651 Research Article Received: 4 January 2010 Accepted: 21 April 2010 Published online in Wiley Interscience: 12 May 2010 (www.interscience.com) DOI 10.1002/jms.1756 Differentiation of Boc-protected α ,δ -/δ ,α - and β ,δ -/δ ,β -hybrid peptide positional isomers by electrospray ionization tandem mass spectrometry G. Raju, a V. Ramesh, a R. Srinivas, a∗ G. V. M. Sharma b∗ and B. Shoban Babu b Two new series of Boc-N-α,δ-/δ,α- and β ,δ-/δ,β -hybrid peptides containing repeats of L-Ala-δ 5 -Caa/δ 5 -Caa-L-Ala and β 3 -Caa-δ 5 - Caa/δ 5 -Caa-β 3 -Caa (L-Ala = L-alanine, Caa = C-linked carbo amino acid derived from D-xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MS n spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc-group, and the side chain. The dipeptide positional isomers are differentiated by the collision-induced dissociation (CID) of the protonated peptides. The loss of 2-methylprop-1-ene is more pronounced for Boc-NH-L-Ala-δ-Caa-OCH 3 (1), whereas it is totally absent for its positional isomer Boc-NH-δ-Caa-L-Ala-OCH 3 (7), instead it shows significant loss of t-butanol. On the other hand, second isomeric pair shows significant loss of t-butanol and loss of acetone for Boc-NH-δ-Caa-β -Caa-OCH 3 (18), whereas these are insignificant for its positional isomer Boc-NH-β -Caa-δ-Caa-OCH 3 (13). The tetra- and hexapeptide positional isomers also show significant differences in MS 2 and MS 3 CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed through five-membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However, b 1 + ion is formed in case of δ,α-dipeptide that may have a six-membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di-, tetra-, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers. Copyright c 2010 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this article. Keywords: electrospray ionization; tandem mass spectrometry; nonnatural amino acids; hybrid peptides; positional isomers Introduction Over the past few years, there has been growing interest in peptides derived from nonnatural amino acids because of their importance in pharmaceutical and foldamer chemistry. [1 – 3] Such efforts have invariably been to understand their conformational behavior and develop them into biomolecules with novel properties. There are considerable number of reports on the β -peptide class of foldamers, which exhibited several novel secondary structures. [4,5] A 12/10-mixed helix unique to the β -peptides was discovered for the first time by Seebach et al. [6] in β 2 /β 3 -dipeptide repeats. In a later study, Sharma et al. [7] demonstrated the presence of robust right-handed mixed 10/12- and 12/10-helices in hetero-chiral dipeptide repeats from carbo-β 3 -amino acid (β 3 -Caa) with alternating chirality at the Cβ carbon. It was also shown in their studies on peptides with alternating Caa and β -h-Gly, the formation of right- and left- handed 10/12- and 12/10-helices. [8] As a part of an ongoing program toward developing new oligomers [7 – 11] of L-Ala and carbo-δ 5 -amino acids with novel secondary structures, one of us [12] has recently reported the synthesis of oligomers with dipeptide repeats of L-Ala and carbo-δ 5 -amino acid and demonstrated novel 13/11-mixed helices. There have been several reports on mass spectrometry (MS) of natural amino acid peptides [13 – 15] and tandem mass spectrometry (MS/MS) of protonated [16 – 20] and deprotonated peptides [21 – 25] in electrospray ionization (ESI) and matrix-assisted laser desorption/ionization [26 – 28] has been used for structure elucidation of peptides. There are very few reports in the literature on the mass spectral study of peptides derived from nonnatural amino acids. [29 – 38] We have reported ESI MS/MS of a series of Boc- protected carbopeptides earlier, [29 – 37] and differentiated both positional and diastereomeric isomers. [29 – 36] Recently, we have reported a study on the effect of N-terminal β - and γ -carbo amino acids on the fragmentation of γ -amino butyric acid (γ -Abu) containing hybrid peptides. [35] We have also shown that aminoxy peptide bond has profound influence on the fragmentation of ∗ Correspondence to: R. Srinivas, National Centre for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad 500 007, India. E-mail: [email protected]G. V. M. Sharma, Organic Chemistry Division III, D 211, Discovery Labora- tory, Indian Institute of Chemical Technology, Hyderabad 500 007, India. E-mail: [email protected]a NationalCentreforMassSpectrometry,IndianInstituteofChemicalTechnology, Hyderabad 500 007, India b Organic Chemistry Division III, D 211, Discovery Laboratory, Indian Institute of Chemical Technology, Hyderabad 500 007, India J. Mass. Spectrom. 2010, 45, 651 – 663 Copyright c 2010 John Wiley & Sons, Ltd.
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65
1
Research ArticleReceived: 4 January 2010 Accepted: 21 April 2010 Published online in Wiley Interscience: 12 May 2010
(www.interscience.com) DOI 10.1002/jms.1756
Differentiation of Boc-protected α,δ-/δ,α-and β,δ-/δ,β-hybrid peptide positional isomersby electrospray ionization tandem massspectrometryG. Raju,a V. Ramesh,a R. Srinivas,a∗ G. V. M. Sharmab∗ and B. Shoban Babub
Two new series of Boc-N-α,δ-/δ,α- and β,δ-/δ,β-hybrid peptides containing repeats of L-Ala-δ5-Caa/δ5-Caa-L-Ala and β3-Caa-δ5-Caa/δ5-Caa-β3-Caa (L-Ala = L-alanine, Caa = C-linked carbo amino acid derived from D-xylose) have been differentiated by bothpositive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MSn spectra of protonatedisomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc-group, and the side chain.The dipeptide positional isomers are differentiated by the collision-induced dissociation (CID) of the protonated peptides. Theloss of 2-methylprop-1-ene is more pronounced for Boc-NH-L-Ala-δ-Caa-OCH3 (1), whereas it is totally absent for its positionalisomer Boc-NH-δ-Caa-L-Ala-OCH3 (7), instead it shows significant loss of t-butanol. On the other hand, second isomeric pairshows significant loss of t-butanol and loss of acetone for Boc-NH-δ-Caa-β-Caa-OCH3 (18), whereas these are insignificantfor its positional isomer Boc-NH-β-Caa-δ-Caa-OCH3 (13). The tetra- and hexapeptide positional isomers also show significantdifferences in MS2 and MS3 CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed throughfive-membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However,b1
Supporting information may be found in the online version of this article.
Keywords: electrospray ionization; tandem mass spectrometry; nonnatural amino acids; hybrid peptides; positional isomers
Introduction
Over the past few years, there has been growing interest inpeptides derived from nonnatural amino acids because of theirimportance in pharmaceutical and foldamer chemistry.[1 – 3] Suchefforts have invariably been to understand their conformationalbehavior and develop them into biomolecules with novelproperties. There are considerable number of reports on theβ-peptide class of foldamers, which exhibited several novelsecondary structures.[4,5] A 12/10-mixed helix unique to theβ-peptides was discovered for the first time by Seebachet al.[6] in β2/β3-dipeptide repeats. In a later study, Sharmaet al.[7] demonstrated the presence of robust right-handed mixed10/12- and 12/10-helices in hetero-chiral dipeptide repeats fromcarbo-β3-amino acid (β3-Caa) with alternating chirality at theCβ carbon. It was also shown in their studies on peptides withalternating Caa and β-h-Gly, the formation of right- and left-handed 10/12- and 12/10-helices.[8] As a part of an ongoingprogram toward developing new oligomers[7 – 11] of L-Ala andcarbo-δ5-amino acids with novel secondary structures, one of us[12]
has recently reported the synthesis of oligomers with dipeptiderepeats of L-Ala and carbo-δ5-amino acid and demonstrated novel13/11-mixed helices.
There have been several reports on mass spectrometry(MS) of natural amino acid peptides[13 – 15] and tandem mass
spectrometry (MS/MS) of protonated[16 – 20] and deprotonatedpeptides[21 – 25] in electrospray ionization (ESI) and matrix-assistedlaser desorption/ionization[26 – 28] has been used for structureelucidation of peptides. There are very few reports in the literatureon the mass spectral study of peptides derived from nonnaturalamino acids.[29 – 38] We have reported ESI MS/MS of a series of Boc-protected carbopeptides earlier,[29 – 37] and differentiated bothpositional and diastereomeric isomers.[29 – 36] Recently, we havereported a study on the effect of N-terminal β- and γ -carboamino acids on the fragmentation of γ -amino butyric acid (γ -Abu)containing hybrid peptides.[35] We have also shown that aminoxypeptide bond has profound influence on the fragmentation of
∗ Correspondence to: R. Srinivas, National Centre for Mass Spectrometry, IndianInstitute of Chemical Technology, Hyderabad 500 007, India.E-mail: [email protected]
G. V. M. Sharma, Organic Chemistry Division III, D 211, Discovery Labora-tory, Indian Institute of Chemical Technology, Hyderabad 500 007, India.E-mail: [email protected]
a National Centrefor MassSpectrometry, Indian InstituteofChemical Technology,Hyderabad 500 007, India
b Organic Chemistry Division III, D 211, Discovery Laboratory, Indian Institute ofChemical Technology, Hyderabad 500 007, India
(1) a = 1; R = CH3 (7) b = 1; R = CH3(2) a = 1; R = H (8) b = 1; R = H(3) a = 2; R = CH3 (9) b = 2; R = CH3(4) a = 2; R = H (10) b = 2; R = H(5) a = 3; R = CH3 (11) b = 3; R = CH3(6) a = 3; R = H (12) b = 3; R = H
(13) c = 1; R = CH3 (18) d = 1; R = CH3(14) c = 1; R = H (19) d = 1; R = H(15) c = 2; R = CH3 (20) d = 2; R = CH3(16) c = 2; R = H (21) d = 2; R = H(17) c = 3; R = CH3 (22) d = 3; R = CH3
β δ
b
Scheme 1. Structures of the Boc-protected α,δ-(1–6)/δ,α-(7–12) peptides and β ,δ-(13–17)/δ,β-(18–22) peptides.
O
H
NH
CH
O
H
O NH
CH
O
H
H H2NCH
H
McLafferty typerearrangement -CO2
[M+H]+ [M+H-C4H8]+ [M+H-Boc+H]+
R
RR
Scheme 2. Proposed McLafferty-type rearrangement.
aminoxy hybrid peptides containing repeats of β-hAla-(R)-Ama-and β-Caa-(R)-Ama- using ESI MS/MS.[37]
In continuation of our studies, here we report an ESI tandemmass spectrometric study on differentiation of two new seriesof positional isomers of hybrid peptides containing repeats ofL-Ala-δ5-Caa (α,δ-)/δ5-Caa-L-Ala (δ,α-) and β3-Caa-δ5-Caa (β ,δ-)/δ5-Caa-β3-Caa (δ,β-) (Scheme 1).
Experimental
Mass spectrometry
ESI mass spectra of di- to hexapeptides (Scheme 1) wererecorded using a liquid chromatography quadrupole ion trap massspectrometer (Thermo Finnigan, San Jose, CA, USA), equippedwith an ESI source. The data acquisition was under the control ofXcalibur software (Thermo Finnigan). The typical source conditionswere: spray voltage, 5 kV; capillary voltage, 15–20 V; heatedcapillary temperature, 200 ◦C; tube lens offset voltage, 20 V; sheathgas (N2) pressure, 30 psi; and helium was used as damping gas.For the ion trap mass analyzer, the automatic gain control settingswere 2 × 107 counts for a full-scan mass spectrum and 2 × 107
counts for a full product ion mass spectrum with a maximum ioninjection time of 200 ms. In the full-scan MS2 and MS3 modes,the precursor ion of interest was first isolated by applying anappropriate waveform across the end-cap electrodes of the iontrap to resonantly eject all trapped ions, except those of them/z ratio of interest. The isolated ions were then subjected toa supplementary ac signal to resonantly excite them and socause collision-induced dissociation (CID). The collision energiesused were 20–38 eV. The excitation time used was 30 ms. All thespectra were recorded under identical experimental conditionsfor isomers and average of 25–30 scans. Some of the product ionpeaks in the spectra were magnified for clarity. All the sampleswere infused into the ESI source at a flow rate of 5 µl/min by usinginstrument’s syringe pump.
Materials
Solvents and chemicals used in this study were purchased fromMerck (Mumbai, India) and were used without further purification.Stock (1 mM) solutions of peptides were diluted with high-performance liquid chromatography-grade methanol to achievea final concentration of 10 µM each.
Differentiation of Boc-protected α,δ-/δ,α- and β ,δ-/δ,β-hybrid peptide positional isomers
Synthesis of the peptides
The syntheses of the peptides studied in this work haverecently been reported.[12] All the peptides were prepared fromcorresponding monomers of L-Ala, carbo-β3-, and δ5-aminoacids (derived from D-xylose) by conventional methods using 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride and1-hydroxy benzotriazole hydrate in dichloromethane.
Results and Discussion
Two new series of positional isomeric Boc-protected di-, tetra-, andhexapeptides containing repeats of L-Ala-δ5-Caa (α,δ-)/δ5-Caa-L-Ala (δ,α-) and β3-Caa-δ5-Caa (β ,δ-)/δ5-Caa-β3-Caa (δ,β-) (L-Ala =L-alanine,β3-Caa= (S)-C-linked carbo-β3-amino acid, andδ5-Caa=(S)-C-linked carbo-δ5-amino acid, derived from D-xylose) studied inthis work are shown in Scheme 1. The positive ion ESI mass spectraof all these peptides (1–22) show abundant [M+H]+ , [M+Na]+ ,and [M+H-Boc+H]+ ions. As reported earlier,[29 – 37] the formationof [M+H-Boc+H]+ ions can be explained by a McLafferty-typerearrangement involving a γ -H migration from the t-butyl groupto the carbonyl oxygen in the Boc-N- moiety followed by theloss of 2-methyl-prop-1-ene (C4H8, 56 Da) and subsequent lossof CO2 from the [M+H]+ (Scheme 2). The [M+H-Boc+H]+ canalso be formed by a stepwise mechanism involving the loss ofCO2 from [M+H-C4H8]+ as evidenced by the MS/MS spectra ofthe latter ions in some of these peptides. Fragmentation of thesepeptides can be explained by using the nomenclature that wasoriginally proposed for α-peptides by Roepstorff, Fohlmann, andlater modified by Biemann.[13 – 15] The negative ion ESI mass spectraof hybrid peptide acids (Scheme 1) show abundant [M−H]− ,[M−H-C(CH3)3OH]−, and [2M−H]− ions.
CID of isomeric dipeptides (1/7 and 13/18)
The ESI MS/MS spectra of [M+H]+ ions of positional isomericpeptides 1 (Boc-NH-L-Ala-δ-Caa-OCH3 (α,δ-)), 7 (Boc-NH-δ-Caa-L-Ala-OCH3 (δ,α-)), 13 (Boc-NH-β-Caa-δ-Caa-OCH3 (β ,δ-)), and 18(Boc-NH-δ-Caa-β-Caa-OCH3 (δ,β-)) show significant differences.The isomer 1 displays prominent [M+H-C4H8]+ ions (m/z 419) andlow abundant [M+H-Boc+H]+ ions (m/z 375), whereas the formeris totally absent and the latter is the base peak for 7 (Fig. 1a andb). Besides, 7 shows an additional ion at m/z 401 correspondingto [M+H-C(CH3)3OH]+. The absence of m/z 419 and increased theabundance of m/z 375 in 7 may be attributed to the N-terminussugar group that presumably acts as a catalyst to transfer theproton from the carbonyl group after the initial McLafferty-typerearrangement, to the amine function leading to the concomitantloss of C4H8 and CO2 to form the intense [M+H-Boc+H]+ ions. Thisis consistent with our earlier report on (S)-βα-dipeptide[30] thatshowed abundant [M+H-Boc+H]+ and no [M+H-C4H8]+ ions,whereas (R)-βα-dipeptide[30] exhibited both [M+H-C4H8]+ and[M+H-Boc+H]+ ions. The formation of [M+H-C(CH3)3OH]+ ions(m/z 401) in 7 can be explained by a ‘H’ migration from one of the–CH2 –groups of δ-Caa to the ‘O’ of the butyloxy group followedby the loss of t-butanol and the absence of this ion in 1 may bedue to the presence of L-Ala at the N-terminus.
To further examine the fragmentation of the peptides, the MS3
CID spectra of these isomeric pairs were examined. These spectraalso showed significant differences. The MS3 spectrum of [M+H-Boc+H]+ (m/z 375) of 1 (α,δ-) mainly shows (Fig. 2a) peaks atm/z 357 (loss of H2O), m/z 343 (loss of CH3OH), m/z 317 (loss of
[M+
H]+
[M+
H-C
4H8]
+
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oc+
H]+
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Figure 1. ESI MS2 of [M+H]+ ions of dipeptides of (a) 1, (b) 7, (c) 13, and(d) 18 at 22 eV.
(loss of acetone + methanol), m/z 272 (y1+−methanol), m/z 267
(loss of H2O from m/z 285), m/z 214 (y1+−acetone + methanol),
m/z 201 (loss of xylose), and m/z 196 (loss of H2O from m/z 214). Onthe other hand, intense b1
+ (m/z 272), m/z 317 (loss of acetone),m/z 214 (b1
+−acetone), low abundant m/z 182 (loss of CH3OHfrom m/z 214), and m/z 164 (loss of H2O from m/z 182) ions areobserved for 7 (Fig. 2b).
The ESI MS2 spectra of [M+H]+ ions (m/z 647) of the secondisomeric pair 13 (β ,δ-) and 18 (δ,β-) also exhibit significantdifferences. While the former shows only [M+H-Boc+H]+ (m/z547) ion, the latter displays [M+H-Boc+H]+, [M+H-C(CH3)3OH]+
(m/z 573), and [M+H-acetone]+ (m/z 589) ions (Fig. 1c andd). Formation of these ions appears to be in line with thefragmentation observed for the isomeric pair 1/7 wherein theN-terminus δ-Caa in 7, gave rise to intense [M+H-Boc+H]+ and[M+H-C(CH3)3OH]+ . The absence of [M+H-C4H8]+ and abundantformation of [M+H-Boc+H]+ in 13 is consistent with our earlierreport[30] as discussed above. Thus, these positional isomers canclearly be distinguished from one another by their characteristicfragmentation.
The MS3 CID spectra of [M+H-Boc+H]+ ions (m/z 547) of 13/18show distinct differences in their fragmentation pattern (Fig. 3).Isomer 13 shows abundant m/z 529 (loss of H2O), m/z 489 (loss
of acetone), m/z 457 (loss of acetone + methanol), m/z 439(loss of H2O from m/z 457), m/z 373 (loss of xylose), m/z 346(loss of Su–CH NH), m/z 304 (y1
+), m/z 256 (loss of acetone +methanol from m/z 346), low abundant m/z 515 (loss of CH3OH),m/z 471 (loss of H2O from m/z 489), m/z 246 (y1
+−acetone),and m/z 196 (y1
+− acetone + methanol + H2O) (Fig. 3a). Theformation of an intense ion of m/z 346 by the loss of imine,a retro-Mannich reaction,[30,33,34,37,38] is highly characteristic forthe presence of β-amino acid (β-Caa) at the N-terminus and thisfragmentation is absent when δ-Caa is present at the N-terminus.On the contrary, isomeric peptide 18 gives a simple spectrumwithout much fragmentation. The abundant fragment ions arem/z 276 (y1
+), m/z 218 (y1+−acetone), and low abundant ions
are m/z 186 (loss of methanol from m/z 218), m/z 168 (loss ofH2O from m/z 186), and m/z 530 (loss of NH3) (Fig. 3b). Mostof the secondary fragmentations observed in MS3 CID of theisomeric pairs have been confirmed by further MSn experimentson respective precursor ions. Thus, the MS3 CID spectra also
provide additional information for distinguishing these dipeptideisomers.
CID of isomeric tetrapeptides (3/9 and 15/20)
The MS/MS CID spectra of [M+H]+ ions (m/z 817) of positionalisomeric tetrapeptides 3 (α,δ,α,δ-) and 9 (δ,α,δ,α-) presentsignificant differences. Both the spectra show the base peak at m/z717 ([M+H-Boc+H]+) and other common ions at m/z 759 ([M+H-acetone]+) and m/z 659 (loss of acetone from m/z 717) (Fig. 4).The direct formation of [M+H-Boc+H]+ ions and the absence of[M+H-C4H8]+ ions in the tetrapeptide (α,δ,α,δ-) can be explainedby a plausible mechanism that may involve a 1,5-H migrationfrom the t-butyl group to the –NH–leading to concomitant loss ofCO2 and C4H8 (Scheme 3). The preferential migration of ‘H’ to the–NH–instead of butyloxy carbonyl group may presumably due tononavailability of the latter group following its participation in the13-mer hydrogen bonding.[12] The secondary structures involving
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Figure 3. ESI MS3 of [M+H-Boc+H]+ ions (m/z 547) of (a) 13 and (b) 18 at 30 eV.
Differentiation of Boc-protected α,δ-/δ,α- and β ,δ-/δ,β-hybrid peptide positional isomers
Boc-group leading to helical structures in higher oligomers 3 and5 in solution phase have been reported by one of us.[12] The majordifferences observed between 3 and 9 are that the former displaysadditional fragment ions at m/z 304 (y1
+) and m/z 514 (b3+), which
are absent for 9 and m/z 375 (y2+) and m/z 743 (loss of t-butanol)
ions that are prominent for the latter are absent for the former. Inaddition, m/z 759 and 659 are more abundant for 3 than for 9.
To further examine the fragmentation of these isomericpeptides, the MS3 CID spectra of [M+H-Boc+H]+ ions (m/z 717)of 3 and 9 were compared (Fig. 5). Both the spectra show m/z 699(loss of H2O), m/z 659 (loss of acetone), m/z 627 (loss of acetone+ methanol), and m/z 609 (loss of H2O from m/z 627). The ionsm/z 659 and 627 are more abundant for 3 than for 9. In addition,the former shows abundant b3
+ (m/z 414), y1+ (m/z 304), m/z 543
(loss of xylose) ions and low abundant ions at m/z 569 (loss ofacetone from m/z 627), m/z 272 (y1
+− methanol), and m/z 246(y1
+− acetone), which are absent for the latter. Whereas, the lattershows significant yn
+ ions (n = 2, 3) at m/z 375 and 446 and lowabundant ions at m/z 414 (y3
+− methanol) and m/z 356 (y3+−
acetone + methanol), which are absent for the former.The MS/MS CID spectrum of [M+H]+ ions (m/z 1161) of second
isomeric pair 15 (β ,δ,β ,δ-)/20 (δ,β ,δ,β-) displays highly abundant[M+H-Boc+H]+ ions (m/z 1061) and other fragment ions at m/z1103 (loss of acetone), m/z 1087 (loss of t-butanol), and m/z 1003(loss of acetone from m/z 1061) (Fig. 6). It can be noted that the lossof t-BuOH, which is insignificant for the β ,δ-dipeptide, is presentfor the tetrapeptide (15), albeit, of low abundance. This may bedue to the increased chain length of the peptide that allows theβ-methylene participate to a minor extent in the loss of t-BuOH.Loss of acetone may also be rationalized due to increased chainlength. The differences observed between 15 and 20 are that theformer shows significant b3
+ ion (m/z 858), which is absent forthe latter. Whereas, the latter displays abundant y2
+ ion (m/z 547)and low abundant y3
+ ion (m/z 790). In addition, the ion at m/z1087 is also more abundant for 20 than for 15. This indicates
that –CH2 –adjacent to C O participates predominantly in theloss of t-butanol. Thus, these positional isomeric tetrapeptidescan be clearly differentiated from one another by their MS/MSspectra.
The MS3 spectra of the [M+H-Boc+H]+ ions (m/z 1061) of 15and 20 also show different fragmentation (Fig. 7). The formershows N-terminal peptide sequencing bn
+ (n = 2, 3) ions atm/z 515 and 758, and C-terminal characteristic yn
+ (n = 2, 3)ions at m/z 547 and 818. Other side-chain fragment ions are alsoobserved at m/z 1043 (loss of H2O), m/z 1003 (loss of acetone),m/z 971 (loss of acetone + methanol), m/z 953 (loss of H2Ofrom m/z 971), m/z 935 (loss of H2O from m/z 953), m/z 895(loss of acetone from m/z 953), m/z 887 (loss of xylose), m/z860 (loss of Su–CH NH), m/z 802 (loss of acetone from m/z860), m/z 700 (b3
+− acetone) and m/z 407 (loss of2 methanol + H2O from m/z 489). On the contrary, the MS3
spectrum of [M+H-Boc+H]+ of 20 displays simple spectrum andshows predominantly y3
+ ion at m/z 790. In addition, the spectrumalso shows low abundant ions at m/z 732 (y3
+− acetone) and m/z547 (y2
+), respectively. It appears that the initially protonatedδ-Caa at the N-terminus undergoes fast dissociation prior to thetransfer of a mobile proton across the peptide backbone leadingto predominant y3
+ and the absence of other fragmentation.This is very similar to the behavior of γ ,α-32 and γ ,β-peptides[34]
which also showed y3+ as the major ion in the MS3 spectrum. It is
interesting to observe that the peptide ions having β-Caa at theN-terminus, instead of δ-Caa, exhibit extensive fragmentation.Thus, these results clearly distinguish the presence and theposition of β-Caa versus δ-Caa at the N-terminus of these hybridpeptides.
CID of isomeric hexapeptides (5/11 and 17/22)
The MS/MS CID spectrum of [M+H]+ ions (m/z 1159) of isomerichexapeptide esters 5 (α,δ,α,δ,α,δ-) and11 (δ,α,δ,α,δ,α-) shows
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e A
bund
ance
717
817
304759
659514
x10
y1+
(a)
b3+
Figure 4. ESI MS2 of [M+H]+ ions (m/z 817) of tetrapeptides (a) 3 and (b) 9 at 22 eV.
O
HNH CH
O
R H
H2NCH
R
H
-C4H8+CO2
[M+H]+ [M+H-Boc+H]+
Scheme 3. Proposed mechanism for the concomitant loss of C4H8 and CO2.
abundant [M+H-Boc+H]+ (m/z 1059) and moderately abundantm/z 1101 (loss of acetone) ions (Fig. 8). Similar to the tetrapeptide3 (α,δ,α,δ-), [MH-C4H8]+ is absent for the hexapeptide 5 that isalready discussed in the context of tetrapeptide. This peptideshows highly abundant b5
+ (m/z 856) and moderately abundantyn
+ (n = 1, 3) ions at m/z 304 and 646, and low abundant m/z 756([b5−Boc+H]+) ions, which are absent for 11. Whereas, the isomer11 displays low abundant m/z 1085 (loss of t-butanol), b4
+ (m/z785), and yn
+ (n = 2, 4) ions at m/z 375 and 717, which are absent
for 5. It can be noted that protonated di-, tetra-, and hexapeptideshaving δ-Caa at the N-terminus lose t-BuOH.
The MS3 CID spectra of [M+H-Boc+H]+ ions (m/z 1059)of isomeric pair 5/11 also show distinct differences in theirfragmentation pattern (Fig. 9). Isomer 5 shows abundant b5
(loss of acetone + methanol) ions and low abundant ions at m/z885 (loss of xylose), m/z 738 (loss of H2O from m/z 756), and m/z698 (loss of acetone from b5
+). Whereas, the isomer 11 shows
200 300 400 500 600 7000
10
20
30
40
50
60
70
80
90
100 659
304
627414
543
246 609569 699454 717272
y1+(a)
b3+
Rel
ativ
e A
bund
ance
200 300 400 500 600 700
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
717
446
375659356 414 699609
(b)
y2+
y3+
Figure 5. ESI MS3 of [M+H-Boc+H]+ ions (m/z 717) of (a) 3 and (b) 9 at 30 eV.
500 600 700 800 900 1000 11000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1061
1103
1161
858 1003
x10
(a)
b3+
500 600 700 800 900 1000 1100
m/zm/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1061
1103
1161
1003547
x10
790
y2+
(b)
y3+ 1087
Figure 6. ESI MS2 of [M+H]+ ions (m/z 1161) of tetrapeptides of (a) 15 and (b) 20 at 22 eV.
Differentiation of Boc-protected α,δ-/δ,α- and β ,δ-/δ,β-hybrid peptide positional isomers
300 400 500 600 700 800 900 1000
m/zm/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
790
732547 1061
y2+
(b)
y3+
300 400 500 600 700 800 900 10000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1061
1003
758971
953818 895547 935
515 650 860407
b2+
(a)
y2+
b3+
y3+
1043700 802
Figure 7. ESI MS3 of [M+H-Boc+H]+ ions (m/z 1061) of (a) 15 and (b) 20 at 30 eV.
200 300 400 500 600 700 800 900 1000 11000
10
20
30
40
50
60
70
80
90
100 856
1059
1159
646
304
756
x3
Rel
ativ
e A
bund
ance
1101y1+
(a)
y3+
b5+
200 300 400 500 600 700 800 900 1000 1100
m/zm/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1059
375
1101
717
1159785
x3
(b)
y2+
y4+
b4+
Figure 8. ESI MS2 of [M+H]+ ions (m/z 1159) of hexapeptides (a) 5 and (b) 11 at 22 eV.
significant yn+ ions (n = 2, 4, 5) at m/z 375, 717, and 788, b4
+ion at m/z 685 and other low abundant ions at m/z 1001 (loss ofacetone), 969 (loss of acetone + methanol), 951 (loss of H2O fromm/z 969), and 730 (loss of acetone from y5
+), respectively.The MS/MS product ion spectra of the [M+H]+ ions (m/z 1675)
of the isomeric pair 17 (β ,δ,β ,δ,β ,δ-) and 22 (δ,β ,δ,β ,δ,β-) showhighly abundant [M+H-Boc+H]+ (m/z 1575) ions and other ionsat m/z 1617 (loss of acetone), 1601 (loss of t-butanol), and /z1517 (loss of acetone from [M+H-Boc+H]+) (Fig. 10). The lossof t-butanol from 17 may be attributed to the participation ofβ-CH2 due to increased length of the peptide as explained for thetetrapeptide 15. Besides, the former shows low abundant b5
+ (m/z1372) and y3
+ (m/z 818) ions. On the contrary, the latter shows thecharacteristic yn
+ ions (n = 4, 5) at m/z 1061, 1304 and b4+ ion at
m/z 1129.The MS3 CID spectrum of [M+H-Boc+H]+ ions (m/z 1575) of
17 displays both the N- and C-terminal fragment ions, i.e. bn+
(n = 2–5) ions at m/z 515, 758, 1029, and 1272 and yn+ ions
(n = 2–5) at m/z 547, 818, 1061, and 1332 (Fig. 11a). Other side-chain fragment ions appear at m/z 1557 (loss of H2O), 1517 (lossof acetone), 1485 (loss of acetone + methanol), 1467 (loss of H2Ofrom m/z 1485), 1450 (loss of NH3 from m/z 1467), 1409 (loss ofacetone from m/z 1467), 1351 (loss of acetone from m/z 1409),1214 (b5
+−acetone), 1164 (b5+− acetone + methanol + H2O),
1003 (y4+− acetone), 971 (b4
+−acetone), 921 (b4+−acetone
+ methanol + H2O), 740 (b3+-H2O), and 650 (b3
+−acetone+ methanol + H2O). Formation of both bn
+ and yn+ ions in
the peptides with β-Caa at the N-terminus suggests that eithermobile proton migration occurs through a larger ‘whole’ peptidecyclic intermediate or the fragmentation proceeds through apeptide with a mobile proton protonated at amide ‘O’ atomsfrom the C-terminus.[20] As the β ,δ-peptides contain β-Caa and
δ-Caa at alternative positions from the N-terminus, N-terminusbn
+ ions and the resulting yn+ ions occur at a mass difference
of 271 and 243 Da alternatively, corresponding to successivelosses of δ-Caa and β-Caa, respectively. For example, in Fig. 11a,the difference between b3
+ (m/z 758) and b4+ (m/z 1029), y3
+
(m/z 818) and y2+ (m/z 547), is 271 Da. Similarly, the difference
between b2+ (m/z 515) and b3
+ (m/z 758), y4+ (m/z 1061) and
y3+ (m/z 818), is 243 Da. On the other hand, the isomer 22
shows highly abundant y5+ ions (m/z 1304) and insignificant ions
at m/z 1517 (loss of acetone), m/z 1246 (y5+−acetone), m/z 1214
(y5+−acetone + methanol), m/z 1061 (y4
+), m/z 790 (y3+), and m/z
547 (y2+). Similar to the tetrapeptide isomer containingδ-Caa at the
N-terminus (20), the isomer 22 does not yield bn+ ions. As the
δ,β-peptides contain δ-Caa and β-Caa at alternative positionsfrom the N-terminus, the peptide sequencing yn
+ ions occur at amass difference of 271 and 243 Da, corresponding to successivelosses of δ-Caa and β-Caa, respectively. For example, in Fig. 11b,the difference between y4
+ (m/z 1061) and y3+ (m/z 790) ions
is 271 Da, y5+ (m/z 1304) and y4
+ (m/z 1061), y3+ (m/z 790)
and y2+ (m/z 547), is 243 Da. Thus, both MS2 and MS3 CID
differentiate the positional isomers of hybrid hexapeptides fromone another.
Structure of bn+ ions
Over the years, there have been several studies focused on thestructure and mechanism of the formation of bn
+ ions fromnatural peptides.[20,39] This has been mainly due to its complexand variable structures. With a view to examine the effect ofpresence of δ-Caa in the peptide backbone on the formation of b-ions from these hybrid carbopeptides, the structures of these ionsare discussed here. As discussed earlier, the MS3 CID spectrum of
Figure 9. ESI MS3 of [M+H-Boc+H]+ ions (m/z 1059) of (a) 5, (b) 11 at 28 eV and MS4 of b5+ (m/z 756) ions of 5 at 28 eV.
[M+H-Boc+H]+ of hexapeptide 5 (α,δ,α,δ,α,δ-) shows an intenseb5
+ ion and other bn+ ions are totally absent (Fig. 9a). This can be
rationalized that the formation of b5+ ion may proceed through
an energetically favorable five-membered cyclic transition state toform the oxazolone structure as illustrated in Scheme 4. A similarmechanism, that has been reported for the natural peptides,[39] isproposed here. The absence of b5
+ ion from the isomeric peptide11 (δ,α,δ,α,δ,α-) can be ascribed to an unfavorable cyclizationprocess involving eight-membered cyclic ring due to the presenceof δ-Caa at the C-terminus of b5
+ ion (Fig. 9b). The formationof oxazolone structure has been supported by the CID spectrum
of b5+ from 5 which shows the loss of CO (m/z 728), b4
+ (m/z685), b3
+ (m/z 414), and b2+ ions (m/z 343) (Fig. 9c). As expected,
the b2+ and b4
+ are of lower abundance as compared to thatof b3
+. This, again, can be attributed to the five-membered ringcyclization for the latter as compared to larger ring cyclizationfor the former ions. Similarly, the absence of b3
+ ion from thetetrapeptide 9 (δ,α,δ,α-) can be attributed to larger size b3
+ iondue to the presence of δ-Caa at the C-terminus. However, incase of the dipeptide 7 (δ,α-) an intense b1
+ ion is noticed. Theformation of this ion can be explained by an intramolecularnucleophilic attack by the –NH2 of the δCaa on its carbonyl
Differentiation of Boc-protected α,δ-/δ,α- and β ,δ-/δ,β-hybrid peptide positional isomers
600 800 1000 1200 1400 16000
10
20
30
40
50
60
70
80
90
100R
elat
ive
Abu
ndan
ce
1575
1601
1517
1675
1372
818 1467
x10
y3+
(a)
b5+
600 800 1000 1200 1400 1600
m/zm/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1575
1601
1675
1517
10611129 1304
x10
(b)
y4+
y5+b4
+
Figure 10. ESI MS2 of [M+H]+ ions (m/z 1675) of hexapeptides (a) 17 and (b) 22 at 25 eV.
NH
NH
H3N
OSuOHN
HN
NH
OCH3
O O
O O
Su
Su
Proton Tranfer
NH
NH
H2N
OSuOHN
HN
NH2
OCH3
O O
O O
Su
Su
NH
NH
H2N
OSuOHN
O Su O
HN
NH
NH
H2N
OSuOHN
O Su O
N
O
H2N OCH3
OSu
H2N OCH3
OSu
O
H
NH
NH
H2N
OSuOHN
O Su O
HN
O
H2N OCH3
OSu
b5+
Scheme 4. Pathway to form b5+ from protonated hexapeptide 5 through the oxazolone mechanism.
group leading to a stable six-membered cyclic ring with theelimination of alanine (Scheme 5). This mechanism is similar tothat reported for direct formation of stable b1
+ ions of variousamino acids such as Arg, Lys, His, from protonated dipeptideswith the basic residue in the N-terminal positions.[40] It was alsoreported that most stable structure proposed is six-memberedcyclic structure.[39]
Negative ion CID of isomeric peptide acids
In contrast to the formation of abundant [M+H-Boc+H]+ ionsunder positive ion conditions, the negative ion ESI mass spectraof isomeric peptide acids (Scheme 1) show abundant [M−H-C(CH3)3OH]− ions.[29,30,32 – 34] The formation of these ions maypresumably due to a 1,3-H migration from –NH–to the ‘O’ of thebutyloxy group leading to the elimination of t-butanol by charge-
Figure 11. ESI MS3 of [M+H-Boc+H]+ ions (m/z 1575) of (a) 17 and (b) 22 at 30 eV.
remote fragmentation.[35] The MS2 CID spectra of [M−H]− ions ofisomeric di- (2, 8), tetra- (4, 10), and hexa- (6, 12) peptides also giveabundant [M−H-C(CH3)3OH]− ions. The participation of –NH–isproposed, because the loss of t-butanol is highly abundant evenfrom the hybrid peptides containing L-Ala at the N-terminus.
The MS3 CID spectra of [M−H-C(CH3)3OH]− of 2, 4, and 6containing repeats of α,δ-carbo amino acids are found to besignificantly different from those of 8, 10, and 12 with repeatsof δ,α-carbo amino acids. Figure 12 shows the MS3 CID spectraof [M−H-C(CH3)3OH]− (m/z 1069) of hexapeptide isomers 6 and12. The spectrum of 6 displays bn
− (n = 5–3) ions at m/z 780,709, and 438, corresponding to successive losses of δ,α,δ aminoacids, respectively. Similarly, yn
− (n = 2–5) ions appear at m/z359, 630, 701, and 972 corresponding to alternative losses of α,δamino acids, respectively. Other side-chain fragment ion peaksappear at m/z 1051 (loss of H2O), m/z 1025 (loss of CO2), m/z 1011(loss of acetone), m/z 961 (loss of acetone + methanol + H2O),m/z 935 (loss of acetone + methanol from m/z 1025), and m/z917 (loss of CO2 from m/z 961) (Fig. 12a). The MS3 spectrum of[M−H-C(CH3)3OH]− ions (m/z 1069) of isomer 12 shows peptidesequencing yn
− ions (n = 3–5) at m/z 430, 701, and 772,b4
− ion at m/z 709. Other side-chain fragment ions are observedat m/z 1051 (loss of H2O), m/z 1025 (loss of CO2), m/z 1011 (lossof acetone), m/z 961 (loss of acetone + methanol + H2O), m/z935 (loss of acetone + methanol from m/z 1025), m/z 917 (loss ofCO2 from m/z 961), m/z 798 (loss of Su CH–N C O from m/z1025), m/z 754 (y5
−-H2O), m/z 736 (loss of H2O from m/z 754), m/z683 (y4
−-H2O), m/z 665 (loss of H2O from m/z 683), m/z 438 (loss
of Su CH–N C O from m/z 665), m/z 412 (y3−-H2O), and m/z
394 (loss of H2O from m/z 412) (Fig. 12b).Similarly, the MS3 CID spectrum of [M−H-C(CH3)3OH]− (m/z
1071) ions of other isomeric tetrapeptides 16 (β ,δ,β ,δ-) and 21(δ,β ,δ,β-) shows different fragmentation from one another (Fig. 13).The former displays [M−H-C(CH3)3OH-HNCO]− ion (m/z 1028) asthe base peak and low abundant ions at m/z 996 (loss of methanolfrom m/z 1028), m/z 938 (loss of acetone + methanol from m/z1028), m/z 802 (y3
− ion), and m/z 514 (z2− ion) (Fig. 13a). Loss of
HNCO from [M−H-C(CH3)3OH]- can be explained by a ‘H’ migrationfrom the active β-methylene group to the –NCO. The absence ofthis fragmentation in 21 can be attributed to the presence of δ-Caa which contains the active methylene group at δ-position withrespect to the NCO. The latter (21) also shows peptide sequencingcn
− ions (n = 2, 3) at m/z 556 and 827, and a2− ions (m/z 513),
yn− ions (n = 2, 3) at m/z 531 and 774 and zn
− ions (n = 2, 3)at m/z 514 and 757. It is known that c- and z-ions are difficultto form from natural peptides and specific techniques such aselectron capture dissociation has been introduced to generatethese ions.[41] Formation of these ions from 21 may be due to theeffect of carbo amino acids in the peptide backbone that probablyinduces the N–C cleavage giving rise to these ions. However, itis difficult to comment on the mechanism of formation of theseions from this study. Other side-chain fragment ions are observedat m/z 1027 (loss of CO2), m/z 937 (loss of acetone + methanolfrom m/z 1027), m/z 919 (loss of H2O from m/z 937), m/z 800 (lossof Su CH–N C O from m/z 1027), m/z 737 (c3
−− acetone +methanol), m/z 667 (z3
−− acetone + methanol), m/z 623 (loss
300 400 500 600 700 800 900 10000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
701
736630
961 1025
9171069780359
455593
1011
935
y2-
y3-
y4-
(a)
438b3
-b5
-
972
Figure 12. ESI MS3 of [M−H-C(CH3)3OH]− ions (m/z 1069) of hexapeptides (a) 6 and (b) 12 at 38 eV.
Differentiation of Boc-protected α,δ-/δ,α- and β ,δ-/δ,β-hybrid peptide positional isomers
300 400 500 600 700 800 900 1000
m/z
0
10
20
30
40
50
60
70
80
90
100 772
1025709
394701
917
1011798
430
575 736 981665 1051646
Rel
ativ
e A
bund
ance
y3-
y4-
y5-
(b)
b4-
754
Figure 12. (Continued).
400 500 600 700 800 900 10000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1028
1071996938
802514
x10
(a)
z2- y3
-
400 500 600 700 800 900 1000
m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1071
937
827
774
1027
513
556423
466800737623
x10
919
757
573531
(b)
c3-
y3-z3
-
c2-
y2-
514z2
-
a2-
Figure 13. ESI MS3 of [M−H-C(CH3)3OH]− ions (m/z 1071) of tetrapeptides of (a) 16 and (b) 21 at 30 eV.
of acetone + 2 CO from m/z 737), m/z 605 (loss of H2O fromm/z 623), m/z 573 (loss of methanol + H2O from m/z 623), m/z481 (a2
−-methanol), m/z 466 (c2−−acetone + methanol), and
m/z 423 (a2−−acetone + methanol) (Fig. 13b). Thus, the negative
ion ESI MS/MS of these hybrid peptides provides complementaryinformation and allows the differentiation of the positional isomersof the hybrid peptides.
Conclusions
Positive and negative ion ESI MS/MS has been shown to be veryuseful for obtaining sequencing information and for differentiatingpositional isomers of hybrid peptides containing repeats of α,δcarbo amino acids from the peptides with repeats of δ,α- andβ ,δ from δ,β-hybrid peptides. In case of positive ions, di-, tetra-,
Scheme 5. Proposed mechanism for the formation of b1+ ion from δ,α-
dippetide (7).
and hexapeptide isomers have been clearly distinguished bythe CID of their [M+H]+ ions. In addition, the CID spectra of[M+H-Boc+H]+ produce product ions that are different for thepositional isomers of di-, tetra-, and hexapeptides. All the peptideshaving N-terminus δCaa produce intense [M+H-C(CH3)3OH]+ ion,whereas this ion is of insignificant or low abundance whenβCaa is at the N-terminus. The peptides with L-Ala at the N-terminus do not eliminate t-BuOH. The fragmentation of α,δ-and β ,δ-peptides yields most of the yn
+ and bn+ ions and
side-chain fragmentation. Whereas, δ,α- and δ,β-peptides displayabundant yn
+ ions except in δ,α-dipeptide due to low masscut off of ion trap instruments. In case of negative ions, theMS3 CID spectra of [M−H-C(CH3)3OH]− ions of α,δ- and δ,α-peptides are different from one another. The β ,δ-isomers exhibit[M−H-C(CH3)3OH-HNCO]− , whereas δ,β-peptides show [M−H-C(CH3)3OH-CO2]− .
Acknowledgements
The authors thank Dr J. S. Yadav, Director, IICT, Hyderabad, forfacilities. G. R. and B. S. thank UGC, New Delhi, for the award ofJunior and Senior Research Fellowships, and V. R. is thankful toCSIR, New Delhi, for the award of Senior Research Fellowship.
Supporting information
Supporting information may be found in the online version of thisarticle.
References
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