaches, which combined CE fraction collection and desorption mass spectrometry (plasma desorption and matrix-assisted laser desorption ionization) for peptide and protein analyses, have been demonstrated (5-8). The advantage of off-line approaches is the possibility of independently optimizing both CE separation and mass spectral analysis. Nanoelectrospray (nES) is a new technique for characterizing biomolecules in small volumes (0.5-2 |il) at low picomole levels (9-11). In nES, signals from a single sample loading typically last more than 30 minutes, which permits optimization of instrument parameters and MS/MS sequencing with high sensitivity. Both nES/MS and nES/MS/MS data can be obtained from a single sample loading. These features make nES an attractive off-line technique for sequencing peptides collected from CE. In this paper, an off-line approach, which combines nES/MS analysis and Edman sequencing of peptide/protein fractions collected from CE, is presented. Automatic peak collection was accomplished using a computer-controlled Beckman P/ACE 5000 instrument (12). Several different samples containing 5lOpicomoles of material, including a peptide mixture (angiotensin-I, methionine enkephalin and substance-P), a tryptic digest of cytochrome-C and proteins like myoglobin, insulin and lysozyme, were used to demonstrate this method. TECHNIQUES IN PROTEIN CHEMISTRY VIII Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.
37
38
Mark D. Bauer et al
II. Materials and Methods A.
Peptides and Proteins
Angiotensin-I, methionine enkephalin, substance-P, bovine trypsin, horse cytochrome-C, horse myoglobin, bovine insulin and egg-white lysozyme were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. B.
Enzymatic
Digestion
Both trypsin and cytochrome-C were dissolved separately to 1 mg/ml in 100 mM NH4AC, pH 8.1. 1 |Lil of the trypsin solution was added to 100 |Lil of the cytochrome-C solution. The reaction mixture was incubated at 37°C for 2 hours. The reaction was stopped by freezing. C.
Capillary
Electrophoresis
A Beckman P/ACE 5000 (Schaumberg, IL) was used for all CE separations. The background electrolyte (BGE) was CH3CN/H2O/HCOOH (50:45:5). Washing solution was 1% NH4OH. All untreated fused-silica capillaries were obtained from Polymicro Technologies (Phoenix, AZ). Column dimensions were 97 cm x 75 |LiM. Samples ranging from 0.3 mg/ml to 2 mg/ml (each analyte) were pressure injected for 10 to 30 sec with the goal to load 5 - 1 2 picomoles of each analyte. During separation, the voltage was maintained at 30kV except during fraction collection when the voltage was reduced to 7.5 kV. To collect a CE fraction, the following stepwise procedures which were used on the Beckman P/ACE 5000 instrument: (1) tum off the voltage just before the peak entered the UV window, (2) switch to the collection vial, (3) tum on the voltage to 25% of the maximum until the peak eluted from the capillary, and (4) turn off the voltage and switch back to the original outlet vial and resume the run under initial voltage conditions. The distance from the flow cell to the capillary outlet end is 7 cm. The fraction collection window is 5-10 minutes, depending on the peak width of interest. Fraction collection was accomplished by using the outlet carousel on the P/ACE 5000 to switch to a collection vial which contained 100 |il BGE. Each fraction was dried in-vacuo. Samples were redissolved in 4 |LI1 of CH3OH/H2O/HCOOH (50:50:0.1) and subjected to nES/MS and nES/MS/MS analysis and Edman sequencing. Protein samples collected from CE were subjected to Edman sequencing without drying the fractions. D.
Nano-electrospray
Mass
Spectrometry
All nES/MS and nES/MS/MS measurements were made on a PerkinElmer Sciex API-in triple quadrupole mass spectrometer (Thomhill, Canada) equipped with the nanoelectrospray source designed by Matthias Wilm and Matthias Mann at the European Molecular Biology Laboratory, Germany (9). The long signal duration allowed the instrumental parameters to be optimized for each peptide interrogated by nES/MS/MS. Argon gas was used as the collision gas. The colHsion gas thickness was 100-200x10 atoms/cm .
Nano-electrospray MS and Edman Sequencing Using CE
E.
39
Edman Sequencing
A PE-ABD 494 Precise protein sequencer was used for Edman sequence analysis of the collected CE fractions. Sample was transferred into a ProSpin (PE-ABD) pre-wetted by 20 L| L1 of methanol. A gentle nitrogen stream was applied to reduce the concentration of acetonitrile. The volume was then brought up to 400 |il with water. The ProSpin unit was centrifuged to dryness at 5,600g. The PVDF disc was cut out and washed three times with water before being placed in a sequencing cartridge. A modified sequencing program based on NORMAL-BLOT was used.
III. Results and Discussion Capillary electrophoresis is a rapid, reliable method for separating complex mixtures according to differences in the charge-to-mass ratio of each component. Fraction collection of the components of interest is necessary for further structural characterization by other analytical methods, e.g. mass spectrometry and Edman sequencing for peptides and proteins. Because of the need to maintain a high voltage across the capillary, fraction collection of analytes is relatively difficult by CE. One approach is to electrokinetically collect fractions into separate tubes by taking advantage of the automated outiet carousel available on the Beckman P/ACE 5000 CE system (12). First, it is necessary to establish reproducible electropherograms using solvent systems that are compatible with the desired subsequent MS analysis. Buffers such as Tris, phosphate or borate, though common for CE separations, are less compatible with electrospray MS. In addition, NaOH washes of the capillary introduce high levels of sodium into the system. Therefore, only volatile solvents and buffers were used in our initial experiments. The background electrolyte (BGE) contained only acetonitrile, water and formic acid, while 1% NH4OH was used as the capillary wash solution. These restrictions on the BGE composition limited the CE separation efficiency. Nonetheless, peptides and proteins could still be separated from one another under these conditions. The collected fraction was dried in-vacuo in preparation for nES/MS analysis and Edman sequencing. Figure 1 depicts the nano-electrospray source. nES/MS offers several features that recommend it for use with fractions collected by CE. First, nES requires small loading volumes at low picomole levels. This minimizes the amount of dilution which the sample must undergo to be effectively introduced into the nES source. Second, the signal obtained by nES lasts for 30 minutes or longer. This feature allows for the extensive interrogation of the sample by MS/MS on appropriately equipped instruments. As a result, all of the data can be generated from one CE run and one nES experiment in which both MS and MS/MS data are obtained. The nES needles, which are gold-coated, pulled glass capillaries, are generally treated as disposable so once loaded all of the data is acquired at one time. Sometimes a needle is defective or is damaged early in the nES experiment. This can result in the loss of the collected CE fraction.
Mark D. Bauer et al
40 syringe for pressure 0 rings ^ ^>w
\
Au coated pulled Capillary
\
600 / ^ ^ s , / 800 V ^
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Figure 1. Nano-electrospray source assembly. The sample capillaries are disposable. The capillary tip is touched against the interface to initiate flow. Voltage is then applied and die capillary is positioned in front of the skinamer cone to obtain signal.
A peptide mixture (angiotensin-I, substance-P and met-enkephalin) was used in our initial experiment. After reproducible CE runs were obtained, each peptide in the nfiixture was collected individually. Figure 2 shows the electropherogram of CE-UV of the peptide mixture and the nES mass spectrum of peak "a" collected from a single CE run. The peaks at m/z 649.2 and 433.2 correspond to the doubly- and triply-charged ions of angiotensin-I, respectively. A fairly high background in the low-mass region was observed in every sample eluted from CE. To reduce the background in nES/MS analysis, the base wash by 1% NH4OH can be eliminated between CE runs. Figure 3 depicts the MS/MS of the doubly- and triply-charged precursor ions of angiotensin-I (DRVYIHPFHL). These spectra were all obtained from a single sample loading. Although the doubly- and triply-charged ions of angiotensin-I showed relatively weak peaks in Figure 2, their corresponding MS/MS spectra showed good signalto-noise ratios. Because the arginine residue was located near the N-terminus, the a- and b-series ions were predominent in both spectra (for nomenclature see ref 13). Two other peptides, substance-P and met-enkephalin, were also analyzed by nES/MS. Figure 4 shows the nES/MS and nES/MS/MS results for metenkephalin (peak c) collected from the CE (see inset Figure 2). Note that the Cterminal metiiionine was oxidized to the sulfoxide (m/z 590.2) during CE sample preparation. The methionine oxidation to the sulfoxide was also observed in substance-P CE fraction. The oxidation reaction of the methionine-containing peptides could occur during the sample drying process. Figure 5 shows that the signal from met-enkephalin lasted for at least 26 minutes with no loss in signal intensity. All three peptides were also successfully sequenced by Edman degradation using half of the material from the same collected CE fractions.
Nano-electrospray MS and Edman Sequencing Using CE
41
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Figure 2. nES mass spectrum of the peak collected at 11.2 minutes. D-R-V-Y-l-H-P-F-H-L
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Figure 3. Off-line CE-nES/MS/MS of both the doubly and triply-charged precursor ions of angiotensin-1.
Mark D. Bauer et al
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700
800
m/z Figure 5. nES/MS spectra of 2 jul met-enkephalin collected from CE showing no loss of signal for 26 minutes.
Nano-electrospray MS and Edman Sequencing Using CE
43
Once the standard peptide mixture was successfully analyzed by off-line CE-nES/MS, the technique was attempted on a more complicated peptide mixture. More than 15 peptides were presented in the 2-hour tryptic digest of cytochromeC. CE separation of the peptides was achieved witiiin 30 minutes. Peaks of interest were collected in 100 |Lil BGE, dried and redissolved in 4 |i,l of the nES solvent. Figure 6 shows the nES mass spectrum of the CE peak at 21.9 minutes (inset) from the tryptic digest. Only one peptide was detected in the CE fraction. Both the singly- and doubly-charged ions of the peptide (m/z 964.5) corresponding to residues 92-99 (EDLIAYLK) of cytochrome-C were observed. Figure 7 is the nES/MS/MS of the doubly-charged precursor ion at m/z 483.0 of the peptide (residues 92-99), yielding a complete series of "y" ions. Fragmentation of the doubly-charged ion was much easier than the singly-charged ion. Both nES/MS and nES/MS/MS spectra of the peptide were obtained from a single loading of a 2-jil sample solution. Edman sequencing of the peak at 21.9 minutes, collected from a separate run, further verified the peptide sequence. Based on both MS and Edman sequencing data, there was no carryover between closely separated peaks.
I00n
92 99 E-D-L-l-A-Y-L-K +2 483.0
^ 50^
Figure 6. nES/MS of the peak (21.9 min) collected from the CE separation of a tryptic cytochrome-C.
Mark D. Bauer et al.
44 482.9
100
U2
75
92 99 E..D--L--I--A--Y--L--K
M
o 50.
> y5 25
ye
JU
i[Lyill..m..iyL.iiLlJlilii.L..., L 200
400 400
600
y7 7'
I
800
m/z Figure 7. nES/MS/MS of the doubly-charged ion at m/z 482.9. 100
m/z
1400
Figure 8a. nES spectrum of myoglobin collected from CE. 12 pmoles of protein were loaded onto the CE.
Proteins like insulin, myoglobin and lysozyme were also loaded onto the non-coated CE capillary and collected for nES/MS analysis and Edman sequencing. Because the peak width of proteins is larger than that of the peptides on the non-coated column, the window is relatively wide (about 10 minutes) under 7.5 kV for fraction collection. Figure 8a is the nES/MS spectrum of
Nano-electrospray MS and Edman Sequencing Using CE
45
myoglobin collected from CE using about 6 picomoles of material. It was noticed that the multiply-charged state produced in nES/MS for myoglobin was shifted to a higher values, compared to the normal electrospray MS. Again, the measured mass was 16 Da higher than the calculated mass (16951.5 Da), corresponding to one oxygen added to the protein. Figure 8b is the Edman sequencing data from the myoglobin CE fraction using about 6 picomoles of protein. The samples collected from a CE run were usually not suitable for direct Edman sequencing. High background interference was often observed which may result in a wrong sequence call. ProSpin can effectively eliminate small molecule contamination. Since the collected CE fraction contained 50% acetonitrile, the sample was partially pre-dried using a nitrogen stream before the centrifugation process. As Figure 8b shows, twelve N-terminal cycles of myoglobin were obtained from a single CE fraction with a quite clean background.
Figure 8b. Edman sequencing data of the CE fraction of myoglobin showing the N-terminal 12 cycles.
IV.
Conclusion
An off-line approach that is simple and useful for peptide/protein sequencing using 5-10 picomoles of material has been demonstrated. Peptide and protein samples were first separated by capillary electrophoresis. Selected peaks were fraction collected and analyzed by both nano-electrospray mass spectrometry and Edman sequencing. A standard peptide mixture, a tryptic-digested protein and intact proteins were used to illustrate this method. Successful fraction collection of each component required reproducible electropherograms, the ability to automatically switch the outlet buffer vessel and the ability to maintain electrophoretic integrity while eluting a peak of interest into a small outlet buffer
46
Mark D. Bauer et al
volume. Successful MS and MS/MS required the use of electrospray-compatible buffers in the initial CE separation along with the nES source which provided signals of sufficient duration to fully interrogate the ions of interest. Recently, Matthias Wilm et al. (10) reported a simple technique for peptide analysis isolated from polyacrylamide gel electrophoresis, using perfusion sorbent for sample clean-up before nano-electrospray MS analysis. This approach might be very useful for sample clean-up of CE fractions, allowing the use of different CE buffers and different types of capillary columns. Work is under way using coated amine capillaries for better CE separation of proteins.
Acknowledgments The authors gratefully acknowledge Dr. Thomas W. Keough and Dr. Kenny Morand for their help with the nano-electrospray source installation.
References 1. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12. 13.
Cai, J. and Henion, J. (1995), J. Chromatography, 703, 667. Pleasance, S., Thibault, P. and Kelly, J. (1992), J. Chromatography, 591, 325. Locke, S.J. and Thibault, P. (1994), Anal Chem., 66, 3436. Sun, Y., Bauer, M.D. and Wang, P., "Analysis of Peptides and Proteins by On-line CE-ES/MS and Off-line CE-nES/MS", Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR (1996). Herold, M. and Wu, S. (1994), LC-GC, 12, No. 7, 531. Takigiku, R., Keough, T., Lacey, M. P., Schneider, R. E. (1990), Rapid Commun. Mass Spectrom., 4(1), 24. Keough, T., Takigiku, R., Lacey, M. P., and Purdon, M. (1992), Anal. Chem., 64, 1594. Licklider L., Kuhr, W. G., Lacey, M. P., Keough, T. Purdon, M. P., and Takigiku, R., (1995), Anal Chem., 67, 4170. Wilm, M.S. and Mann, M. (1994), International J. Mass Spectrom. and Ion Processes, 136, 167. Wilm, M.S., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T. and Mann, M. (1996), Nature, 379, 466. Shevchenko, A., Wilm, M.S., Vorm, O. and Mann, M. (1996), Anal Chem., 68, 850. Biehler, R. and Schwartz, H.E., Beckman Instruments technical bulletin, TIBC-105. Roepstorff, P. and Fohlman, J., 1984, 11, 601.