Deuterium Scrambling during Gas

Jul 31, 2008 - Addition of a proteolysis step between the exchange reaction and mass ... pentapeptide are first deuterated by dissolving it in deuteri...
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Anal. Chem. 2008, 80, 6785–6790

A Simple Test To Detect Hydrogen/Deuterium Scrambling during Gas-Phase Peptide Fragmentation Yoshitomo Hamuro,* Justine C. Tomasso, and Stephen J. Coales ExSAR Corporation, 11 Deer Park Drive, Suite 103, Monmouth Junction, New Jersey 08852 Amide hydrogen/deuterium (H/D) exchange coupled with mass spectrometry has become a powerful tool to study protein dynamics. Addition of a proteolysis step between the exchange reaction and mass analysis can be used to localize the positions of deuterium and improve overall resolution. The resolution can be further enhanced by the fragmentation of digested peptides in the gas phase if scrambling of exchangeable hydrogens and deuteriums on the peptides does not occur. Although some laboratories reported successful localization of deuteriums by gas-phase fragmentations, others described total scrambling. Here we propose a simple method to detect the presence or absence of scrambling using a commercially available small peptide, neurotensin (9-13; RPYIL). All exchangeable hydrogens on this pentapeptide are first deuterated by dissolving it in deuterium oxide. The deuterated peptide is loaded onto a reversed-phase column, and then washed with copious amounts of cold acidic aqueous buffer. This washing exchanges all deuteriums on both the terminals and the side chains back to hydrogens. Now only three deuteriums are attached on the pentapeptide, one on each of the amide nitrogens of Y, I, and L. After the partially deuterated peptide is eluted from the column with 95% acidic acetonitrile, collisioninduced dissociation (CID) generates a series of b ions, which are analyzed by mass spectrometer. In the absence of scrambling, no deuterium should be observed in the b2 ion, as neither R nor P have amide hydrogens. On the other hand, in the event of scrambling, b2 should carry about half of the deuteriums of the parent pentapeptide. In theory, complete scrambling should distribute deuteriums equally among all of the exchangeable hydrogens. The b2 portion of neurotensin (9-13) has 6 exchangeable hydrogens, whereas the +1 charge state of neurotensin (9-13) has 12 exchangeable hydrogens. We demonstrated that CID caused complete scrambling of hydrogens and deuteriums with an LCQ (a ion trap machine). Hydrogen/deuterium (H/D) exchange coupled with mass spectrometry (MS) is an increasingly popular technology to measure protein dynamics,1-6 protein-ligand interactions,7-11 and * Corresponding author. E-mail: [email protected]. Phone: 732-438-6500. Fax: 732-438-1919. (1) Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2, 522–531. 10.1021/ac800645f CCC: $40.75  2008 American Chemical Society Published on Web 07/31/2008

protein-protein interactions.12-14 A typical experimental procedure involves incubation of a protein solution in deuterated buffer (usually neutral pH) for labeling, followed by quenching of the reaction by the addition of a cold acidic buffer and analysis by MS. Digestion by acid-stable protease(s) after the acid quenching can localize the positions of deuterium incorporations to obtain more detailed information. The resolution of the information obtained by the proteolysis is typically limited by the size of the proteolytic fragments generated. The resolution can potentially be further improved by a gasphase fragmentation technique, such as collision-induced dissociation (CID).15,16 CID can generate a series of b and y ions, and the subtraction of deuteration levels in analogous ions can sublocalize the deuterated sites. A potential problem of this approach is an intramolecular randomization (scrambling) among the exchangeable hydrogens and deuteriums upon gas-phase fragmentation. Scrambling would lead to the loss of sublocalized information and make this approach useless. Anderegg’s group reported the first attempt of deuterium sublocalization by tandem mass spectrometry (MS/MS) in 1994.17 Smith’s15 and Deinzer’s16 groups demonstrated successful sublocalization of deuterium by CID combined with pepsin digestion. Their results are consistent with the previous NMR data. (2) Engen, J. R.; Smith, D. L. Anal. Chem. 2001, 73, 256A–265A. (3) Ehring, H. Anal. Biochem. 1999, 267, 252–259. (4) Hamuro, Y.; Coales, S. J.; Southern, M. R.; Nemeth-Cawley, J. F.; Stranz, D. D.; Griffin, P. R. J. Biomol. Tech. 2003, 14, 171–182. (5) Hamuro, Y.; Burns, L. L.; Canaves, J. M.; Hoffman, R. C.; Taylor, S. S.; Woods, V. L., Jr J. Mol. Biol. 2002, 321, 703–714. (6) Hoofnagle, A. N.; Resing, K. A.; Ahn, N. G. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 1–25. (7) Codreanu, S. G.; Ladner, J. E.; Xiao, G.; Stourman, N. V.; Hachey, D. L.; Gilliland, G. L.; Armstrong, R. N. Biochemistry 2002, 41, 15161–15172. (8) Frego, L.; Davidson, W. Protein Sci. 2006, 15, 722–730. (9) Hamuro, Y.; Coales, S. J.; Morrow, J. A.; Molnar, K. S.; Tuske, S. J.; Southern, M. R.; Griffin, P. R. Protein Sci. 2006, 15, 1. (10) Yan, X.; Broderick, D.; Leid, M. E.; Schimerlik, M. I.; Deinzer, M. L. Biochemistry 2004, 43, 909–917. (11) Kim, M. Y.; Maier, C. S.; Reed, D. J.; Ho, P. S.; Deinzer, M. L. Biochemistry 2001, 40, 14413–14421. (12) Burns-Hamuro, L.; Hamuro, Y.; Kim, J.; Sigala, P.; Fayos, R.; Stranz, D.; Jennings, P.; Taylor, S., Jr Protein Sci. 2005, 14, 2982–2992. (13) Horn, J. R.; Kraybill, B.; Petro, E. J.; Coales, S. J.; Morrow, J. A.; Hamuro, Y.; Kossiakoff, A. A. Biochemistry 2006, 45, 8488–8498. (14) Hamuro, Y.; Anand, G.; Kim, J.; Juliano, C.; Stranz, D.; Taylor, S., Jr J. Mol. Biol. 2004, 340, 1185–1196. (15) Deng, Y.; Pan, H.; Smith, D. L. J. Am. Chem. Soc. 1999, 121, 1966–1967. (16) Kim, M. Y.; Maier, C. S.; Reed, D. J.; Deinzer, M. L. J. Am. Chem. Soc. 2001, 123, 9860–9866. (17) Angeregg, R. J.; Wagner, D. S.; Stevenson, C. L. J. Am. Soc. Mass Spectrom. 1994, 5, 425–433.

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Figure 1. Deuteration of neurotensin (9-13) in the scrambling test.

Akashi’s18-20 and Kaltashov’s21-23 groups also reported localization of deuterium in an intact protein using nozzle skimmer CID. On the other hand, other groups found extensive scrambling during gas-phase fragmentation.24-29 These somewhat conflicting results may imply that the extent of scrambling is dependent on the nature of analyte ions and various fragmentation parameters. Rand and Jorgensen recently developed synthetic peptides for the detection of scrambling in gas-phase fragmentation.28 Their peptides have faster intrinsic exchange rates at the N-terminal and slower intrinsic exchange rates at the C-terminal.30 After a certain period of H/D exchange reaction, the peptide can be deuterated at the N-terminal and not deuterated at the C-terminal. They found complete scrambling using these peptides with their system. More recently, the same group reported nonscrambling during electron capture dissociation (ECD) of this model peptide.31 (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31)

Akashi, S.; Naito, Y.; Takio, K. Anal. Chem. 1999, 71, 4974–4980. Akashi, S.; Takio, K. Protein Sci. 2000, 2497–2505. Akashi, S.; Takio, K. J. Am. Soc. Mass Spectrom. 2001, 12, 1247–1253. Eyles, S. J.; Speir, P.; Kruppa, G.; Gierasch, L. M.; Kaltashov, I. A. J. Am. Chem. Soc. 2000, 122, 495–500. Hoerner, J. K.; Xiao, H.; Dobo, A.; Kaltashov, I. A. J. Am. Chem. Soc. 2004, 126, 7709–7717. Xiao, H.; Kaltashov, I. A. J. Am. Soc. Mass Spectrom. 2005, 16, 869–879. Johnson, R. S.; Krylov, D.; Walsh, K. A. J. Mass Spectrom. 1995, 30, 386– 387. Demmers, J. A. A.; Rijkers, D. T. S.; Haverkamp, J.; Killian, J. A.; Heck, A. J. R. J. Am. Chem. Soc. 2002, 124, 11191–11198. Jorgensen, T. J.; Gardsvoll, H.; Ploug, M.; Roepstorff, P. J. Am. Chem. Soc. 2005, 127, 2785–2793. Bulleigh, K.; Howard, A.; Do, T.; Wu, Q.; Anbalagan, V.; Stipdonk, M. V. Rapid Commun. Mass Spectrom. 2006, 20, 227–232. Rand, K. D.; Jorgensen, T. J. D. Anal. Chem. 2007, 79, 8686–8693. Ferguson, P. L.; Pan, J.; Wilson, D. J.; Dempsey, B.; Lajoie, G.; Shilton, B.; Konermann, L. Anal. Chem. 2007, 79, 153–160. Bai, Y.; Milne, J. S.; Mayne, L. C.; Englander, S. W. Proteins: Struct., Funct., Genet. 1993, 17, 75–86. Rand, K. D.; Adams, C. M.; Zubarev, R. A.; Jorgensen, T. J. J. Am. Chem. Soc. 2008, 130, 1341–1349.

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EXPERIMENTAL CONCEPT We propose to use a commercially available neurotensin (913, RPYIL) for the detection of scrambling. Our approach is based on the fact that deuterium will be equally distributed among all exchangeable hydrogen positions including side chains, N-, and C-terminal (Figure 1) in addition to all amide hydrogen positions in the molecule upon scrambling. When neurotensin (9-13) is dissolved in deuterium oxide, all 11 hydrogens on hetero atoms are deuterated (Figure 1b). The deuterated pentapeptide is washed with a copious amount of cold acidic aqueous buffer after being loaded onto a reversed-phase liquid chromatography (LC) column. The washing of the deuterated peptide should remove all deuteriums except three on the amides of Y, I, and L (Figure 1c). It takes more than 1.5 h for half of the deuterium on Y to exchange with bulk hydrogen under the conditions employed for the aqueous acid washing (pH 2.3 and 0 °C) according to HXPEP (can be downloaded from Walter Englander’s Web site: http:// hx2.med.upenn.edu/download.html).30 It also takes more than 5 h for half of the deuterium on I or L to be lost. In the absence of scrambling neither b1 nor b2 ions should carry any deuteriums. On the other hand, both b1 and b2 ions should carry a significant amount of deuteriums in the event of scrambling (Figure 1d and Table 1). There are 12 exchangeable hydrogens in the +1 charge state of monoprotonated neurotensin (9-13). There are six hydrogens on either the b1 or b2 ion. If the +1 charge state of monoprotonated neurotensin (9-13) is subject to fragmentation in the gas phase and the hydrogens and deuteriums scramble completely, either the b1 or b2 ion should carry half of the deuteriums of the parent ion. After deuteration and subsequent aqueous washing in quenching condition, the deuteration level in the b1 or b2 ion of neurotensin (9-13) should give the extent of scrambling in the tested condition and instrument.

Table 1. Deuterium Incorporation on Fragmented Ions of Neurotensin (9-13) with Activation Energies of 27% and 30%

a

calcd D without scrambling calcd D with scramblinga calcd D without scramblingb calcd D with scramblingb obsd D at 27% obsd D at 30%

b1

b2

b3

b4

b5

parent

0.00 1.50 0.00 1.42 not obsd not obsd

0.00 1.50 0.00 1.42 1.42 ± 0.03 1.38 ± 0.15

1.00 2.0 0 0.95 1.89 1.95 ± 0.06 1.82 ± 0.07

2.00 2.25 1.89 2.13 2.04 ± 0.11 2.07 ± 0.03

3.00 2.50 2.84 2.37 2.27 ± 0.03 2.31 ± 0.01

3.00 3.00 2.84 2.84 2.84 ± 0.04 2.84 ± 0.04

a The numbers in the top two calculated D rows are when neurotensin is deuterated in 100% D2O without back exchange. There should be three deuteriums on the parent ion after the deuteration and aqueous washing. There are 12 exchangeable hydrogen sites on the parent ion. As the b1 ion carries six exchangeable hydrogen sites, the number of deuteriums on b1 with scrambling is 1.50 ) 3 × 6/12. b As the fully deuterated neurotensin (9-13) was prepared in 98% D2O and there was about 4% of back exchange during the analysis, the numbers in the top two rows cannot be compared with the observed numbers. The numbers in the third and forth rows were obtained by multiplying the numbers in the top two rows by 0.947 to directly compared with the observed numbers.

Figure 2. Isotope envelopes and centroid values of the parent ion of neurotensin (9-13) with various isolation widths. (a) The dotted line is the isotope envelope of neurotensin (9-13) in MS. The black line is the isotope envelope of neurotensin (9-13) in MS/MS with an isotope envelope of 8 (parent mass range of 661-669). The gray line is the isotope envelope of neurotensin (9-13) in MS/MS with an isotope envelope of 7 (parent mass range of 661.5-668.5). (b) Centroid values of the parent ion of neurotensin (9-13) with various isolation widths.

MATERIALS AND METHODS Materials. Neurotensin (9-13) was purchased from Bachem (King of Prussia, PA). Deuterium oxide and all other reagents were obtained from Sigma (St. Louis, MO). Infusion Experiments for Optimization of MS/MS Isolation Width and Activation Energy. An amount of 0.1 mg/mL neurotensin (9-13) prepared in 19% acetonitrile, 0.04% trifluoroacetic acid (TFA) was infused into the mass spectrometer at a flow rate of 20 µL/min. MS/MS was acquired in profile mode with a parent mass of 665 (calculated monoisotopic mass of +1 neurotensin is 661.4). For the optimization of MS/MS isolation width, the activation energy was set to 0% to prevent fragmentation and the MS/MS isolation width of the parent ion was varied between 4 and 11. For the optimization of activation energy, the isolation width was set to 10 (parent mass range, 660-670) and the activation energy was varied between 20% and 35%. Deuterium Incorporation to the b Ions of Neurotensin (913). To a 20 µL solution of 0.1 mg/mL nondeuterated (prepared in 100% water) or deuterated (prepared in 98% deuterium oxide) neurotensin (9-13) was added 30 µL of a chilled 0.8 M guanidine hydrochloride (GuHCl), 0.8% formic acid solution to practically stop the amide hydrogen exchange reactions. The mixture was loaded onto a reversed-phase column chilled at 0 °C and washed with 400 µL of a chilled 0.05% aqueous TFA solution at 200 µL/ min to remove all deuteriums from N- and C-terminal as well as the side chains. The peptide was then eluted by chilled 95% acetonitrile, 5% water, 0.0025% TFA at 10 µL/min. The elution was analyzed by an LCQ (Thermo Fisher Scientific, San Jose, CA) with a capillary temperature of 175 °C. Typical MS/MS parameters are the following: parent mass, 665; isolation width, 10 (these make

the mass range of 660-670); activation energy, 27% or 30%; activation time 30 ms. RESULTS AND DISCUSSION Optimization of Isolation Width. Before performing MS/MS, the parent ion has to be isolated in a mass spectrometer. Although a narrower isolation width is preferred to avoid the introduction of impurities, the complete isotope envelope of the parent ion has to be covered to obtain unbiased deuteration levels of daughter ions. In order to monitor the quality of parent ion isolation, the isotope envelope of nondeuterated neurotensin (9-13) was monitored in MS/MS. The parent mass was set at 665 which is 4 m/z higher than the monoisotopic peak (661.4) of neurotensin (9-13). Gradually increasing isolation width (and thus mass range) should gradually decrease the centroid value (average mass) of peaks in the MS/MS spectrum. The centroid value is decreased because a lighter part of the parent ion isotope envelope is included. The activation energy was set to 0% to avoid any fragmentation for this set of experiments. In this way, the centroid value of the parent peak in MS/MS can be followed to monitor the isolation of parent ion. When the parent mass range is 661-669 (parent mass ) 665 and isolation width ) 8), the shape of the isotope envelope in MS/MS and that in MS are identical, implying that all ions in this mass range were isolated uniformly (Figure 2a). On the other hand, when the parent mass range is 661.5-668.5 (parent mass ) 665 and isolation width ) 7), the monoisotopic peak lowered, implying some lightweight ions were not isolated. The centroid value of this isotope envelope was constant when the isolation Analytical Chemistry, Vol. 80, No. 17, September 1, 2008

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Figure 3. MS/MS spectrum of neurotensin (9-13) with an activation energy of 30%.

width was 8 or higher (Figure 2b), indicating that the entire isotopic envelope was covered when the isolation width was 8. Just to be on the safe side, the combination of parent mass 665 and isolation width 10 (parent mass range of 660-670) was used for the rest of the MS/MS experiments with both nondeuterated and deuterated neurotensin (9-13). Fragmentation Pattern of Neurotensin (9-13). Neurotensin (9-13) was fragmented with an activation energy of 30% and parent mass range of 660-670 (Figure 3). The tallest peak was the b5 ion (monoisotopic m/z ) 643), and the second highest peak was the parent ion (monoisotopic m/z ) 661) in the MS/MS spectrum. Other b series ions, b2 (monoisotopic m/z ) 254), b3 (monoisotopic m/z ) 417), and b4 (monoisotopic m/z ) 530) ions were observed in good quality, although only one y ion (y4, monoisotopic m/z ) 505) was observed in reasonable quality. The y4 ion was not usable for the scrambling test, as it is very close to another peak at 502 and the peak overlapping with that peak upon deuteration prevents accurate determination of the centroid value. Taken together, the deuteration levels of b2, b3, b4, b5, and parent ions of neurotensin (9-13) can be monitored in MS/MS to detect scrambling, whereas none of the y ions can be monitored. In theory, y ions should contain at least some scrambling as the formation of y ions requires proton transfer. In practice, both Smith’s group15 and Deinzer’s group16 reported more reliable nonscrambling data with b ions than y ions. Therefore, the detection of scrambling in b ions is of greater interest than that in y ions. Optimization of Activation Energy: Effects of Activation Energy on Isotope Envelopes. Although low activation energy does not give enough fragmentation to monitor all b ions, unnecessarily high activation energy may have detrimental effects, 6788

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such as undesired fragmentations. The fragmentation pattern of nondeuterated neurotensin (9-13) was monitored with the activation energy between 20% and 35%. First, the effects of activation energy on overall fragmentation were analyzed. Although the parent ion was the tallest peak in the MS/MS spectrum with the activation energy of 28% or less, the b5 ion became the most dominant peak with the activation energy of 29% or higher. The number of peaks observed with the activation energies of 27% and 35% are very similar. The population of not-well-populated peaks, such as b2 ion, relative to the dominant peak, b5, increases as the activation energy became higher. The minimum activation energy necessary to observe strong enough b ion series was 27%. The effects of activation energy on the isotope envelope in the MS/MS spectra of nondeuterated neurotensin (9-13) were studied. Interestingly, as activation energy becomes higher, the M + 1 peak (the peak with one 13C) in the parent ion in MS/MS decreases and the M + 1 peak in the b5 ion increases (Figure 4, parts a and b). As a result, when the activation energy gets higher the centroid value of the b5 ion becomes larger and that of the parent becomes lower (Figure 4, parts c and d). The centroid values of parent and b5 ions were stable with the activation energy of 27% or lower (Figure 4, parts c and d). This implies that there may be some isotopic effects during the fragmentation and that high activation energy may skew the isotope envelope of daughter ions. Deuterium Incorporation to the b Ions of Neurotensin (913). Both nondeuterated and deuterated neurotensin (9-13) were fragmented by CID with parent mass range of 660-670, activation energy of 27%, and activation time of 30 ms. The isotope envelope of each b ion in nondeuterated or deuterated neurotensin (9-13)

Figure 4. Isotope envelopes and centroid values of the parent ion and b5 ion of neurotensin (9-13) in MS/MS with various activation energies. (a) Isotope envelopes of the b5 ion. (b) Isotope envelopes of the parent ion. (c) Centroid values of the b5 ion. (d) Centroid values of the parent ion.

Figure 5. Isotope envelopes of the parent ion and b ions of neurotensin (9-13) with an activation energy of 27%. The dotted line is the nondeuterated sample, and the solid line is the deuterated sample.

was monitored (Figure 5), and the centroid value of each ion in each sample preparation was determined. The deuterium incorporation to each b ion is calculated by the subtraction of the centroid value of the nondeuterated isotope envelope from that of the deuterated isotope envelope. The difference between the b2 ions of deuterated and nondeuterated neurotensin (9-13) was 1.42 Da, which is exactly what is expected with complete scrambling (Table 1 and black diamonds in Figure 6). As described in the Experimental Concept section, the b2 ion should not carry any deuterium without scrambling. Also, since the deuterium incorporation in the parent ion and b5 should be identical in the absence of scrambling, the deuteration levels as well as the shape of the isotope envelopes of these two ions should be very similar. The lower deuterium incorporation in the b5 ion (Table 1) and difference in the deuterated isotope envelopes of the two ions (Figure 5) also imply the presence of scrambling.

All other b ions also incorporated the number of deuteriums expected for complete scrambling (Table 1). The x-axis of Figure 6 is the calculated deuterium incorporation to each b ion in 100% deuterium oxide with complete scrambling (unlike most articles which use the number of residues), and the y-axis is the observed deuterium incorporation in each b ion. With complete scrambling of hydrogens and deuteriums among all the exchangeable positions, a straight line can be expected in Figure 6. On the other hand, without scrambling, the jagged line is expected. Deuterium incorporation to the b ions with higher activation energy conditions was also tested, since Kaltashov’s group found higher activation energy minimized scrambling in his system.22 One condition is activation energy of 30% and activation time 30 ms (white diamonds in Figure 6), and the other is activation energy of 60% and activation time 2 ms (data not shown). Both conditions led to complete scrambling in the current test. Analytical Chemistry, Vol. 80, No. 17, September 1, 2008

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scrambling. (ii) The proline at the second residue can eliminate the ambiguity about the deuteration level. The exchange rate of amide hydrogen in the second residue is about 25 times faster than the rest of the amide hydrogens according to HXPEP (http:// hx2.med.upenn.edu/download.html).30 The deuteration level at the second residue can be not as high as the rest of main chain amides but may not be negligible like the terminals and the side chains. (iii) The CID of neurotensin (9-13) can generate a series of b ions except b1 ion.

Figure 6. Deuterium incorporation to the b ions of neurotensin (913). The x-axis is the calculated deuterium incorporation to each b ion in 100% deuterium oxide with complete scrambling. When the complete scrambling occurs, the plot becomes linear. On the other hand, in the absence of scrambling, the plot should appear as the jagged line with ×. The black diamonds are the observed deuterium incorporation with an activation energy of 27%, and the white diamonds are that with an activation energy of 30%.

Selection of the Peptide for the Scrambling Test. The proposed test utilizes two facts: (i) Washing with a chilled acidic aqueous buffer removes all deuteriums from a deuterated peptide except those on main chain amides. (ii) Complete scrambling distributes the deuteriums equally not only among main chain amides but also all exchangeable positions, such as N- and C-terminals as well as side chains. Therefore, a peptide other than neurotensin (9-13) can be used for this purpose, if it fragments well in the gas phase generating a series of b ions, especially small ones. Neurotensin (9-13) was chosen for this study for the following reasons: (i) The difference between the presence and absence of scrambling should be large in small b ions, since the N-terminal arginine of neurotensin (9-13) can carry extra deuteriums upon

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CONCLUSION A simple test to detect scrambling during gas-phase peptide fragmentation using a commercially available pentapeptide, neurotensin (9-13), is proposed. The deuteration level of the b2 ion of the pentapeptide should give clear indication of the extent of gas-phase scrambling. In the absence of scrambling, the b2 ion from the fully deuterated pentapeptide should carry no deuteriums. On the other hand, the b2 ion from the fully deuterated pentapeptide can carry up to 1.5 deuteriums in the presence of scrambling. This test utilizes the two facts: (i) scrambling equally distributes deuteriums to all exchangeable hydrogen sites and (ii) the arginine side chain has many exchangeable hydrogens (Figure 1). Plotting the deuteration level of each b ion against the number of exchangeable hydrogen sites of the b ion also helps in recognizing the presence of scrambling (Figure 6). With two different sets of collision energy and activation time, the test showed complete scrambling during CID in our mass spectrometer, an LCQ. It is interesting that both Smith’s15 and Deinzer’s16 groups used CID in similar instruments to successfully sublocalize deuteriums. The test is applicable to other types of mass spectrometers and fragmentation techniques and should also help in optimizing fragmentation parameters. Received for review March 31, 2008. Accepted June 30, 2008. AC800645F