a carbonyl oxygen migration in electrospray ionization mass spectrometry and its application in...

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 2002; 37: 934–939Published online 24 July 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.352

A carbonyl oxygen migration in electrospray ionizationmass spectrometry and its application in differentiatinga- and b-alanyl peptides

Jing Chen, Yi Chen, Yan-Lin Niu, Hua Fu and Yu-Fen Zhao∗

Key Laboratory for Bioorganic Phosphorus Chemistry, Ministry of Education, Department of Chemistry, School of Life Sciences and Engineering,Tsinghua University, Beijing 100084, China

Received 7 January 2002; Accepted 9 May 2002

A novel rearrangement reaction with a carbonyl oxygen migration was observed in the electrosprayionization tandem mass spectra of N-diisopropyloxyphosphoryl dipeptides and their analogues. A possiblemechanism was proposed and supported by the MS/MS study, derivatization of different functional groupsand deuterium labeling experiments. It was found that metal ions could catalyze the rearrangement througha five-membered ring intermediate. A strong affinity between the phosphoryl group and oxygen atomin the gas phase was proposed to result in this kind of rearrangement reaction, which might providesome basic information on the nature of phosphorylation in biochemistry. The replacement of N-terminala-alanine by b-alanine stopped the migration, which provides a simple method for differentiating the a-and b-alanine residues at the N-terminus of peptides. Copyright 2002 John Wiley & Sons, Ltd.

KEYWORDS: ˛- and ˇ-alanyl peptide differentiation; rearrangement reaction; electrospray ionization tandem massspectrometry; phosphorylation

INTRODUCTION

As a common constituent in living systems, phosphorusplays very important role in nucleic acids, coenzymes, phos-pholipids and almost all the metabolic processes.1 Muchresearch work has been carried out to clarify the func-tion of phosphorylation in biomolecules. Although moststudies were performed in the liquid phase, the reactionof phosphoryl molecules in the gas phase could providesome basic information about the nature of phosphory-lation. Mass spectrometry offers a suitable tool for thispurpose. Since its introduced by Yamashita and Fenn in1984,2 electrospray ionization mass spectrometry (ESI-MS)has been widely used in biological research. More specifi-cally, ESI-MS has been used in the sequencing of proteins3

and polynucleotides,4 the elucidation of protein foldingpathways,5 detection of non-covalent complexes,6 quan-titative study of enzyme-catalyzed reactions7 etc. In ourprevious work, phosphorylated amino acids and peptideswere analyzed using several kinds of mass spectrometer.8,9

In this paper, we report a special rearrangement reaction ofthe sodium adducts of N-diisopropyloxyphosphoryl (DIPP)dipeptide methyl esters (DIPP-X-Y-OMe, where X and Y

ŁCorrespondence to: Yu-Fen Zhao, Key Laboratory for BioorganicPhosphorus Chemistry, Ministry of Education, Department ofChemistry, School of Life Sciences and Engineering, TsinghuaUniversity, Beijing 100084, China.E-mail: [email protected]/grant sponsor: Chinese National Natural ScienceFoundation; Contract/grant number: 20175026.Contract/grant sponsor: Ministry of Science and Technology.Contract/grant sponsor: Chinese Education Ministry.Contract/grant sponsor: Tsinghua University.

mean amino acid residues) and their analogues using ESI-MS/MS.

EXPERIMENTAL

ChemicalsAll peptides and their derivatives were synthesized inthis laboratory by literature methods and phosphorylatedaccording to our published methods.10 Chemicals for syn-theses were purchased from Sigma or Glsynthesis (Shanghai,China).

Mass spectrometryHigh-resolution mass spectra were obtained using a BrukerAPEX II Fourier transform ion cyclotron resonance massspectrometer with an ESI source. Other mass spectra wereacquired using a Bruker ESQUIRE-LC ion trap spectrometerequipped with a gas nebulizer probe, capable of analyzingions up to m/z 6000. Nitrogen was used as drying gas with aflow-rate of 4 l min�1. The nebulizer pressure was 7 psi. Thecapillary was typically held at 4 kV. The samples dissolvedin methanol were ionized by ESI and continuously infusedinto the ESI chamber at a flow-rate of 4 µl min�1 with a Cole-Parmer Model 74900 syringe pump. The [M C Na]C ions wereanalyzed by multistage tandem mass spectrometry throughthe collision with helium.

RESULTS AND DISCUSSION

Dipeptide methyl esters were phosphorylated through asimple one-step procedures developed by Zhao10 and co-workers with a yield of about 70%10 (Scheme 1).

Copyright 2002 John Wiley & Sons, Ltd.

ESI-MS of ˛- and ˇ-alanyl peptides 935

PHO

OO

H2N PeptideTEA, EtOH, CCl4, H2O

+ice bath, 30min

R

R

PO

OO

NH PeptideR

R

R=methyl DMPHR=isopropyl DIPPH

R=methyl DMP-PeptideR=isopropyl DIPP-Peptide

Scheme 1. General strategy for peptide phosphorylation.

Table 1. Fragment ions observed in the tandem mass spectra from different DIPP-X-Y-OMe (X, Y D amino acid) and theiranalogues (BA means benzylamine): m/z with relative abundance (%) in parentheses

No. CompoundPrecursor

(m/z) Species [M � 42]C [M � 102]C [M � 164]CRearrangement

product

1 DIPP-Leu-Leu-OMe 455 (42) [M C Na]C 403 (78) 343 (34) 281 (100) 163 (47)2 DIPP-Leu-Leu-OMe 423 (31) [M C H]C 391 (100) 321 (10) — —3 DIPP-Ile-Ala-OMe 403 (100) [M C Na]C 361 (97) 301 (34) 239 (83) 163 (40)4 DIPP-Leu-Ala-OMe 403 (16) [M C Na]C 361 (100) 301 (40) 239 (94) 163 (28)5 DIPP-Leu-Val-OMe 431 (34) [M C Na]C 389 (100) 329 (29) 267 (79) 163 (47)6 DIPP-Ala-Val-OMe 389 (28) [M C Na]C 347 (100) 287 (23) 225 (69) 163 (17)7 DIPP-Leu-Phe-OMe 479 (22) [M C Na]C 437 (97) 377 (40) 315 (100) 163 (16)8 DIPP-Phe-Ile-OMe 479 (22) [M C Na]C 437 (60) 377 (18) 315 (100) 163 (13)9 DIPP-Ala-Phe-OMe 437 (22) [M C Na]C 395 (82) 335 (37) 273 (100) 163 (6)

10 DIPP-ˇ-Ala-Phe-OMe 437 (20) [M C Na]C 395 (89) 335 (28) 273 (100) —11 DIPP-Ala-BA 365 (42) [M C Na]C 323 (100) 263 (21) 201 (57) 163 (50)12 DIPP-ˇ-Ala-BA 365 (22) [M C Na]C 323 (100) 263 (31) 201 (38) —13 DIPP-Ala-BA 381 (10) [M C K]C 339 (100) 279 (31) 217 (18) 179 (33)14 DIPP-Ala-BA 349 (23) [M C Li]C 307 (100) 265 (26) 185 (6) 147 (3)15 DIPP-Cys-BA 397 (100) [M C Na]C 355 (76) 295 (18) 233 (40) 163 (27)16 DMP-Ala-Phe-OMe 381 (52) [M C Na]C — — — 149 (26)17 DIPP-Ala-OMe 290 (41) [M C Na]C 248 (100) 188 (6) 126 (18) —18 DIPP-Phe-OMe 366 (61) [M C Na]C 324 (100) — 202 (99) —

A rearrangement reaction of the sodium adducts ofDIPP amino acids–alcohol in ESI had been discovered inthis laboratory, in which the strong oxygen atom affin-ity to the phosphoryl group resulted in the migrationof the hydroxyl group to the phosphoryl group to pro-duce the phosphoric acid isopropyl ester (PAIE, m/z163).11 Interestingly, it was found that the fragment ofm/z 163 was also observed in the ESI-MS/MS study ofDIPP-Leu-Leu-OMe. To study this further, the phospho-rylation products of several dipeptide methyl esters andtheir analogues were prepared. The sodium adducts wereselected and analyzed by ESI-MS/MS. The fragment ionof m/z 163 was observed in all of them, implying thatit could be formed in the MS/MS of any DIPP dipep-tide methyl ester with an ˛-amino acid residue at theN-terminus. (Fig. 1 and Table 1, Nos 1, 3, 4, 5, 6, 7, 8, 9,11, 15).

To clarify the constituent of the fragment of m/z 163, DIPPalanylbenzylamine (DIPP-Ala-BA) was analyzed by high-resolution ESI-MS/MS. The exact mass was 163.0129630,corresponding to C3H9O4PNa, which is consistent withthat of PAIE. The formation of PAIE is also supported bythe fragmentation of sodiated PAIE, with the appearanceof a peak at m/z 121, which resulted from the loss ofpropylene. From the element composition study of DIPP-Ala-BA (Scheme 2), it was found that there are only four

oxygen atoms including the carbonyl oxygen atom involvedin this molecule, which means that the carbonyl oxygenparticipated in the rearrangement reaction to form the ion ofm/z 163.

P

O

O

O

NH CH C

CH3

NH

O

CH2

Scheme 2. Structure of DIPP-Ala-BA.

The fragmentation route of DIPP dipeptide methyl estersis presented in Scheme 3. Almost every peak could be easilyidentified.

To test the possible role of sodium ion in the rearrange-ment, potassium and lithium ions were used to substitutefor sodium in 11 (Table 1, Nos 13 and 14). The peak at m/z179 or 147 was detected, respectively, which should be thecorresponding PAIE–metal ion adduct. It should be noted,however, that when a proton was substituted for the sodium(2 and 1), the corresponding rearrangement peak was notobserved. This result strongly suggested that the metal ionwas essential for the migration.

To explain all these results, a possible mechanism wasproposed (Scheme 4). First, a metal ion such as sodium

Copyright 2002 John Wiley & Sons, Ltd. J. Mass Spectrom. 2002; 37: 934–939

936 J. Chen et al.

Figure 1. ESI-MS/MS of DIPP-Leu-Leu-OMe and DIPP-Ile-Ala-OMe.

[M+Na]+

MSMS

PO

O

OH

OH

Na+

m/z 163

P

O

HOOH

OH

Na+

m/z 121

PO

OO

NH CHR1

C

Na+

O

NH CHR2

C

O

OCH3

−102

P N CH

O

OH

R1

C

Na+

O

NH CH

R2

C

O

OCH3 NH2 CH

R1

C

Na+

O

NH CH

R2

C

O

OCH3

−164

P

O

OOH

NH CH

R1

C

Na+

O

NH CH

R2

C

O

OCH3

−42

−42

PO

HOOH

NH CHR1

C

Na+

O

NH CHR2

C

O

OCH3

[M+Na-102]+ [M+Na-164]+

[M+Na-42]+

[M+Na-84]+

−18

PO

OHNC H

R1

C

Na+

O

NH CHR2

C

O

OCH3

[M+Na-102]+

−80

NH2 CH

R1

C

Na+

O

NH CH

R2

C

O

OCH3

[M+Na-164]+

Scheme 3. Fragmentation pathway of DIPP-X-Y-OMe.



-

P

OH

O

OH

O

Na+

m/z=163

P

O

O

O

NH CH

CNH

Na+

[M+Na]+

R1

O

CH

R2

C

O

OCH3

P

O

O

OH

NH CH

C

Na+

R1

O

NH CH

R2

C

O

OCH3

P

O

O

OHNH

CH

CONa+ - +

[M+Na-42]+

N

R2

CH C

O

OCH3

R1

H

P

O

O

OHNH

CH

CHONa

+ -+

N CH

R2

C

O

OCH3

R1

- NH

N CH

R2

C

O

OCH3

I

II

III

IV

Scheme 4. Possible rearrangement mechanism for DIPP-X-Y-OMe in mass spectrometry.

Figure 2. (A) ESI-MS/MS of the sodium adduct of deuterium-labeled DIPP-Ala-BA. (B) ESI-MS/MS/MS of the sodium adduct ofdeuterium-labeled DIPP-Ala-BA.

ion might coordinate with the phosphoryl oxygen andcarbonyl oxygen simultaneously to form a seven-memberedring. Then the intramolecular carbonyl oxygen attacks thephosphorus to form a five-membered pentacoordinatedphosphorus intermediate, which is very similar to thepentacoordinated phosphorus compounds that we trappedin previous work.12 Finally, with the migration of protonfrom imine to the oxygen, the five-membered ring mightbreak down by elimination of a neutral molecule to yieldthe sodium adduct of PAIE. To test the proton migration,DIPP-Ala-BA was deuterium labeled through exchange withCH3OD, and its sodium adduct was subjected to ESI-MSn.

The formation of an ion of m/z 164 in ESI-MS/MS/MS(Fig. 2(B)) showed that one deuterium atom was includedin the rearrangement product. This result is consistent withthe transfer of the active hydrogen atom in the amide groupto the phosphoryl group. The fact that no m/z 163 ion wasdetected in the MS/MS study of DIPP-Ala-OMe (17) andDIPP-Phe-OMe (18) also supported the proton migration,since no active proton could transfer to the phosphorylgroup in these cases.

Finally, to clarify the influence of the isopropyl group,DIPP alanylphenylalanine methyl ester (DMP-Ala-Phe-OMe,16), from which it would be difficult to lose a neutral molecule


938 J. Chen et al.

analogous to the propylene lost from the phosphoryl groupin the DIPP compounds, was analyzed by ESI-MS/MS. Theformation of a sodiated adduct of phosphoric acid dimethylester (PAME, m/z 149) revealed that the required eliminationof propylene before rearrangement was not due to theparticipation of the already formed hydroxyl group in therearrangement reaction, but reflected the need to reduce theinfluence of steric hindrance to facilitate the migration of thehydroxyl group (Fig. 3, Table 1).

To our knowledge, this is the first report of such a rear-rangement reaction in which carbonyl oxygen atom migratesto the phosphoryl group to form a hydroxyl group. Here,the strong affinity between phosphorus and oxygen atoms,which we proposed in a previous paper,11 might play an

important role in this specific rearrangement. Phosphoryla-tion in nature normally occurs on the hydroxyl group of theside-chain of serine, threonine and tyrosine, not the aminogroup. The strong affinity between phosphorus and oxy-gen shown here might provide some basic information forinterpretation.

With the introduction of ˇ-Ala to the N terminus ofdipeptides, DIPP-ˇ-Ala-Phe-OMe (8) and DIPP-ˇ-Ala-BA(10) were analyzed by ESI-MS/MS and compared with the˛-alanyl peptides. PAIE was not observed in the fragmentsof ˇ-alanyl peptides (Fig. 4), which seems reasonable sincethe increasing distance between the phosphoryl group andcarbonyl group made the attack of carbonyl oxygen onphosphorus difficult. In previous work, we found that the

Figure 3. ESI-MS/MS of the sodiated adduct of DMP-Ala-Phe-OMe.

Figure 4. Differentiation of ˛- and ˇ-alanine in the N-terminus of peptides.



˛- and ˇ-alanyl peptides presented the same fragmentationpattern in ESI-MS/MS, so it was difficult to differentiatethem with the normal MS/MS method. Using other methodssuch as NMR will face the difficulty of purification and therequirement for many more samples. The method presentedhere could be a solution for this purpose and might havesome applications in future work.

In conclusion, several dipeptide methyl esters and theiranalogues were derived from DIPP and analyzed by ESI-MS/MS. Under the activation of a metal ion, such assodium, potassium and lithium, the corresponding fragmentof the metal ion adduct of phosphoric acid isopropyl ester(PAIE) was formed, possibly via a specific migration of thecarbonyl group to the phosphoryl group through a five-membered ring intermediate. No migration was observed inthe absence of metal ions or when a ˇ-alanine was at theN-terminus. This could be used to develop a novel methodfor differentiating stereoisomers when either ˛- or ˇ-alanineis at the N-terminus of peptides. The strong affinity betweenphosphorus and oxygen in the gas phase, which has beenshown in this rearrangement reaction, might provide somebasic information on the nature of phosphorylation.

AcknowledgementsThe authors thank the Chinese National Natural Science Foundation(No. 20175026), the Ministry of Science and Technology, the visiting

scholar fund of the Chinese Education Ministry and TsinghuaUniversity for financial support.

REFERENCES1. Todd L. Science and Scientists: Essays by Biochemists, Biologists and

Chemists, 1 Japan Scientific Societies Press: Tokyo and Reidel:Dordrecht, 1981.

2. Yamashita M, Fenn JB. J. Phys. Chem. 1984; 88: 4671.3. Bauer MD, Sun YP, Wang F. J. Protein Chem. 1999; 18: 337.4. Wu HQ, Aboleneen H. Anal. Biochem. 2001; 290: 347.5. Miranker A, Robinson CV, Radford SE, Dobson CM. FASEB J.

1996; 10: 93.6. Krebs JF, Siuzdak G, Dyson HJ, Stewart JD, Benkovic SJ.

Biochemistry 1995; 34: 720.7. Bothner B, Chavez R, Wei J, Strupp C, Phung Q, Schneemann A,

Siuzdak G. J. Biol. Chem. 2000; 275: 13455.8. Chai WG, Yan L, Wang GH, Liang XY, Zhao YF, Ji GJ. Biomed.

and Environ. Mass Spectrom. 1987; 14: 331.9. Jiang Y, Fu H, Xu L, Lu Q, Wang JZ, Zhao YF. Rapid Commun.

Mass Spectrom. 2000; 14: 1491.10. Ji GJ, Xue CB, Zeng JN, Li LP, Chai WG, Zhao YF. Synthesis 1988;

6: 444.11. Chen J, Chen Y, Jiang Y, Fu H, Xin B, Zhao Y-F. Rapid Commun.

Mass Spectrom. 2001; 15: 1936.12. Fu H, Li ZL, Zhao Y-F, Tu GZ. J. Am. Chem. Soc. 1999; 121: 291.


a carbonyl oxygen migration in electrospray ionization mass spectrometry and its application in...

Documents