Anal. Chem. 1998, 70, 1569-1575
MALDI Mass Spectrometry Combined with Avidin-Biotin Chemistry for Analysis of Protein Modifications David C. Schriemer, Talat Yalcin, and Liang Li*
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
A general mass spectrometric method that combines purification and analysis in one step is described for the rapid and sensitive determination of protein modification that involves covalent attachment of a modifying group. In this method, the modifying group is first labeled with a biotin moiety, and the covalent interaction of this group with the targeted protein results in a biotinylated product. The modified protein can then be subjected to enzymatic digestion, followed by the isolation of the biotinylated peptide based on a previously described MALDI method incorporating the avidin-biotin interaction (Schriemer, D. C.; Li, L. Anal. Chem. 1996, 68, 3382-3387). To illustrate the validity of the method, a study of a model system was undertaken, involving the interaction between avian skeletal muscle troponin C and a sulfhydryl-specific biotinylation reagent. It is shown that isolation of a modified peptide with an immobilized avidin product could be achieved, even in the presence of an excess of contaminating protein. Exoproteases could be added to the crude tryptic digest to generate peptide ladders, each containing biotin, which could be analyzed by the avidinbiotin/MALDI method for sequence information. Complementary sequence information could be obtained from the application of this technique in a tandem sector/time-offlight mass spectrometer for MALDI MS/MS analysis, which allowed for the identification of the modification site.
With the development of techniques such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization, mass spectrometry (MS) plays an increasingly important role in the structural analysis of modified proteins. New methods based on MS are being rapidly developed for this purpose.1 A sensitive MS procedure can facilitate the analysis and minimize the consumption of precious materials. Aside from the extensive method development in the area of elucidating protein posttranslational modifications (e.g., glycosylation, phosphorylation), studies have appeared in which the covalent attachment of ligands, suicide (1) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1996, 68, 599R651R. S0003-2700(97)01034-2 CCC: $15.00 Published on Web 03/18/1998
© 1998 American Chemical Society
inhibitors, and affinity labels have been investigated, through either electrospray2-9 or MALDI.10-14 The location and exact nature of the modification must be mapped out to develop an understanding of its functional manifestation as well as to provide insight for the rational design of the ligand or inhibitor. The general strategy developed for protein primary structure analysis by MS involves enzymatic or chemical digestion into a set of smaller peptides. For a simple amino acid substitution, a mass spectral map can be obtained from the unfractionated mixture. The peptide map is compared with the map arising from the unmodified protein to identify the altered peptide(s). More extensive modifications such as glycosylation require purification/ fractionation and further enzymatic processing prior to mass spectral analysis and identification of the altered peptide(s).15,16 An exact identification of the modification site often requires the sequencing of the modified peptide. Sequencing could be performed in the conventional fashion by Edman degradation or (2) Alpin, R. T.; Robinson, C. V.; Schofield, C. J.; Waley, S. G. J. Chem. Soc., Chem. Commun. 1993, 121-123. (3) Withers, S. G.; Aebersold, R. Protein Sci. 1995, 4, 361-372. (4) Tull, D.; Miao, S.; Withers, S. G.; Aebersold, R. Anal. Biochem. 1995, 224, 509-514. (5) Staedtler, P.; Hoenig, S.; Frank, R.; Withers, S. G.; Hengstenberg, W. Eur. J. Biochem. 1995, 232, 658-663. (6) DiIanni, C. L.; Stevens, J. T.; Bolgar, M.; O’Boyle, D. R., II; Weinheimer, S. P.; Colonno, R. J. J. Biol. Chem. 1994, 269, 12672-12676. (7) Salto, R.; Babe, L. M.; Li, J.; Rose, J. R.; Yu, Z.; Burlingame, A. L.; De Voss, J. J.; Sui, V.; Ortiz de Montellano, P.; Craik, C. S. J. Biol. Chem. 1994, 269, 10691-10698. (8) Caldera, P. S.; Yu, Z. H.; Knegtel, R. M. A.; McPhee, F.; Burlingame, A. L.; Craik, C. S.; Kuntz, I. D.; Ortiz de Montellano, P. Bioorg. Med. Chem. 1997, 5, 2019-2027. (9) Costello, C. A.; Kelleher, N. L.; Abe, M.; McLafferty, F. W.; Begley, T. P. J. Biol. Chem. 1996, 271, 3445-3452. (10) Jespersen, S.; Ploemen, J. H. T. M.; Vanbladere; Niessen, W. M. A.; Tjaden, U. R.; Vandergreef, J. J. Mass Spectrom. 1996, 31, 101-107. (11) Roberts, E. S.; Ballou, D. P.; Hopkins, N. E.; Alworth, W. L.; Hollenberg, P. F. Arch. Biochem. Biophys. 1995, 323, 303-312. (12) Girault, S.; Sagan, S.; Bolbach, G.; La Vielle, S.; Chassaing, G. Eur. J. Biochem. 1996, 240, 215-222. (13) Kurian, E.; Prendergast, F. G.; Tomlinson, A. J.; Holmes, M. W.; Naylor, S. J. Am. Soc. Mass Spectrom. 1997, 8, 8-14. (14) Roberts, E. S.; Hopkins, N. E.; Zaluzec, E. J.; Gage, D. A.; Alworth, W. L.; Hollenberg, P. F. Arch. Biochem. Biophys. 1995, 323, 295-302. (15) Apffel, A.; Chakel, J.; Udiavar, S.; Hancock, W. S.; Souders, C.; Pungor, E. J. Chromatogr. A 1995, 717, 41-60. (16) Kussmann, M.; Lassing, U.; Sturmer, C. A. O.; Przybylski, M.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 483-493.
Analytical Chemistry, Vol. 70, No. 8, April 15, 1998 1569
with exoproteases, although modifications can prohibit sequencing and the sample requirements can be excessive (>100 pmol).17,18 These methods require the purification/isolation of the peptide to be sequenced. More recently, tandem mass spectrometry has allowed for peptide sequencing on unfractionated peptide mixtures.19-21 Little sample handling is involved, and sample requirements are low. The appropriate peptide ion is mass selected and dissociated, followed by mass analysis of the resulting fragments. The fragmentation patterns generated by the dissociation step are rich in information; however, determining the primary sequence from the pattern can be quite difficult due to the complexity of fragmentation processes.22 As a result, complete sequencing is not always realized. Both tandem mass spectrometry and the conventional approaches are often used to provide complementary sequence information.23-26 A detailed structural analysis usually arises from such an approach, yet the conventional sequencing approach can remain the limiting step in terms of sample requirements. Many of the approaches cited above require that the modified protein be presented for analysis in highly purified form. For example, to generate a noticeable difference in the peptide maps of a modified and unmodified protein requires the isolation of each form. Furthermore, in comparative approaches, a high degree of sequence coverage is usually required, so that modified sites are not missed in the peptide map. Laborious purification of the modified peptide fragment is often a necessity for its full characterization. Several groups have illustrated the combination of noncovalent affinity interactions with MALDI MS for sample purification.27-31 In a previous paper, we described the incorporation of the strong avidin-biotin interaction into a MALDI method, using commercially available avidin-biotin chromatographic products.32 The procedure allowed for the rapid purification and identification of biotinylated peptides or proteins from mixtures, and the merits of various commercially available avidin-biotin products were assessed for their ease of incorporation into the MALDI experiment. A related approach has appeared in the (17) Mathews, C. K.; van Holde, K. E. Biochemistry; The Benjamin/Cummings Publishing Co.: Redwood City, CA, 1990. (18) Smillie, L. B.; Carpenter, M. R. In HPLC of Peptides and Proteins: Separation, Analysis, and Conformation; Mant, C. T., Hodges, R. S., Eds.; CRC Press: Boca Raton, FL, 1991; pp 875-894. (19) Yates, J. R.; McCormack, A. L.; Link, A. J.; Schieltz, D.; Eng, J.; Hays, L. Analyst 1996, 121, R65-R76. (20) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (21) James, P. Biochem. Biophys. Res. Commun. 1997, 231, 1-6. (22) Papayannopoulos, I. A. Mass Spectrom. Rev. 1995, 14, 49-73. (23) Roberts, G. D.; Johnson, W. P.; Burman, S.; Anumula, K. R.; Carr, S. A. Anal. Chem. 1995, 67, 3613-3625. (24) Burgisser, D. M.; Siegenthaler, G.; Kuster, T.; Hellman, U.; Hunziker, P.; Birchler, N.; Heizmann, C. W. Biochem. Biophys. Res. Commun. 1995, 217, 257-263. (25) Berger, B.; Hunziker, P. E.; Hauer, C. R.; Birchler, N.; Dallinger, R. Biochem. J. 1995, 311 (Part 3), 951-957. (26) Taylor, J. A.; Johnson, R. S. Rapid Commun. Mass Spectrom. 1997, 11, 10671075. (27) Hutchens, T. W.; Yip, T.-T. Rapid Commun. Mass Spectrom. 1993, 7, 576580. (28) Zhao, Y.; Chait, B. T. Anal. Chem. 1994, 66, 3723-3726. (29) Papac, D. I.; Hoyes, J.; Tomer, K. B. Anal. Chem. 1994, 66, 2609-2613. (30) Nelson, R. W.; Krone, J. R.; Bieber, A. L.; Williams, P. Anal. Chem. 1995, 67, 1153-1158. (31) Brockman, A. H.; Orlando, R. Anal. Chem. 1995, 67, 4581-4585. (32) Schriemer, D. C.; Li, L. Anal. Chem. 1996, 68, 3382-3387.
1570 Analytical Chemistry, Vol. 70, No. 8, April 15, 1998
literature, using the weaker-binding biotinyl sulfone and streptavidin on magnetic particles.12,33 In this study, we report the analysis of a system designed to demonstrate the combination of MALDI MS with common avidinbiotin purification chemistry for the rapid identification of the site of covalent modification. A covalent interaction between avian skeletal muscle troponin C (TnC) and a biotinylation reagent was analyzed. The site-specific biotinylation of this protein was used as a model system for irreversible enzyme inhibition.34 Enzymatic sequencing and tandem mass spectrometry protocols, in conjunction with the avidin-biotin/MALDI technique, were developed to sequence the biotinylated peptides resulting from enzymatic digestion of the modified TnC. With this model system, we show that the analysis of very impure samples containing the covalently modified protein can yield rich structural information. EXPERIMENTAL SECTION Biotinylation of Troponin C. The reagent N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide (biotin-HPDP, structure I) was purchased from Pierce (Rockford, IL) and used to achieve sulfhydryl-specific biotinylation. A 4 mM solution of biotin-HPDP
was prepared in N,N-dimethylformamide (DMF). Avian skeletal muscle troponin C (TnC) was dissolved in phosphate-buffered saline (PBS, 50 mM phosphate, 0.5 M NaCl, 1 mM EDTA, pH 7.4) to a concentration of 1 mM. The biotinylation reagent and the protein were combined in equimolar amounts and diluted with PBS such that the final solution contained 5% DMF (v/v). The final protein concentration was 200 µM. The reaction was carried out at room temperature for 2 h and then stored at -20 °C. Enzymatic Digestion. Enzymatic digestions using bovine trypsin (TPCK-treated), yeast carboxypeptidase Y (Sigma, St. Louis MO), and porcine leucine aminopeptidase M (1 mg/mL slurry in 3.8 M ammonium sulfate, Fluka BioChemika, Buchs, Switzerland) were undertaken in the analysis of the model system. Digestions were carried out on various combinations of TnC, biotinylated TnC, bovine serum albumin (BSA), and equine cytochrome c. Tryptic digests were carried out at room temperature in PBS or water for 3 h, using approximately equimolar amounts of trypsin to digest a protein sample. Soybean trypsin inhibitor (Sigma) was added to terminate the digestion after the specified time. To obtain sequencing information, leucine aminopeptidase M (LAP) or carboxypeptidase Y (CPY) digests of the unfractionated tryptic digests were undertaken.35-37 For Nterminal sequencing, LAP and trypsin-digested protein (in PBS) (33) Girault, S.; Chassaing, G.; Blais, J. C.; Brunot, A.; Bolbach, G. Anal. Chem. 1996, 68, 2122-2126. (34) Schriemer, D. C.; O’Callaghan, K.; Anders, M.; Vederas, J. C.; Li, L. Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 12-16, 1996; p 1001. (35) Thiede, B.; Liebold-Wittman, B.; Bienert, M.; Krause, E. FEBS Lett. 1995, 357, 65-69.
were combined. Ratios are cited in the Results and Discussion. After a 75-min digestion at room temperature, tetrameric avidin immobilized on cross-linked, beaded agarose (Pierce, Rockford, IL; catalog no. 20219) was added to extract the biotinylated peptides for analysis by MALDI, according to a previously described procedure.32 For C-terminal sequencing, ∼30 pmol of CPY was combined with ∼1 nmol of trypsin-digested total protein (in water) and digested for variable periods of time (see Results and Discussion). After digestion, the biotinylated peptides were isolated with a small quantity of immobilized tetrameric avidin, washed with PBS and water, and then analyzed by MALDI. Instrumentation. Mass spectra were collected on a linear time-lag focusing MALDI time-of-flight (TOF) mass spectrometer. Its construction was based on the design of our prototype instrument.38 The new instrument has a flight tube length of ∼1.15 m and has been designed for operation up to 30 kV. A single dc power supply was used to set the potential on the repeller and first extraction plate and the potential on the second extraction plate through a voltage divider. Under normal operation, the power supply was set to 20 kV dc. A high-voltage pulser built in-house was used to generate the delayed extraction pulse. The extraction pulse and time lag were varied as required to focus the mass range of interest. A nitrogen laser (VSL-337ND, Laser Science, Inc., Newton, MA) with a 3-ns pulse width was used for desorption at 67.5° to the probe surface normal. A neutral-density filter was used to attenuate the laser energy that reached the sample surface. A mass filter was added to the system. The mass filter prevented saturation of the microchannel plate detector by deflecting matrix ions out of the usual ion trajectory, reducing the ion current from low-mass ions. All spectra were externally calibrated with well-characterized peptides, resulting in mass accuracy typically better than (70 ppm. The spectra presented in this study represent the summation of ∼50 laser shots. MALDI collisionally induced dissociation (CID) spectra were collected on a ZabSpec orthogonal acceleration (oa) time-of-flight instrument from Micromass (Manchester, UK). A nitrogen laser (337 nm) was operated at 10 Hz for desorption. The precursor ions were accelerated by a voltage of 8 kV and mass selected with the EBE mass spectrometer. The ions were introduced into the collision cell floated to 7200 kV, imparting 800 eV to the precursor ions. Xenon was used as the collision gas, and, on average, the intensity of the precursor ion pulses was attenuated by ∼30%. The ions exiting the collision cell were guided into the oa-time-of-flight spectrometer and pulsed into the flight tube for mass separation. Ions were detected with a microchannel plate detector. MALDI Sample Preparation. The matrixes used in this work were R-cyano-4-hydroxycinnamic acid (HCCA) and sinapinic acid (SA). Both matrixes were purchased from Aldrich (Milwaukee, WI). A layer of matrix (dissolved in acetone, 25 mg/mL) was first applied to the MALDI probe tip, forming a dense layer of small crystals.39 One microliter of an agarose bead slurry (in water) was then added and allowed to dry partially. An aliquot of a second matrix solution could then be applied. The composition (36) Patterson, D. H.; Tarr, G. E.; Regnier, F. E.; Martin, S. A. Anal. Chem. 1995, 67, 3971-3978. (37) Woods, A. S.; Huang, A. Y. C.; Cotter, R. J.; Pasternack, G. R.; Pardoll, D. M.; Jaffee, E. M. Anal. Biochem. 1995, 226, 15-25. (38) Whittal, R. M.; Li, L. Anal. Chem. 1995, 67, 1950-1954. (39) Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281-3287.
Figure 1. MALDI mass spectra of (A) unlabeled TnC (expected [M + H]+ ) 18 257 u) and (B) biotinylated TnC (expected [M + H]+ ) 18 686 u).
of this second matrix solution varied, with the proviso that it be saturated with the matrix to prevent dissolution of the bottom layer. Upon complete drying, the beads were blown off with a stream of dry nitrogen. Elution of the biotinylated peptide from the solid support does not appear to be dependent upon the composition of the second matrix solution, and it is surmised that dehydration of the agarose particles plays a role.32 All solution samples were analyzed in a similar fashion; the analyte was diluted with the second matrix solution and deposited on the first matrix layer. The direct analysis of tryptic digest solutions was achieved using HCCA saturated in 2-propanol/formic acid/water as the matrix preparation. The analysis of intact TnC involved the twolayer method, using SA as the matrix. The second matrix solution consisted of SA saturated in 25% CH3CN/0.1% trifluoroacetic acid. RESULTS AND DISCUSSION Biotinylation of TnC. Troponin C plays a regulatory role in vertebrate striated muscle contraction and relaxation by interacting with Ca2+.40 Skeletal muscle TnC is a protein consisting of 162 amino acids, with a molecular weight of 18 256.4 u. It contains only one cysteine.40 To mimic an inhibitor-enzyme interaction, a biotinylation reagent was selected that would selectively form a covalent linkage with the cysteine residue. The biotinylation reaction could be monitored by MALDI. Figure 1A shows the MALDI spectrum obtained from the pure protein. The measured [M + H]+ value of 18 257 u agrees with the expected value, within acceptable error. A sulfhydryl-specific biotin reagent (biotinHPDP, I) was chosen to react with TnC, resulting in a modified (40) Gagne, S. M.; Tsuda, S.; Li, M. X.; Smilie, L. B.; Sykes, B. D. Nature Struct. Biol. 1995, 2, 784-789.
Analytical Chemistry, Vol. 70, No. 8, April 15, 1998
1571
protein and the loss of pyridine-2-thione, as shown in the schematic below.
A spectrum was collected from the reaction mixture and is shown in Figure 1B. The measured [M + H]+ value of 18 685 u indicates a mass shift of 428 u, which agrees well with the expected mass shift from biotinylation (428.6 u). The absence of a peak at m/z 18 257 indicates the high efficiency of the reaction. Therefore, this represents a model system in which a biotin group is attached to a site-specific modifier (pyridyldithio group), tagging the protein in a single location of the primary sequence. Similar systems could be designed, such as the biotinylation of a mechanism-based inhibitor, followed by inhibition of the target enzyme.34 Mass Spectra of Tryptic Digests. A small amount of the biotinylated TnC sample was purposely mixed with other proteins as contaminants, to mimic less than ideal situations such as the low conversion of protein to its modified form. This can be seen as a model of the final state of a mechanism-based enzyme inhibition, for example. A mixture containing 1 nmol of TnC and 10 pmol of biotin-TnC was prepared and digested with trypsin in PBS for 3 h. The digest was then sampled for MALDI analysis, generating the mass spectrum displayed in Figure 2A. Approximately 80% sequence coverage was observed from complete cleavage products. Peaks arising from incomplete cleavage were also observed at higher masses, as well as trypsin autolysis products. Note that some measure of methionine oxidation occurred during digestion, as some tryptic fragments were represented in the spectrum at a mass 16 u higher than expected. One-tenth of this crude mixture was purified and analyzed using the avidin-biotin/MALDI procedure. Approximately onefifth of the settled beads were sampled for mass analysis. The spectrum arising from this procedure is shown in Figure 2B, indicating monoisotopic peaks at m/z 1625.8 and 1197.5. The mass difference of 428.3 u agrees with the expected mass difference arising from biotinylation. The peak at m/z 1625.8 would, therefore, seem to indicate a biotinylated peptide, with the peak at m/z 1197.5 representing the loss of the biotin moiety. The lower mass peak likely arose from the partial reduction of the disulfide in the spacer arm of the modifying group, due to the higher acid content of the matrix preparation, rather than from nonspecific binding of the corresponding unlabeled peptide. A number of experiments were conducted to support the above conclusions. A control experiment was performed in which the digested biotinylated TnC was absent; no peaks at m/z 1197.5 and 1625.8 were observed. Also, the solution containing both 1572 Analytical Chemistry, Vol. 70, No. 8, April 15, 1998
Figure 2. (A) Mass spectrum from a sample of the tryptic digest of a 100:1 blend of TnC and biotinylated TnC (molar ratio). Trypsin autolysis peaks are identified by stars; a single peptide due to incomplete cleavage was identified at m/z 2289.7. Inset shows an expansion of the region from m/z 1400 to 1900. (B) Purification and analysis of the tryptic digest using the avidin-biotin/MALDI procedure, representing at most 200 fmol of peptide applied to the probe tip.
digested TnC and biotinylated TnC was applied to avidin-agarose beads presaturated with free biotin; again, no peaks at m/z 1197.5 and 1625.8 were observed. As a positive control experiment, 1 µL of a 50 mM dithiothreitol solution was added to the bead slurry on the probe tip.32 This resulted in the more extensive reduction of the disulfide linker and an increase in the intensity of the peak at m/z 1197.5. A search of the possible protonated tryptic fragments of TnC identified a candidate with a monoisotopic mass of 1197.5 u. This would correspond to a mass error of 42 ppm. The next candidate, at 1198.5 u, would indicate an unreasonably high error of 650 ppm. The sequence of the candidate peptide is SEEELANCFR. The presence of the only cysteine in this sequence supports the labeling of the peak at m/z 1625.8 as the biotinylated SEEELANCFR (expected [M + H]+ of 1625.7 u) and the peak at m/z 1197.5 as its reduced analogue. From the inset of Figure 2A, note that the spectrum does not show a peak at m/z 1625.7. The concentration of the corresponding peptide in solution was simply too low to be observed. To further illustrate the isolation power of the avidin-biotin system, a more complex digest solution was prepared and analyzed. Figure 3 represents the spectrum arising from the purification of a tryptic digest of a solution containing 10 nmol of BSA, 10 nmol of cytochrome c, 10 nmol of trypsin, 1 nmol of TnC, and 100 fmol of biotinylated TnC in 1 mL of PBS. Approximately one-fourth of the settled beads were sampled for mass analysis to generate this spectrum, which reveals a peak at m/z 1625.8. Note that peaks due to nonspecifically adsorbed species are noticeable at this low level of biotin-peptide loading. In these cases, control experi-
Figure 3. Mass spectrum obtained from a tryptic digest of a solution containing BSA, cytochrome c, TnC, and a trace amount of biotinylated TnC. Purification and analysis were achieved using the avidinbiotin/MALDI procedure. Spectrum represents at most 20 fmol of peptide applied to the probe tip.
ments are essential to determine the identity of the appropriately labeled peptide. Although a high acid matrix preparation was used, a peak at m/z 1197.5 representing the reduced peptide was not observed. With a second sample of the settled beads, an aliquot of aqueous dithiothreitol was added to the bead slurry to achieve on-probe disulfide reduction, but a slight loss of sensitivity resulted in the loss of the peak at 1625.8, and no peak was detectable at m/z 1197.5. A separate experiment, in which the biotinylated TnC was excluded from the tryptic digestion, resulted in the disappearance of the peak at m/z 1625.8. Ladder Sequencing of the Modified Peptide. Identification of the peptide containing the modification site based solely on the mass of a single peptide becomes virtually impossible when the parent protein is large, as so many possibilities can exist for a certain mass given the accuracy of MALDI TOF mass determinations. At this point, partial or even full sequencing of the modified peptide becomes necessary. Sequencing based on Edman degradation or exoproteases requires the peptide to be purified to a high degree. Simplification of purification results if the modified peptide can be complexed to a certain species through a high-affinity interaction. To illustrate, both N-terminal and C-terminal enzymatic sequencing of the biotinylated peptide represented at m/z 1625.8 was undertaken. Ideally, partial and successive cleavage of amino acids would generate a series of peptides, all containing the biotin moiety. LAP Digestions. A solution consisting of 1 nmol of TnC and 10 pmol of biotinylated TnC in 100 µL of PBS was digested with trypsin and the presence of the biotinylated peptide confirmed. For N-terminal sequencing, the solution was split into two equal portions (∼45 µL each). Approximately 400 pmol of LAP slurry was added to one portion, and 1.2 nmol to the other. After equivalent digestion times, one-fourth of each sample was purified and analyzed using the avidin-biotin/MALDI procedure. Figure 4A shows the spectrum from the digestion using 400 pmol of LAP. Note the two smaller peaks in this spectrum, in addition to the expected peaks at m/z 1625.7 and 1197.5. These smaller peaks reflect the cleavage of amino acids from the N-terminus of the biotinylated peptide. Mass accuracy is sufficient to identify the -87.0 u as arising from the loss of serine, and the -129.1 u arising from the loss of glutamic acid. Incubating the rest of the digest
Figure 4. Mass spectra obtained from 75-min LAP digestions of an unpurified tryptic digest containing trace amounts of a biotinylated peptide, using (A) 400 pmol and (B) 1.2 nmol of LAP. Biotinylated species were isolated and analyzed using the avidin-biotin/MALDI procedure.
for longer periods of time gave no further sequence information. However, the digestion solution containing 1.2 nmol of LAP demonstrated more extensive cleavage (Figure 4B). The peaks at m/z 1625.8 and 1197.7 were less intense, and two new peaks arose at lower mass. A large gap of 371.1 u was observed between the peaks at m/z 1038.7 and 1409.8. This would correspond to at least 3 amino acids and is indicative of the different cleavage rates for exoproteolysis.35-37 Clearly, the enzyme processes this part of the sequence very quickly and efficiently. The two new, lower mass peaks differ by 71.1 u, indicating the presence of alanine at least five amino acids removed from the N-terminus. Again, incubation for a longer period of time gave no further sequence information. Note that all these tests, including confirmation of biotinylation, were performed on 10 pmol of biotinylated TnC in the presence of substantial contamination. CPY Digestions. A similar approach was followed for C-terminal sequencing using CPY as the exoprotease. A tryptic digest of 1 nmol of TnC and 10 pmol of biotinylated TnC in 100 µL of water was prepared and the presence of the biotinylated peptide confirmed. CPY was added to the mixture and the digestion monitored with time. One-tenth of the sample was removed in each case. Figure 5A-C shows the spectra obtained at 10, 40 and 120 min, respectively. Rapid cleavage of the first amino acid was indicated by the intense peak in Figure 5A. The difference of 156.1 u between the two peaks of this spectrum indicates that arginine is the C-terminal amino acid. At 40 min, the parent peptide represented by m/z 1625.7 was completely digested; however, no new sequence information was obtained (Figure 5B). After 120 min, a small peak corresponding to the loss of phenylalanine (147.0 u) was observed. Analytical Chemistry, Vol. 70, No. 8, April 15, 1998
1573
Figure 5. Mass spectra obtained from a CPY digestion of an unpurified tryptic digest containing trace amounts of a biotinylated peptide. The digestion was sampled at (A) 10, (B) 40, and (C) 120 min. Biotinylated species were isolated and analyzed using the avidin-biotin/MALDI procedure.
Partial, successive cleavage of amino acids from a peptide such as described above has been dubbed “ladder sequencing” and has been demonstrated as useful for the identification of protein/ peptide modifications.41 If a biotin can be incorporated in the modifying group, Figures 4 and 5 show that such sequencing can also be performed quickly on crude mixtures. A current shortcoming with this technique relates to the difficulty in achieving efficient, successive enzymatic digestions. As no purification was imposed on the sample from the initial tryptic digests onward, only one buffer system for all digestions could be used. CPY digests do not work well in phosphate buffers; therefore, the initial tryptic digest was carried out in pure water (CPY digests work well in pure water36). Such a compromise will not always be tolerable. An unsuccessful attempt was made to enzymatically sequence the bound biotinylated tryptic fragment. The tight complexation with avidin would allow for buffer exchange by washing the beads prior to administering the next enzyme. No ladder sequencing was obtained in this case, presumably because the enzymes had restricted access to the bound peptide. A further drawback with the current procedure is that an amount of digestion enzyme must be chosen that is sufficient to cleave all components in the mixture, including impurities. This can lead to a high consumption of expensive digestion enzymes. Nevertheless, Figures 4 and 5 illustrate that the incorporation of biotin allows a quick and sensitive ladder sequencing of the modified peptide in a crude mixture, which provides useful, although not necessarily complete, sequencing information. (41) Chait, B. T.; Wang, R.; Beavis, R. C.; Kent, S. B. H. Science 1993, 262, 89-92.
1574 Analytical Chemistry, Vol. 70, No. 8, April 15, 1998
Based on an accurate mass determination of the modified peptide, enzymatic ladder sequencing, and foreknowledge of the modification chemistry, a definite confirmation of peptide identity and the site of modification can be made. However, if the chemistry is not known (or if there exists some ambiguity in the protein structure), complete sequencing is needed to determine the exact location in the peptide where the modification has occurred. MS/MS Spectra of the Modified Peptide. In an attempt to independently determine the binding site in the model study, MS/ MS spectra from the biotinylated peptide were collected, using a tandem sector/time-of-flight instrument. The ZabSpec used in this work was outfitted with a MALDI source to allow for a sample preparation identical to that used in the linear time-of-flight analysis. Sample requirements were somewhat higher for the ZabSpec. Usually a loading of ∼10 pmol of peptide on the probe tip was necessary to obtain intense fragment ion signals. A high-energy CID spectrum of the peptide corresponding to m/z 1625.7 was collected on the ZabSpec, under conditions favoring single collisions.42 The full spectrum is shown in Figure 6A, with expansions shown in Figure 6B-D. In the ensuing discussion, it will be assumed that the sequence of the modified peptide has been determined, but not the modification site or chemistry. The spectrum shown in Figure 6 indicates a high degree of fragmentation, and little useful sequence information between m/z 500 and the precursor ion peak. Figure 6B is an expansion of the immonium ion region of the spectrum, and the positively identified amino acids are represented by their one-letter code. Note that the peak at m/z 86.0 could correspond to leucine or isoleucine; one cannot distinguish between these amino acids on the basis of immonium ions. The peak at m/z 87.0 is indicative of both arginine and asparagine. Peaks due to arginine also arise at m/z 70.2, 100.1 and 112.0. The y1 peak at m/z 175.1 (Figure 6C) confirms that arginine resides at the C-terminus. The presence of arginine in the spectrum casts doubt upon the assignment of m/z 87.0 as being at least partially due to asparagine. However, since its presence in the sequence is known, this assignment is reasonable. Note that no immonium ion corresponding to cysteine was observed (expected at m/z 76.0). All of the other amino acids appear to be represented, supporting an initial hypothesis that the modifying group was bound to cysteine. This hypothesis should receive reinforcement from the sequence information present in the spectrum. Figure 6C,D shows expansions of higher m/z portions of the spectrum. A series of b ions were observed, up to b4. This identifies a portion of the N-terminal sequence as SEEE. Recall from the LAP enzymatic sequencing (Figure 4) that a sequence jump of 371.1 u was observed. The b ion information from the high CID spectrum reveals that EE accounts for part of the jump. This leaves a mass of 112.9 u, which indicates leucine or isoleucine. A y2 ion was identified in Figure 6C, corresponding to FR. Other y ions could not be positively identified. (42) Hayes, R. N.; Gross, M. L. In Methods in Enzymology: Mass Spectrometry; MaCloskey, J. A., Ed.; Academic Press: San Diego, CA, 1990; Vol. 193, pp 237-263.
The presence of the modifying group also appears to adversely affect the intensity of higher order fragments that might normally be present in a similar analysis of an unmodified peptide. Nevertheless, with the aid of a high-energy CID spectrum and enzymatic sequencing, cysteine can be identified as the strongest candidate for modification. While the modification chemistry was assumed to be unknown, the suspected reactivity of the modifying group correlates well with the identification of cysteine as the altered amino acid.
Figure 6. MS/MS spectrum of m/z 1625.7 biotinylated peptide, obtained with high-energy CID on the ZabSpec. (A) Full spectrum; (B) expansion of the immonium ion region; (C) m/z 165-335 region; and (D) m/z 335-500 region. The biotinylated peptide was isolated and analyzed from a tryptic digest using the avidin-biotin/MALDI procedure.
At this point, a combination of enzymatic sequencing and tandem mass spectrometry has identified that the modifying group does not reside on the following amino acids in the peptide (indicated by underscoring): SEEELANCFR. Figure 6D provides additional insight. A strong cluster of peaks is visible in the spectrum. Note that the peak at m/z 429.1 coincides with the protonated biotinylation reagent, minus the pyridine-2-thione leaving group. The strong peak at m/z 431.1 could indicate the acquisition of two protons for ion stabilization.22 None of the peaks in this cluster seem to correspond to a rational cleavage of the peptide itself. Also note that a smaller cluster 32.0 u higher suggests that the modifying group is bound to a sulfur in the peptide. These peaks as well do not correspond to a rational cleavage of the peptide. Note that no immonium ion for the modified amino acid is evident in the spectrum at higher m/z.
CONCLUSIONS Rapid identification of appropriately modified proteins can be achieved on picomole quantities of protein in contaminated solutions. Using TnC as the model system, it was shown that incorporating biotin in the modifying group allows for successive enzymatic processing for sequence information, without purification between digestions. Purification occurred in the final step by simply incubating the crude digest in the presence of tetrameric avidin immobilized on beaded agarose. These beads could then be sampled for MALDI analysis. An attractive feature is the ability to monitor the progress of a digestion, by sampling small amounts of the total digest over time, for isolation with immobilized avidin. Using this approach, buffer exchange was not possible, necessitating the use of a single buffer system for different enzymatic digestions. Incorporating a chemically cleavable spacer between the biotin and the protein provided an easy method for confirmation of modification, although a disulfide group would not be generally appropriate when protein denaturing is required prior to digestion. Tandem mass spectrometry was performed on the biotinylated tryptic fragment for the generation of complementary sequence information and modification site information. This also allowed for confirmation of modification by generating ions characteristic of the modifying group. The combination of enzymatic sequencing and tandem mass spectrometry on a peptide with a group such as biotin represents a rapid and sensitive method for the investigation of protein modification. The specific biotinylation of TnC and its subsequent analysis represents a good model for similar investigations of enzyme/inhibitor or protein/ ligand interactions, provided that biotinylation of the modifying group does not affect its interaction with the protein in question. For example, the approach described in this work could be applied to the study of irreversible inhibition, where there is very limited knowledge of the inhibition chemistry.34 ACKNOWLEDGMENT This work was supported in part by the Natural Sciences and Engineering Research Council of Canada (NSERC). The TnC used in this study was a generous gift from Drs. M. X. Li, L. Smillie, and B. Sykes of the Biochemistry Department at the University of Alberta. D.C.S is a Killam Trust predoctoral fellow.
Received for review September 17, 1997. February 4, 1998.
Accepted
AC9710341
Analytical Chemistry, Vol. 70, No. 8, April 15, 1998
1575
