Anal. Chem. 1993, 65, 1703-1708
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Simplification of High-Energy Collision Spectra of Peptides by Amino-Terminal Derivatization John T. Stults,' Johnny Lai, Shannon McCune, and Ronald Wetzelt Protein Chemistry Department, Genentech, Inc., 460 Point Sun Bruno Boulevard, South San Francisco, California 94080-4990
Four-sector tandem mass spectrometry proves extremely useful for providing sequence information for peptides. The complexity of ion fragmentations, however, makes data interpretation difficult and time consuming. Attachment of a fixed positive chargeto the peptideaminoterminus forces production of only N-terminal fragment ions to yield simplified,predictablefragmentation. Reaction of a peptide at pH 6 with iodoacetic anhydride selectively modifies the N-terminus by exploitingthe pK,differences between the a-amino group and any lysine side-chain e-amino groups. The iodoacetyl peptide can react with many reagents to form a fixed positive charge. We find reaction with dimethyloctylamine forms a quaternary ammonium derivative with good surface activity properties and concomitant increased sensitivity. The high-energy CAD fragment ion spectra of the N-terminally derivatized peptides show predominantly a,,and dnions. The abundant d, ions permit ready distinction of leucine and isoleucine. Fewer fragment ions make data interpretation simpler and lead to more intense peaks since the ion intensity is spread among fewer peaks. The method is particularly useful for peptides which do not otherwise yield sufficient fragmentation to provide either the complete sequence or the locations of modified amino acids.
INTRODUCTION
Mass spectrometry is an integral tool in the protein sequencing laboratory. In particular, tandem mass spectrometry (MS/MS) proves extremely useful for sequencing of peptides. Four-sector tandem double-focusing instruments, utilizing high-energy (typically >1 keV in the laboratory frame of reference) collisional-activated dissociation (CAD),routinely provide sequence for peptides of up to about 2000 Da.14 Triple-quadrupole instruments, utilizing lowenergy (C300 eV) CAD, also provide sequence data for peptides of similar size.s The tandem double-focusing instruments allow selection of the 12C peak of the peptide (M + H)+ ion isotope cluster * To whom correspondence should be addressed. + Present address: Department of Macromolecular Chemistry, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of P m s i a , PA 19406-0939. (1) Biemann, K.; Scoble, H. A. Science 1987, 237, 992-998. (2) Biemann, K.; Martin, S. A. Mass Spectrom. Reu. 1987, 6, 1-76. (3) Anderegg, R. J.; Carr, S.A.; Huang, I. Y.; Hiipakka, R. A.; Chang, C.; Liao, S.Biochemistry 1988,27,4214-4221. (4) Gibson, B. W.; Yu, Z.; Aberth, W.; Burlingame, A. L.; Boss, N. M. J.Biol. Chem. 1988,263,4182-4185. (5) Hunt, D. F.; Yates, J. R., III; Shabanowitz, J.; Winston, S.;Hauer, C. R. Roc. Natl. Acad. Sci. U.S.A. 1986,83,6233-6237. 0003-2700/93/0365-1703$04.00/0
produced by fast atom bombardment (FAB). Fragmentation of this precursor produces CAD spectra with isotopic purity which permit more straightforward interpretation of the spectra. Operation of MS-2at unit resolution permits closely spaced peaks to be assigned to the correct mass accurately, a requirement for differentiation of the acidic amino acids (Glu,Asp) from their correspondingamides (Gln,Am). Highenergy collisions are essential because the fragmentations involving side-chain cleavage (d, and w, ions) that permit distinction of Leu and Ilew occur only at higher energies (>300eV in center of mass framel.9 Collisions in this energy regime may be ahieved with triple-quadrupole and hybrid instruments,lO but are not routinely measured. High-energy CAD spectra often display fairly complex fragmentation patterns, involving ions from a number of different ion fragment series. The fragmentation is influenced by the position of basic amino acid residues within the peptide.8 For example, a basic residue at the amino terminus preferentially yields a,, b,, and d, ions. Similarly, a basic amino acid at the carboxy terminus preferentially yields v,, w,, y,, and z, ions. A basic residue in the center of a peptide may yield a more complex pattern of both N-and C-terminal ions. The variety of ions permits internal verification of the sequence and often aids in precise determination of sites of modification. When one is deducing the sequence of a peptide with unknown structure, however, the complex fragmentations make interpretation difficult. In particular, w, ions are often the most intense peaks in spectra of tryptic peptides. A number of computer algorithms are available for aiding interpretation of high-energy CAD spectra."-18 Only a few are useful for peptides of >loo0 Da. Our experience indicates that while the correct sequence (or partial sequence) is often found in the list of best fits from automated interpretation algorithms, frequently the data require further manual interpretation to determine the correct sequence. This latter (6) Stulta, J. T.; Watson, J. T. Biomed. Enuiron. Mass Spectrom. 1987, 14,583-586.
(7) Johnson, R. S.;Martin, S.A.; Biemann, K.; Stulta, J. T.;Watson, J. T. A d . Chem. 1987,59, 2621-2625. (8) Johnson, R. S.;Martin, 5.A.; Biemann, K. Znt. J.Mass Spectrom. Zon Processes 1988,86,137-154. (9) Martin, S. A.; Johnson, R. S.;Costello, C. E.; Biemann, K. In The Analysis of Peptides and Roteins by Mass Spectrometry; McNeal, C. J., Ed.;Wiley: New York, 1988, pp 135-150. (10) Alexander, A. J.; Thibault, P.; Boyd, R. K. Rapid Common. Mass Spectrom. 1989, 3, 30-34. (11) Scoble, H. A.; Biller, J. E.;Biemann, K. Freseniw' 2.And. Chem. 1987,327, 239-245. (12) Johnson,R. 5.;Biemann, K. Biomed. Enuiron. Mass Spectrom. 1989,18, 945-957. (13) Siegel, M. M.; Bauman, N. Biomed. Enuiron. Mass Spectrom. 1988,15, 333-343. (14) Bartels, C. Biomed Enuiron. Mass Spectrom. 1990,19,363-368. (15) Ishikawa, K.; Niwa, Y. Biomed. Enuiron. Mass. Spectrom. 1986, 13,373-380. (16) Sakurai, T.; Matauo, T.;Matsude, H.; Katakuae, I. Biomed. Mass Spectrom. 1984,11, 396-399. (17) Hamm, C. W.; Wilson, W. E.; Harvan, D. J. Comput. Appl. Biosci. 1986,2, 115-118. (18) Hines, W. M.; Falick, A. M.; Burlingame, A. L.; Gibson, B. W. J. Am. SOC.Mass Spectrom. 1992,3, 326-336.
0 I993 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1, 1993
step requires considerable expertise and can be time consuming. The utility of MSIMS for rapid, routine sequencing of large numbers of peptides is limited as a result. An important use of the four-sector tandem instrument that sets it apart from the triple-quadrupole mass spectrometer is the identification or verification of the structure of modified peptides. The putative sequence of a modified peptide may be known, as in the case of a synthetic peptide or a peptide from recombinant DNA-derived proteins. Amino-terminal sequencing by Edman degradation is not useful in many instances because the N-terminus may be blocked, the modification may be labile under the alternating acidlbase conditions of the Edman chemistry, or the retention time of the modified PTH-amino acid may not provide a clear answer. The precise location of the modification by tandem mass spectrometry requires fragmentation a t and around the modified residue. Furthermore, fragmentation of the amino acid side chain may provide additional data for the identification or verification of the structure of the modification. The four-sector tandem mass spectrometer generally provides more extensive peptide fragmentation, through high-energy collisions, than the triple-quadrupole instrument. Even then, not all peptides provide sufficient fragmentation to locate and identify a modification. In these cases, the ability to enhance or otherwise alter the fragmentation is important. One approach to the problems described above is to derivatize the peptide in order to direct fragmentation and/ or simplify the CAD spectra. A number of derivatization procedures have been developed to alter the FAB ionization1 desorption characteristics for peptides.'%% Similarly, a number of procedures have also been described which demonstrate the feasibility of derivatizing peptides to alter collision spectra.2mO One may, in principle, choose to modify either the N- or C-terminal of the peptide. Derivatives which fix the charge a t specific sites (e.g., a quaternary ammonium moiety) produce the most predictable fragmentations.* Amino-terminal derivatives tend to produce mostly a, and d, ions whereas carboxy-terminal derivatives produce v,, y,, z,, and wn ions. Thus N-terminal modification produces simpler fragmentation. We previously described an amino-terminal modification reaction that is specific for the a-amino group of the amino terminal, leaving any side-chain amino groups unmodified.31
This specificity is important for producing unambiguous spectra of only a single isomeric species. In the present work, the iodoacetyl peptide produced with this reaction is further reacted to produce a quaternary ammonium-derivatized molecule. The resulting peptides yield CAD fragment ion spectra that are straightforward to interpret and provide fragmentation at most or all peptides bonds.
13,405-410. (24) Renner, D.; Spiteller, G. Angew. Chem., Znt. Ed. Engl. 1985,24, 408-409. (25) Busch, K. L.; Unger, S. E.; Vincze, A.; Cooks, R. G.; Keough, T. J. Am. Chem. SOC. 1982,104,1507-1511. (26) Johnson, R. S. Ph.D. Dissertation, Massachusetts Institute of Technology, Cambridge, MA, 1988. (27) Vath, J. E.; Zollinger,M.; Biemann, K. Freseniw' 2.Anal. Chem. 1988,331, 248-252. (28) Krishnamurthy, T.; Szafraniec, L.; Hunt, D. F.; Shabanowitz, J.; Yates, J. R., III; Hauer, C. R.; Carmichael, W. W.; Skulberg, 0.; Codd, G.A.; Missler, S. h o c . Natl. Acad. Sci. U S A . 1989, 86, 770-174. (29) Vath, J. E.; Biemann, K. Znt. J. Mass Spectrom. Ion Processes 1990,100,287-299.
EXPERIMENTAL SECTION Materials. Trimethylamine (TMA) (12.5% in water) and trifluoroacetic acid (TFA) were from Applied Biosystems, Inc. Iodoacetic anhydride, dimethylhexylamine (DMHA), and tetrahydrofuran (THF) were from Aldrich. Dimethyloctylamine (DMOA)was obtained from Ethyl Corp. Formic acid (88%) and m-nitrobenzyl alcohol were obtained from Fluka. 2-(N-morpholino)ethanesulfonicacid (MES)was from Sigma. Water (>18 Ma) was from a Millipore Milli-Q system. Acetonitrile was from Burdick and Jackson. Synthetic peptides were prepared at Genente~h.3~ The tryptic peptides were obtained after digestion of desacetyl-a-thymosinor recombinantmethionyl human growth hormone (hGH) with trypsin (Boeringer-Mannheim) and subsequently purified by microbore reversed-phase HPLC. Preparation of Peptide derivative^.^^ A 1-pL aliquot of each peptide solution (0.1-2 nmol) was placed into 12 p L of 0.30 M MES buffer solution,pH 6, in a 0.5-mL Eppendorf tube. After the mixture was cooled to 0 O C in an ice bath, a 5-pL aliquot of 0.01 M iodoacetic anhydride in dry THF was injected into the mixture. The solution was immediately vortexed for 1min. The tube was returned to the ice bath for another 5 min and then allowed to equilibrate to ambient temperature (2-3 min). To this solution of iodoacetyl peptide was immediately added 5 pL of either (a) 0.5 M dimethylhexylamine in dry THF, (b) 0.5 M octyldimethylamine in dry THF, or (c) 12.5% (w/v) aqueous trimethylamine. The reaction tube was vortexed and heated to 37 "C for 2 h. During this period, the tube waa vortexed every 30 min to ensure adequate mixing. The reaction was allowed to cool to room temperature and was centrifuged to combine any condensedvapor with the solution. The solution was immediately purified or frozen until purified. Purification of Peptide Derivatives. The mixture was purified by HPLC withan AppliedBiosystems (ABI)140Asolvent delivery system. Reversed-phase HPLC separations employed a 2.1 x 100 mm, AB1 Aquapore RP-300, C-8 column. Solvent A was Milli-Q-purified water and solvent B was acetonitrile,with 0.05% trifluoroacetic acid (ABI)included in both solvents. The purified peptides were dried and then reconstituted in 0.05 % trifluoroacetic acid., The concentrations were quantitated by amino acid analysis. Amino Acid Analysis. Aliquota were dried and then vaporphase hydrolyzed with 6 N HCl at 110 O C for 24 h. The hydrolysate was analyzed with a Beckman 6300 amino acid analyzer. Mass Spectrometry. Spectra were obtained with a JEOL JMS-HXllOHF/HXllOHF tandem mass spectrometer. Ions were produced by fast atom bombardment using either 6-keV Xe atoms or 15-keV Cs+ions. FAB spectra were obtained with MS-1, operating at 10-kV accelerating voltage, resolution of 1:3000. Matrices used were m-nitrobenzyl alcohol or monothioglycerol. A cooled-probe (Hill Instruments) was used with monothioglycerol to prolong the matrix lifetime during analysis. Data were collected with a JEOL DA-5000 data system and analyzed with a Kratos MACH3 workstation. CAD spectra were obtained by first setting MS-1 to pass the 12Cpeak of the MH+ion of the peptide of interest. The resolution of MS-1 was set to allow static resolution of the isotope peaks, usually half the resolving power required for The collision gas was helium, introduced at sufficient pressure to attenuate the precursor signal to 30% of ita initial intensity. Fragment ions were analyzed in MS-2, at resolution 500, with a JEOL array detector. The electroopticalmultichannelplate array is positioned in place of the conventional point detector on MS-
Chem. 1990,1, 114-122.
(32) Martin, S. A,; Scoble, H. A.; Coatello,E. E.; Biemann, K. Presented at the 35th Annual Conference on Maea Spectrometryand Allied Topics, Denver, CO, May 24-29,1987; pp 78-79.
(19) Kidwell, D. A.; Ross, M. M.; Colton, R. J. J.Am. Chem. SOC. 1984, 106,2219-2220. (20) Keough, T.; DeStefano, A. J. Presented at the 31st Annual
Conference on Mass Spectrometry and Allied Topics, Boston, MA, May 8. 1983. (21) Kidwell, D. A.; Ross, M. M.; Colton, R. J. Secondary Zon Mass Spectrometry-SZMS V; Benninghoven, A., Colton, R. J., Simons, D. S., Werner, H. W., Eds.; Springer-Verlag: New York, 1986; pp 509-511. (22) Busch,K.L.;Kroha,K. J.;Flurer,R.A.;DiDonato,G.C.Secondary Ion Maes Spectrometry-SIMS V; Benninghoven, A,, Colton, R. J., Simons, D. S., Werner, H. W., Eds.; Springer-Verlag: New York, 1986; pp 512-514. (23)Renner, D.; Spiteller, G. Biomed. Enuiron. Mass Spectrom. 1986,
(30)Wagner, D. S.; Salari, A.; Gage, D. A,; Leykam, J.; Fetter, J.; Hollingsworth,R.; Watson, J. T. Biol. Mass Spectrom. 1991,20,419-425. (31) Wetzel, R.; Halualani, R.; Stulta, J. T.; Quan, C. Bioconjugate
ANALYTICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1, 1993
Scheme I (I-CH,-CO),O
t
i
NH2-CHR-CO----
{p 5-
I-cH,-co-NH-CHR-CO----
+
N(CH,),R'
E +
(cH,),R'N-cH,-co-NH-CHR-CO----
2. It consists of a 3-in. chevron-configurationmicrochannelplate
(MCP),a phosphor, a 3:2 reducing fiber-opticbundle, and a 2-in. photodiode array. The data are acquired by a Princeton Instruments module and processed on a personal computer using Spectra-Calc software from Galactic Industries. The 3-in. MCP permits simultaneousacquisition of 9.5% of the mass range (e.g., 1OOO-1095~).TheMCPvoltagewas 1.2-1.5kVandthephosphor voltage was 4.0 kV. The collision cell was operated at 6 kV (4keV collisions) to permit efficient transfer of the fragment ions through the MS-2 electric sector.
RESULTS AND DISCUSSION Simplification of CAD Fragment Ion Spectra. The derivatization reactions were chosen in order to modify the amino terminus selectively. Selective derivatization of an N-terminal a-amino group in the presence of lysine e-amino groups is possible due to their different pK,'s (a-amino pK, -8, Lys t-amino pK, -10.5). The reaction described here utilizes amide formation with iodoacetic anhyride a t pH 6, which acylates the a-amino group of peptides with 70-90 5% selectivity over an t-amino group.31 The iodoacetyl peptide can then react in a second step with a variety of compounds to form a fixed-charge species (see Scheme I). If cysteine or cystine is present in the peptide, it must be reduced and alkylated prior to these reactions to prevent reaction with the iodoacetic anhydride. The CAD fragment ion spectrum for a tryptic peptide from a-thymosin (sequence SDAAVDTSSEITTK) is shown in Figure la. The complexity of the spectrum for the underivatized peptide is revealed by the presence of a number of different ion series (a,, b,, v,, w,, x,, y,, z,) from both the amino and carboxyl ends of the molecule. The ions are labeled according to Biemann's adaptation33 of the Roepstorff and Folhman ion designations." It would be difficult to determine the amino acid sequence from this spectrum if the sequence were not previously known. Attachment of a fixed charge to the amino terminus significantly simplifies the spectrum and produces a spectrum that contains only N-terminal ions (Figure Ib). In this case, the DMHA-Ac derivative adds a quaternary ammonium functionality to the acetyl group. The a, and d, ions are observed predominantly. The c, series ions also appear, but with lower abundance. The entire sequence may be derived from a, ions. The d, ions are important because they permit distinction of leucine from isoleucine residues based on side-chain cleavages.8 For example, the dll, and dllb ions allow conclusive identification of the eleventh residue aa Ile. The most likely a,, c,, and d, ion structures are shown in Figure 2. Since the positive charge is fixed a t the amino terminus the structural possibilities are limited. Each structure contains one proton less than the corresponding fragment ion from a protonated precursor ion. The fragment (33) Biemann, K. Biomed. Enuiron. Mass Spectrom. 1988,16,99-111. (34) Roepstorff, P.; Fohlmann, J. Biomed. Mass Spectrom. 1984,11, 601.
1705
ions must be formed by remote site fragmentation.36 The mechanism of remote site fragmentation is not yet clearly established, although cyclic transition states, in which the charged site interacts with the fragmentation site, have been proposed.% The peak labeled be in Figure l b is the only b ion observed in this spectrum. A structure for this ion could be drawn, similar to those in Figure 2 and most likely a ketene structure. However, upon closer inspection the ba ion contains an aspartic acid at its C-terminus. The mass (mlz 728.7 in Figure lb) matches a succinimide structure that could form from the Asp side chain. Such a structure is supported by the fact that the only other fragments observed for these derivatives that correspond to b, ions (see below) also occur at aspartic acid residues. The derivatives described here may also be prepared by the gas-phase method developed by Vath and Biemann.29 The solution chemistry presented here has the advantage that the pH is carefully controlled to prevent possible modification of side-chain amine groups, although such byproducts have not been noted for the gas-phase method. The peptide is also not heated in solution, as is required in the solid-state reaction, so special apparatus and careful exclusion of oxygen are not required. A purification step is required after the solution chemistry described here, but the added purity following HPLC separation will ensure optimal precursor signal, which is quite susceptible to attenuation by small quantities of impurities, particularly at low analyte levels. A solution-based derivatization scheme to form an N-terminal ethyltriphenylphosphonium ion has also been described,30337but selective derivatization of the a-aminogroup has not been demonstrated. Furthermore, the derivatizing reagent is not commercially available. These N-terminal derivatives are particularly useful for peptides that do not otherwise yield sufficient fragmentations to provide the complete sequence or locations of modified amino acids. The derivatization procedure described here has also been used successfullyby othersB*39for this purpose. Relative SignalStrengths from Derivatives. Additions of a fixed charge might be expected to increase the signal strength for a peptide due to the formation of a precharged species.19 However, the addition of a fixed charged increases the hydrophilic character of a peptide. Ligon and DornN noted a reduction in signal strength in FAB spectra upon addition of a fixed charge to a molecule, attributed to reduced surface activity of the molecule. A hydrophobic group is needed to overcome this increase in polarity in order to increase the surface activity for improved ionizationefficiency by fast atom bombardment. Figure 3 shows the relative signal strengths for the M+ ions of three dimethylalkylammonium derivatives of the synthetic hexapeptide FGAKQA. The trimethylammonium derivative gives a lower signal, compared to the underivatized peptide. The dimethylhexylammonium and dimethyloctylammonium derivatives,on the other hand, show substantiallybetter signal strengths. For this reason, we use the DMOA-Ac derivative (35) Jansen, N. J.; Tomer, K. B.; Gross, M. L. J. Am. Chem. SOC.1986, 107, 1863-1868. (36) Gross, M. L. Znt. J. Mass Spectrom. Zon Processes 1992,118/119, 137-165. (37) Watson,J.T.;Wagner,D.S.;Chang,Y.-S.;Strahler,J.R.;Hanash, S. M.; Gage, D. A. Znt. J. Mass Spectrom. IonProcesses 1991,111,191209. (38) Herrmann, J.; Leffler, H.; Barondes, S. H.; Burlingame, A. L.
Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics; Tucson,AZ, June 3-8,1990, pp 285-286. (39) Bean, M. F.; Carr, S.A.Proceedings of the 4th ASMS Conference on Mass Spectrometry and Allied Topics; Washington, DC, M a y 31June 5,1992; pp 1825-1826. (40) Ligon, W. V., Jr.; Dorn, S. B. J . Am. Chem. Soc. 1988,110,66846688.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1, 1993 x2
L
4
Thymosin-TI
I
M+H+ = 1424 1
W48
w6
I
I I
z12+1 b12
w5 w4b
b5 y4
w13
v5
600
400
800
1200
1000
1400
a5
30
Flgwe 1. CAD fragment Ion spectrum (a, top) of 200 pmol of a WpUc peptide from a-thymosin, sequence SDAAVDTSSEITTK and CAD fragment Ion spectrum (b, bottom) of the derivatlzed (DMHA-Ac) peptide, approximately 40 pmoi. The ,a, Ion series in (b) provides the complete sequence. The d1la and drlb ions allow identlficatlon of the eleventh residue as Ile, not Leu. The peak labeled .d, is a doublet of .d, and dm, separated by 2 u, which represent losses of OH and CH,I respectively, from the Thr side chain. 5-
e
4-
C
: 3J
n 4
2-
dn: (CH3)3N+- - - - NH - CH CHRna Figure 2. Most likely structures of the a, c,, and d, ions. The charge location is flxed by the derivation procedure.
for most peptides. Poulter et alS4lnoted similar increases in signal strength for hexyl and octyl side chains of aminobenzyl alkyl ester derivatives of reduced carbohydrates. Similarly, Falick and Maltby,showedincreases in signal strength of small peptides upon formation of hexyl esters.42 Even longer chain alkylaminesmay yield greater signals but will be increasingly difficult to form due to their limited solubility in aqueous solution. (41) Poulter,L.; Karrer, R.; Burlingame,A. L.Anal. Biochem. 1991, 195,l-13. (42)Falick, A. M.; Maltby, D.A.Anal. Biochem. 1989,182,165-169.
0 '
I
Underiv
I
I
I
TMA-Ac
DMHA-Ac
DMOA-Ac
Derivative Flgure3. Relative signal intensltlesfor the MHf ionof the hexapeptide FGAKQA and the M+ Ion of Itsthree dlmthylalkylammoniumderivatives. The greater hydrophobic character of the C6 and C8 alkyl Chains of the DMHA and DMOA derivatives, respectively, slgnlflcantly enhances the ion abundance by fast atom bombardment.
Similar enhancements in precursor ion signal were not observed with larger peptides (the a-thymosin and hGH tryptic peptides analyzed here). One would expect a diminished influence from a hydrophobic moiety for larger or more hydrophobic peptides. Such results are in agreement with data presented by Vath and Biemann,28who showed that the
ANALYTICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1, 1993 1707
w7
hGH-T4 M+& = 2342.1
218+1
w10
I I. ws v8
a7
1
I
z8+1
I
2
DMOA-AC-~GH-T~ d3
M+ = 2539.3
C l
C:
500
m/z
m/z
Flguro 4. CAD fragment ion spectrum (a, top) of 100 pmoi of the T4 tryptic peptide of hQH, sequence LHQLAFDTYQEFEEAYIPK. The spectrum displays a variety of different fragment ion serles, none of which cover a substantial portlon of the spectrum. The DMOA-Ac derivative (40 pmoi) (b, bottom) yields a much improved spectrum wlth a greater slgnal-to-noise ratio, fewer peaks, but more complete lon series. The unlabeled high-mass ions correspond to amino acid sldechein losses. The peak labeled dh is a doublet for the Thr sldechain cleavage, as observed In Figure lb. The peak labeled d, is accompanied by a peak 2 u higher than corresponds to a,-C02.
length of the alkyl chain had little effect on the signal strength. The peptide noted in their work (des-Argl-bradykinin)was larger than the peptide measured in Figure 3. The minimum quantity of peptide required for derivatization is presently approximately 100 pmol. This amount permits easy identification of the derivatized component in the chromatogram and does not necessarily reflect the mass spectrometer detection limit. The overall yield for these reactions is typically 60-80% ,based on amino acid analysis of the purified derivative. The major limitations appear to be the purity of the reagents and losses from the purification step. Background in the chromatogram is minimized by using minimal quantities of freshly prepared reagents. The iodoacetic anhydride and amines were reagent grade; further purification of the reagents would likely simplify the chromatograms. Iodoacetic acid formed by hydrolysis of excess anhydride reacts with the amine to form a quaternary species, which in the case of the hexyl and octyl forms elutes with a retention time similar to some derivatizedpeptides. The longchain dimethylalkylamines also elute in a similar region of the chromatogram. A potential limitation of yield is cyclization of the peptide due to reaction of the intermediate iodoacetylpeptide with downstream amino acids that contain
nucleophilic side chains.4 Such byproducts have not been observed, suggesting that cyclization may be negligible if the second reaction is carried out immediately after formation of the iodoacetyl peptide. When a mixture of peptides, such as a tryptic digest, is to be analyzed, the derivatization reactions may be performed on the mixture. A single chromatographic step could then be used for both peptide separation and derivative purification. Alternate purification strategies are also being examined, including selective retention of the quaternary ammonium derivatives on cation exchange at high pH or gel permeation using a highly hydrophilic resin.4 Such approaches should further reduce the amount of peptide required. Improved MS/MS Signals. The large number of fragment ion types observed in Figure l a is detrimental to signal strength. The ion current from the precursor ion produced in MS-1 must be distributed among a great many fragments. In contrast, the limited number of fragment ions series (43)Wood, S. J.; Wetzel, R.Int. J. Pept. Protein Rea. 1992,39,633539. (44) Andrewe,P. C. Preeented at the Symposiumof the ProteinSociety,
San Diego, CA, Aug 11-16, 1990.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1, 1993
observed with the N-terminal derivatives (mainly a, and d, ions) should result in greater signal strength because the precursor ion intensity is distributed among fewer channels. This concept is demonstrated in Figure 4. The CAD spectrum of the underivatized tryptic peptide T4 of hGH, a 19-residue peptide,at the 100-pmollevelgavepoor signal-to-noise (Figure 4a). A large variety of ion series were observed and none covered a substantial, uninterrupted portion of the sequence. The v,, and w, ions were of large abundance, which often further complicates data interpretation. The CAD spectrum of the DMOA-Ac-T4 peptide, in contrast (Figure 4b), gives considerablyfewer ions, but with greater signal strength, even though only 40% as much peptide was used for the measurement. The entire sequence may be deduced from the a, ion series. Such restriction of fragmentation pathways may provide useful data for yet larger peptides on a routine basis. We are presently investigating this hypothesis. High resolving power is one of the advantages of the foursector tandem mass spectrometer. However, when sufficient signal is not available, analysis at lower resolution may be necessary. The limited number of fragmentation pathways for the derivatized peptides allows one to operate the mass spectrometer at lower resolving power, and thus higher transmission, without compromising peak separation. The lack of overlapping N- and C-terminal fragment ions removes the possibility of closely spaced, potentially unresolved, fragment ions. All major peaks must be separated by at least 14 u, the mass difference between the c, ion and the an+lion of glycine. Only minor fragmentations (e.g., dab and a7-CO2 in Figure 4b) would be less well separated, and these should
become apparent as,for example, when a d, ion is observed for threonine. Most often, MS-1 is set to resolvethe precursor ion isotopes. MS-2 may be set for lower resolution. This mode of operation guarantees isotopic purity of the fragment ion peaks (due to 12C selection in MS-l), but permits greater sensitivity. Additional signal could be obtained by reducing the resolution of MS-1 so that the entire isotope cluster of the M+ ion is selected for CAD. This technique is of greater importance for peptides larger than about 1500 u, in which case the 18C peak is of equal or greater abundance than the peak. The fragment ion peaks will no longer correspond to the monoisotopic mass, but to the average mass. Although unit mass resolution would be sacrificed, the mass accuracy is still sufficient to permit distinction of Asn/Asp and Glnl Glu residues. This method of operation is successfully used with triple-quadrupole instrumenta, where the low-energy CAD process provides relatively simple fragmentation.6 The present derivatives preserve the advantages of high-energy CAD but permit operation at low resolution when necessary for high sensitivity.
ACKNOWLEDGMENT Helpful discussions with Dr. John Burnier concerning peptide derivationsare appreciated. The authors are grateful to Clifford Quan for peptide synthesis and Suzy Wong for amino acid analysis.
RECEIVED for review October 1, 1992. Accepted March 9, 1993.
