Peptide sequencing through N-terminal phosphonylation and electrospray ionization mass spectrometry

Article abstract of DOI:10.1002/jms.850

Peptides were phosphonylated at their N-termini by reacting with ethoxyphenylphosphinate in the presence of triethylamine and tetrachloromethane under mild conditions. The phosphonylated peptides were analyzed by tandem electrospray ionization mass spectr

Full text of DOI:10.1002/jms.850

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 2005; 40: 772–776
Published online 9 May 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.850
Peptide sequencing through N-terminal
phosphonylation and electrospray ionization mass
spectrometry
Jiangyin Bao,1 Huiwang Ai,1 Hua Fu,1∗ Yuyang Jiang,1,2 Yufen Zhao1 and Cheng Huang1
1
Key Laboratory for Bioorganic Phosphorus Chemistry of Education Ministry, Department of Chemistry, Tsinghua University, Beijing 100084, P. R.
China
2
Key Laboratory of Chemical Biology, Guangdong Province, Graduate School of Shenzhen, Tsinghua University, Shenzhen 518057, P. R. China
Received 16 December 2004; Accepted 15 February 2005
Peptides were phosphonylated at their N-termini by reacting with ethoxyphenylphosphinate in the
presence of triethylamine and tetrachloromethane under mild conditions. The phosphonylated peptides
were analyzed by tandem electrospray ionization mass spectrometry. N-Terminal phosphonylation
selectively increased the intensities of bn-type ions relative to other ion types. The resulting simplified
mass spectra clearly show the sequential loss of amino acid residues from the C-termini of peptides,
providing a convenient and rapid method for peptide sequencing. Copyright  2005 John Wiley & Sons,
Ltd.
KEYWORDS: peptide sequencing; electrospray ionization mass spectrometry; phosphonamidate peptides; N-terminal
phosphonylation; ethoxyphenylphosphinate
using a carboxyl-group activating reagent and thiocyanate
anion.8,9 C-Terminal sequencing of peptides has also been
achieved through enzymatic digestion of peptides with car-
boxypeptidase Y followed by MALDI-MS analysis.10,11 These
methods, however, require pure peptide samples and chem-
ical or enzymatic degradation prior to mass spectrometry,
making them inconvenient for small amounts of peptides
especially peptide mixtures.
INTRODUCTION
Primary sequences of proteins are traditionally determined
by cleaving a protein into smaller peptide fragments and
sequencing the peptides using a variety of methods.1 The
most widely practiced method has been the Edman method,2
which degrades a peptide sequentially from its N-terminus
to release phenylthiohydantoin derivatives which are sub-
sequently analyzed by HPLC. The advent of soft ionization
techniques such as fast atom bombardment (FAB),3 matrix-
assisted laser desorption/ionization (MALDI)4,5 and electro-
spray ionization (ESI),6 which make it possible to generate
intact macromolecular ions in the gas phase, has led to the
development of numerous mass spectrometric approaches
to obtain sequence and structural information of biolog-
ical molecules. For example, Chait et al.7 have developed
a modified Edman degradation method to convert a pep-
tide to be sequenced into a series of progressively shorter
peptides, which are analyzed by MALDI-MS to obtain the
sequence of the original peptide (peptide ladder sequenc-
ing). Boyd et al. developed a C-terminal peptide sequencing
method analogous to Edman degradation by converting the
C-terminal amino acid of a peptide into a thiohydantoin
Tandem MS equipped with soft ionization methods
provides an increasingly popular alternative approach
for peptide characterization. A peptide mixture can be
separated during the first stage of mass spectrometry; during
the next stages, the selected peptide ion is fragmented
by either collision-induced dissociation (CID) or surface-
induced dissociation (SID).12 The resulting MS/MS spectrum
can be used to deduce the amino acid sequence of the original
peptide. Unfortunately, a protonated peptide is typically
dissociated into a wide variety of fragment ions including
the an, bn, and cn ions that correspond to the N-terminal
fragments and the xn, yn, and zn ions that represent the C-
terminal fragments.13,14 In general, it is difficult to predict
which type of fragment ions will be formed for a given
peptide, and the MS/MS spectra are often so complicated
that de novo sequence determination is impossible. It has
previously been shown that alkali-cationized peptides can
be successively dissociated from their C-termini in magnetic
sector mass spectrometers,15–22 providing opportunities for
peptide sequencing. The alkali-cationized peptides can also
be cleaved sequentially from their C-termini to yield a series
of [bn C Na C OH]C ions in ion-trap instruments using low
collision energies.23,24 This method is analogous to Edman
ŁCorrespondence to: Hua Fu, Key Laboratory for Bioorganic
Phosphorus Chemistry of Education Ministry, Department of
Chemistry, Tsinghua University, Beijing 100084 P. R. China.
E-mail: fuhua@mail.tsinghua.edu.cn
Contract/grant sponsor: Excellent Dissertation Foundation of the
Chinese Ministry of Education; Contract/grant number: No.
200222.
Contract/grant sponsor: Excellent Young Teacher Program of
MOE, P. R. C., and the National Natural Science Foundation of
China; Contract/grant number: Grant No. 20472042.
Copyright  2005 John Wiley & Sons, Ltd.

Peptide N-terminal phosphonylation 773
degradation, except that the cleavage occurs from the C-
terminus instead of the N-terminus, making it suitable
for sequencing N-terminally blocked peptides. However,
some drawbacks such as the lower sensitivity of cationized
peptides and the still-very-complex dissociation pathways
render this method impractical.23–28
Chemistry
31P NMR chemical shifts were reported in ppm downfield
(C) or upfield (–) from external H3PO4 (85%) reference.
Ethoxyphenylphosphinyl and phosphonamidyl peptides
were prepared as previously described.35
Many efforts have been made to simplify the mass
spectra by modifying peptides with either positively or
negatively charged groups. For positive derivatization, a
positively charged group such as trimethylammoniumacetyl
or tris[(2,4,6-trimethoxypheny)phosphonium]acetyl is intro-
duced to the N-terminus of a peptide.29,30 Similarly, a
negatively charged group such as the sulfonyl group and
the 4-aminonaphthalenesulfonyl group has been added to
the N- and C-termini of peptides, respectively.31,32 We have
previously found that the ionization efficiency of peptides
can be greatly improved by introducing a neutral dialky-
loxyphosphoryl group to their N-termini in positive-ion
ESI-MS33 and FAB-MS.34 The dissociation efficiency also
seemed to be higher; unfortunately, a variety of dissoci-
ation pathways of the phosphoryl group complicated the
mass spectra and sequence interpretation. We reasoned that,
since the P-C bond in phosphonamidate is relatively stable
under low-collision activation conditions, an N-terminally
phosphonamidylated peptide should have less fragmenta-
tion and therefore simpler MS/MS spectra. In this work, we
show that peptides can be phosphonylated at their N-termini
and their sequences can be unambiguously determined by
ESI-MS/MS.
Synthesis of ethoxyphenylphosphinate (EPP)
Ethanol (5 mmol) was added dropwise to 2 mmol of
dichloro(phenyl)phosphine dissolved in 4 mL of ethyl ether
at room temperature under nitrogen atmosphere. The
reaction completed within 30 min, and 31P NMR spectrum
showed that DCPP was almost quantitatively converted
intoEPP. The crude product was obtained after removal
of HCl, ethyl ether, and ethanol by distillation and was used
without further purification (1H NMR shows that its purity
is more than 95%). 31P NMR of EPP: 26.99 ppm.
General procedure for synthesis of phosphonamidate
peptides
Peptide (¾1 mg) was dissolved in 100 µL of triethylamine,
800 µL of ethanol and 100 µL of tetrachloromethane and
°
was treated with 1.2–1.5 equiv of EPP (¾20 µL) at ꢀ5 C.
The resulting solution was stirred for 45 min and directly
analyzed by ESI-MS/MS.
Mass spectrometric conditions
Mass spectra were recorded on a Bruker ESQUIRE ¾LC
ion-trap spectrometer equipped with a gas nebulizer probe.
Nitrogen gas was used for drying at a flow rate of 4 L/min.
The nebulizer gas fore-pressure was 7 psi. The electrospray
capillary was typically held at 4 kV. Samples were dissolved
in ethanol and ionized by electrospray ionization. The scan
range was from m/z 100 to 1000 in positive-ion. The selected
ions [M C H]C were analyzed by multistage tandem mass
spectrometry through collision with helium.
EXPERIMENTAL
Materials
All peptides include Ala-Gly-Phe-Leu-ValOH (MW 505),
Leu-Gly-Phe-Ala-ValOH (MW 505), Gly-Leu-Val-Ala-
PheOH (MW 505), Phe-Phe-Phe-Phe-PheOH (MW 753), Phe-
Ala-Ser-Asp-LeuOH (MW 551), Val-Ala-Ser-Phe-LeuOH
(MW 535), Val-Glu-Gln-HisOH (MW 511), Leu-Glu-His-
GlnOH (MW 525) and Val-Arg-Leu-Asp-Ser-PheOH (MW
735) were commercially available from Shanghai Glsyn-
thesis Company (¾80% pure). The peptides were used in
phosphonylation and ESI-MS/MS analysis without further
purification. Dichloro(phenyl)phosphine (DCPP) was pur-
chased from Beijing Chemical Factory (Beijing). Ethanol was
rendered anhydrous by refluxing with Mg.
RESULTS AND DISCUSSION
N-terminally ethoxyphenylphosphinylated peptides were
prepared by treating the free peptides with 1.2 to 1.5
equiv of EPP in a mixed solvent containing triethylamine,
tetrachloromethane and ethanol (Scheme 1).35 The resulting
solution was directly analyzed by ESI-MS/MS.
The mass spectra of the modified peptides showed
intense molecular ions (M C HCꢀ usually as the base
peaks. The protonated molecular ions were selected as
precursor ions for CID fragmentation. ESI-MS/MS showed
Scheme 1. Synthetic route of phosphonamidate peptides.
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 772–776

774
J. Bao et al.
much simpler fragmentation pattern relative to those of
unmodified peptides. The spectra are usually dominated
by the bn ions, corresponding to the modified N-terminal
fragments. This made sequence interpretation trivial and
unambiguous in most cases. For example, Fig. 1 shows
the ESI-MS2 mass spectrum of peptide EPP-Ala-Gly-Phe-
Leu-Val-OH. The spectrum consists of the [M C H]C ion
at m/z 674, the [M C H ꢀ H2O]C ion at m/z 656, b4 ion
([M-Val-16]C) at m/z 557, b3 ion ([M C H ꢀ LeuValOH]C)
at m/z 444, and b2 ion ([M C H ꢀ PheLeuValOH]C) at
m/z 297. The spectrum is essentially devoid of any other
peaks and therefore the peptide sequence Phe-Leu-Val-
OH is unambiguously assigned on the basis of the mass
differences between adjacent bn ions. Next, the b2 ion
at m/z 297 ([M C H ꢀ PheLeuValOH]C) was selected for
further fragmentation to obtain the ESI-MS3 spectrum, which
displayed an intense peak at m/z 240 (data not shown). This
indicates the loss of a glycyl residue and the remaining
fragment ion corresponds to [EPP-Ala]C. Two small satellite
peaks at m/z 416 and 269 were most likely due to loss
of ethylene (from the phosphonyl group) or CO (from
amide) from b3 and b2 ions, respectively (Fig. 1). Therefore,
the complete amino acid sequence of the phosphonylated
pentapeptide was determined as EPP-Ala-Gly-Phe-Leu-Val-
OH.
of LeuOH, AspLeuOH, SerAspLeuOH, and Ala-Ser-Asp-
LeuOH from the original peptide, respectively. We also
observed intense peaks corresponding to [b3 C 1]C at m/z
475 and [b2 C 1]C at m/z 388, although their mechanism
of formation is currently unknown. More impressively, the
ESI-MS2 spectrum of peptide EPP-Val-Glu-Gln-HisOH pro-
duced a complete set of the bn fragment ions at m/z 680, 525,
397, and 268 (corresponding to [M C H]C , b3, b2, and b1 ion,
respectively), but was still relatively free of other fragment
ions (Fig. 3). In addition, an intense ion [EPP C Val ꢀ CO]C
at m/z 240 was observed.
Mass spectra of arginine-containing peptides often show
peculiar results due to the presence of a guanidino group
on its side chain.36 In addition, some investigators reported
that peptides with Asp usually displayed strong signals
resulting from cleavage at the carboxy-side of Asp in mass
spectrometry.24,37 We chose to determine the sequence of
peptide Val-Arg-Leu-Asp-Ser-PheOH, which contains both
arginyl and aspartyl residues. The peptide was N-terminally
phosphonylated and then analyzed by ESI-MS/MS. The ESI-
MS2 spectrum (Figure 4) shows [M C H]C at m/z 904, b5
at m/z 739, b4 at m/z 652, b3 at m/z 537, and b2 at m/z
424. In addition, ions corresponding to [b3-17]C (m/z 520)
and [b2-17]C (m/z 407) were observed, which are formed
due to loss of NH3 from the arginine guanidino group. The
b4 ion (m/z 652) was the base peak, which is in agreement
with the previous reports by other investigators.24,36 The ESI-
MS3 spectrum of the b4 ion (m/z 652) displayed the similar
fragment ions to ESI-MS of the original molecule (Figure 5),
which allowed the entire peptide sequence to be determined.
We also determined the sequences of other peptides con-
taining special amino acid residues, for example, EPP-Phe-
Ala-Ser-Asp-LeuOH and EPP-Val-Glu-Gln-HisOH. Figure 2
shows the ESI-MS2 spectrum of peptide EPP-Phe-Ala-Ser-
Asp-LeuOH. A series of fragment ions resulting from sequen-
tial loss of C-terminal amino acid residues were observed
at m/z 702, 589, 474, 387, and 316, corresponding to loss
557.2
b4
6000
4000
b3
444.2
[M+H]+
656.3
b2
297.1
2000
0
200
250
300
400
450
m/z
500
550
600
650
350
Figure 1. ESI-MS2 spectrum of the protonated molecule [M C H]C at m/z 674 of EPP-Ala-Gly-Phe-Leu-ValOH.
b4
589.1
1250
1000
750
500
250
0
702.3
571.1
b1
b3
b2
474.1
[M+H]+
316.1
388.3
200
300
400
500
m/z
600
700
Figure 2. ESI-MS2 spectrum of the protonated molecule [M C H]C at m/z 720 of EPP-Phe-Ala-Ser-Asp-LeuOH.
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 772–776

Peptide N-terminal phosphonylation 775
240.1
397.3
a1
b2
×104
1.00
b3
525.2
0.75
0.50
0.25
0.00
[M+H]+
b1
268.1
662.3
680.2
200
250
300
350
400
450
m/z
500
550
600
800
600
650
Figure 3. ESI-MS2 of the protonated molecule [M C H]C at m/z 680 of EPP-Val-Glu-Gln-HisOH.
b4
652.3
1500
1000
500
0
[M+H]+
b2
424.2
904.4
b3
537.2
b5
739.4
200
300
400
500
600
m/z
700
900
Figure 4. ESI-MS2 of the protonated molecule at m/z 904 of EPP-Val-Arg-Leu-Asp-Ser-PheOH.
407.1
b4
652.3
250
200
150
100
50
424.2
b2
a1
240.0
b3
537.3
0
250
300
350
400
450
m/z
500
550
650
Figure 5. ESI-MS3 spectrum of b4 ion at m/z 652 in Fig. 4.
2. Wang P, Arabaci G, Pei D. Rapid sequencing of library-derived
peptides by partial Edman degradation and mass spectrometry.
J. Comb. Chem. 2001; 3: 251.
3. Barber M, Bordoli RS, Sedgwick RD, Tyler AN, Bycroft BW. Fast
atom bombardment mass spectrometry of bleomycin A2 and B2
and their metal complexes. Biochem. Biophy. Res. Commun. 1981;
101: 632.
4. Karas M, Bachmann D, Bahr U, Hillenkamp F. Matrix-assisted
ultraviolet laser desorption of non-volatile compounds. Int. J.
Mass Spectrom. Ion Proc. 1987; 78: 53.
5. Hillenkamp, F. Laser desorption mass spectrometry:
mechanisms, techniques, and applications. Adv. Mass Spectrom.
1989; 11A: 354.
CONCLUSION
Peptides with free N-termini were readily converted into the
corresponding N-terminally phosphonamidylated peptides
by treatment with EPP under mild conditions. MS/MS of
the resulting peptide derivatives produced predominantly
the bn-type ions and much simplified spectra that allow
ready sequence determination of the original peptide. This
provides a rapid, convenient, and general method for peptide
sequence determination.
Acknowledgements
This work was supported by the Excellent Dissertation Foundation
of the Chinese Ministry of Education (No. 200222), the Excellent
Young Teacher Program of MOE, P. R. C., and the National Natural
Science Foundation of China (Grant No. 20472042).
6. Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse
CM. Electrospray ionization for mass spectrometry of large
biomolecules. Science 1989; 246: 64.
7. Chait BT, Wang R, Beavis RC, Kent SBH. Protein ladder
sequencing. Science 1993; 262: 89.
8. Samyn B, Hardeman K, Van der Eycken J, Van Beeumen J.
Applicability of the alkylation chemistry for chemical C-terminal
protein sequence analysis. Anal. Chem. 2000; 72: 1389.
REFERENCES
1. Gygi SP, Aebersold R. Mass spectrometry and proteomics. Curr.
Opin. Chem. Biol. 2000; 4: 489.
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 772–776

776
J. Bao et al.
9. BoydVL, Bozzini M, ZonG, Noble RL, Mattaliano RJ. Sequencing
of peptides and proteins from the carboxy terminus. Anal.
Biochem. 1992; 206: 344.
10. Patterson DH, Tarr GE, Regnier FE, Martin SA. C-terminal
ladder sequencing via matrix-assisted laser-desorption mass-
spectrometry coupled with carboxypeptidase-y time-dependent
and concentration-dependent digestions. Anal. Chem. 1995; 67:
3971.
11. Thiede B, Wittmannliebold B, Bienert M, Krause E. Maldi-ms
for C-terminal sequence determination of peptides and proteins
degraded by carboxypeptidase-y and carboxypeptidase-P. FEBS
Lett. 1995; 357: 65.
Sodiated Gas-Phase Peptide Ions Adjacent to Aspartic Acid
Residues. J. Am. Chem. Soc. 1998; 120: 3188.
25. Li HB, Siu KWM, Guevremont R, LeBlanc JCY. Complexes of
silver(I) with peptides and proteins as produced in electrospray
mass spectrometry. J. Am. Soc. Mass Spectrom. 1997; 8: 781.
26. Gonzalez J, Besada V, Garay H, Reyes O, Padron G, Tambara Y,
Takao T, Shimonishi Y. Effect of the position of a basic amino
acid on C-terminal rearrangement of protonated peptides upon
collision-induced dissociation. J. Mass Spectrom. 1996; 31: 150.
27. Nemirovskiy OV, Gross ML. Complexes of iron(II) with cysteine-
containing peptides in the gas phase. J. Am. Soc. Mass Spectrom.
1996; 7: 977.
12. Lee SW, Kim HS, Beauchamp JL. Salt bridge chemistry applied
to gas-phase peptide sequencing: Selective fragmentation of
sodiated gas-phase peptide ions adjacent to aspartic acid
residues. J. Am. Chem. Soc. 1998; 120: 3188.
13. Sundqvist B, Kamensky I, Haakansson P, Kjellberg J, Salehpour
M, Widdiyasekera S, Fohlman J, Peterson PA, Roepstorff
P. California-252 plasma desorption time of flight mass
spectroscopy of proteins. Biomed. Mass Spectrom. 1984; 11: 242.
14. Biemann K. Mass spectrometry of peptides and proteins. Annu.
Rev. Biochem. 1992; 61: 977.
15. Mallis LM, Russell DH. Fast atom bombardment–tandem mass
spectrometry studies of organo-alkali metal ions of small
peptides. Anal. Chem. 1986; 58: 1076.
16. Tang X, Ens W, Standing KG, Westmore JB. Daughter ion mass
spectra from cationized molecules of small oligopeptides in a
reflecting time-of-flight mass spectrometer. Anal. Chem. 1988; 60:
1791.
17. Renner D, Spiteller G. Linked scan investigation of
peptide degradation initiated by liquid secondary ion mass
spectrometry. Biomed. Mass Spectrom. 1988; 15: 75.
18. Grese RP, Cerny RL, Gross ML. Metal ion-peptide interactions
in the gas phase: a tandem mass spectrometry study of alkali
metal cationized peptides. J. Am. Chem. Soc. 1989; 111: 2835.
19. Grese RP, Gross ML. Gas-phase interactions of lithium ions and
dipeptides. J. Am. Chem. Soc. 1990; 112: 5098.
20. Lin T, Payne AH, Glish GL. Dissociation pathways of alkali-
cationized peptides: Opportunities for C-terminal peptide
sequencing. J. Am. Soc. Mass Spectrom. 2001; 12: 497.
21. Teesch LM, Adams J. Intrinsic interactions between alkaline
earth metal ions and peptides: a gas-phase study. J. Am. Chem.
Soc. 1990; 112: 4110.
28. Newton KA, McLuckey SA. Generation and manipulation of
sodium cationized peptides in the gas phase. J. Am. Soc. Mass
Spectrom. 2004; 15: 607.
29. Mamer OA, Just G, Li CS, Preville P, Watson S, Young R, Yergey
JA. Enhancement of mass-spectrometric detection of ltc4, ltd4,
and lte4 by derivatization. J. Am. Soc. Mass Spectrom. 1994; 5: 292.
30. Huang ZH, Wu J, Roth KDW, Yang Y, Gage DA, Watson JT.
A Picomole-Scale Method for Charge Derivatization of Peptides
for Sequence Analysis by Mass Spectrometry. Anal. Chem. 1997;
69: 137.
31. Keough T, Youngquist RS, Lacey MP. A method for high-
sensitivity peptide sequencing using postsource decay matrix-
assisted laser desorption ionization mass spectrometry. Proc.
Natl. Acad. Sci. USA 1999; 96: 7131.
32. Lindh I, Sjo¨vall J, Bergman T, Griffiths WJ. Negative-ion
electrospray tandem mass spectrometry of peptides derivatized
with 4-aminonaphthalenesulphonic acid. J. Mass spectrum. 1998;
33: 988.
33. Chen J, Chen Y, Gong P, Jiang Y, Li YM, Zhao YF. Novel
phosphoryl derivatization method for peptide sequencing by
electrospray ionization mass spectrometry. Rapid Commun. Mass
Spectrom. 2002; 16: 531.
34. Chai WG, Yan L, Wang GH, Liang XY, Zhao YF, Ji GJ.
Improvement in sensitivity of fast atom bombardment mass
spectrometry of amino acids by di-isopropylphosphorylation.
Biomed. Environ. Mass Spectrom. 1987; 14: 331.
35. Ai HW, Fu H, Zhao YF. Novel synthetic method of
phosphonamidate peptides and its application in peptide
sequencing via multistage mass spectrometry. Chem. Commun.
2003;; 2724.
36. Tsaprailis G, Somogyi A, Nikolaev EN, Wysocki VH. Refining
the model for selective cleavage at acidic residues in arginine-
containing protonated peptides. Int. J. Mass. Spectrom. 2000; 195:
467.
22. Teesch LM, Adams J. Fragmentations of gas-phase complexes
between alkali metal ions and peptides: metal ion binding to
carbonyl oxygens and other neutral functional groups. J. Am.
Chem. Soc. 1991; 113: 812.
37. Gu CG, Tsaprailis G, Breci L, Wysocki VH. Selective gas-phase
cleavage at the peptide bond terminal to aspartic acid in fixed-
charge derivatives of asp-containing peptides. Ana. Chem. 2000;
72: 5804.
23. Lin T, Glish GL. C-Terminal Peptide Sequencing via Multistage
Mass Spectrometry. Anal. Chem. 1998; 70: 5162.
24. Lee S-W, Kim HS, Beauchamp JL. Salt Bridge Chemistry Applied
to Gas-Phase Peptide Sequencing: Selective Fragmentation of
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 772–776