Vol. 1 (2012), A0010

Mass SPectrometrY DOI: 10.5702/massspectrometry.A0010

Original Article

N α Selective Acetylation of Peptides Toshiyuki Mikami,*,1 Toshifumi Takao,*,2 Kazunori Yanagi,1 and Hiroshi Nakazawa3 2

1 Genomic Science Laboratories, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, Osaka, Japan 3 Pharmaceutical Business Division, Sumika Chemical Analysis Service, Ltd., Osaka, Japan

A chemical tag at the peptide N-terminus, in combination with MS, can be useful for quantitative analysis, N-terminal peptide identification, or peptide sequencing. Here we report on the Nα selective acetylation of a peptide using acetic anhydride, a popular reagent for the modification of amino groups, without the need for the blocking of lysine side-chain ε-amino groups, which is usually required for Nα selective acetylation. By controlling the amount of acetic anhydride used and running the reaction at 0°C, it is possible to preferentially acetylate the α-amino group. As a typical application of the method, a tryptic digest of an N-terminally blocked protein, cytochrome c, was directly acetylated using the present method. When deuterated acetic anhydride was used as the reagent, the N-terminal blocked peptide could be easily identified as a non-labeled ion peak while the Nα-acetyl groups of all the other peptides were deuterated. Keywords: acetylation, isotope-labeling, peptide, protein (Received September 26, 2012; Accepted October 10, 2012)

INTRODUCTION A chemical tag by labeling with a stable isotope has been widely used for quantitative proteomics and peptide sequencing.1) Various chemical tags that can react with amino groups of peptides and proteins using commercially available reagents such as N-acetoxysuccinamide, 2) acetic anhydride,3–5) chlorosulfonylacetyl chloride,6) etc. have been reported. Among them, acetic anhydride has the advantage of volatility, which allows for the direct MS analysis of a reaction mixture. In addition, Nα selective acetylation has been successfully applied in the area of proteomic analyses.3–5) The α- and ε-amino groups of a protein have slightly different pKa values (9.06 and 10.54, respectively). Thus, ε-amino groups are more likely to be protonated than α-amino groups in aqueous solvent, leading to a lower nucleophilicity of ε-amino groups. It has been reported that Nα selective acetylation can be achieved, when the reaction is run at neutral pH, whereas, at higher or lower pH, both types of amino groups are equally acetylated, since their degrees of protonation are the same.1) In our previous study, however, the concentration of acetic anhydride, as well as the pH of the reaction mixture, was also found to significantly affect the Nα-selectivity of the reaction.7,8) We report herein on the optimizing of Nα selective acetylation using acetic anhydride, resulting in an Nα selectivity of more than 90%.

EXPERIMENTAL Materials

Dynorphin A was purchased from the Peptide Institute

* Correspondence to: Toshiyuki Mikami, Genomic Science Laboratories, Dainippon Sumitomo Pharma Co., Ltd., 3–1–98 Kasugade-naka, Konohanaku, Osaka 554–0022, Japan, e-mail: [email protected]; Toshifumi Takao, Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, 3–2 Yamadaoka, Suita, Osaka 565–0871, Japan, e-mail: [email protected]

© 2012 The Mass Spectrometry Society of Japan

(Osaka, Japan). Sequencing grade-modified trypsin was from Roche Diagnostics (Mannheim, Germany). Cytochrome c, calmodulin, fibrinopeptide B, acetic anhydride-d 6, α-cyano-4-hydroxycinnamic acid (CHCA) and acetonitlile were obtained from Sigma (St. Louis, MO). Phenyl isocyanate was purchased from Wako Pure Chemical Industry, Co. (Osaka, Japan).

N-Terminal selective acetylation

Peptides were dissolved in 12 μL of 0.1% acetic acid (pH 3.3), or 0.1 M pyridine-acetic acid buffer (pH is adjusted to 4.5 and 6.0). After incubation on an ice bath for 1 min, 5 μL of an appropriate concentration of acetic anhydride in anhydrous THF or acetonitrile was added to the peptide solution. The solution was voltexed and incubated on an ice bath for 5 min. The derivatized peptides were then dried by means of a SpeedVac.

Tryptic digestion of a protein

Cytochrome c (200 mg) was dissolved in 200 μL of 50 mM ammonium bicarbonate solution and digested by treatment with 4 mg of chymotrypsin at 37°C for 18 h. An aliquot of the solution was dried using a SpeedVac and used for acetylation.

Mass spectrometry

Fast-atom bombardment (FAB) mass spectra and MS/MS spectra were obtained with a JEOL JMS-HX/HX110A tandem mass spectrometer at an accelerating voltage of 10 kV and a mass resolution of 1 : 1000 (10% valley). Samples were mixed with the matrix (glycerol–thioglycerol, 1 : 1, v/v) and were ionized by bombardment with 6-keV xenon atoms. FAB mass spectra were acquired with MS-1 and processed by a JEOL MP-8020D data system. For tandem mass spectrometry, the precursor ions selected by MS-1 were collided with argon in a collision cell located between MS-1 and MS-2, and the product ions were then analyzed in MS-2 with a JEOL array detector. The collision cell potential was Page 1 of 3 (page number not for citation purpose)

N α SeLectIve AcetYL atIon oF PePtIDes

Fig. 1.

Fig. 2.

Vol. 1 (2012), A0010

Acetylation of a peptide (YGGFLRRIRPKLKWDNQ) with acetic anhydride. The peptide was reacted with various concentrations of acetic anhydride in a solution at pH 6 at 0°C. FAB mass spectra were acquired without A) and after incubation for 5 min with 0.01 M (B), 0.1 M (C) and 1 M (D) of acetic anhydride.

Nα Selectivity for the acetylation of a peptide (YGGFLRRIRPKLKWDNQ) as a function of the concentration of acetic anhydride under acidic conditions at 0°C. The values indicated in the graph denote the pH of the sample solution before the addition of acetic anhydride. Nα Selectivity (%) was calculated as 100×Intensity[M+1Ac+H] /(Int. [M+H] +Int. [M+1Ac+H] +Int. [M+2Ac+H] +Int. [M+3Ac+H] ). +

+

+

+

+

held at 8.0 kV. The collision gas was introduced into the collision chamber at a pressure sufficient to reduce the precursor ion signal by 60%. Electrospray mass spectra and MS/MS spectra were obtained with a Micromass QTOF2 quadruple time-of-flight mass spectrometer (Manchester, UK). Samples were dissolved in a 1 : 1 (v/v) ratio of water and methanol with 1% acetic acid and were infused into the MS using nanospray ion source. The instrument was operated in the positive ion mode with a capillary-skimmer voltage of 1.8 kV.

RESULTS AND DISCUSSION Dynorphin A (YGGFLRRIRPKLKWDNQ), as a Nterminally free peptide with two lysine residues, was used for optimizing the Nα selective acetylation with acetic anhydride. The peptide (1 nmol) was dissolved in 12 μL of © 2012 The Mass Spectrometry Society of Japan

Fig. 3.

Specific detection of a blocked N-terminal peptide in a tryptic digest of cytochrome c by ESI-MS. The digest was subjected to Nα selective acetylation at pH 3.3 with 0.5 M equimolar mixture of d0- and d6-acetic anhydride. All the internal peptides showed doublet peaks with a 3 Da space derived from d3-acetyl groups, while the originally blocked N-terminal peptide, observed at m/z 589.2, remained intact.

0.1 M pyridine-acetate buffer (pH 6.0), and 5 μL of 0.01 M, 0.1 M and 1 M acetic anhydride in anhydrous THF was added. After reaction for 5 min on ice, an aliquot (1 μL) of the solution was subjected to FAB mass spectrometry. It is worth mentioning that the degree of acetylation increased as a function of the concentration of acetic anhydride (Figs. 1 and 2). The use of 0.02 M acetic anhydride at pH 6.0 resulted in a single acetylation with 70% Nα selectivity, the site of which was determined to be the N-terminal position by MS/ MS of the acetylated form (MH+ at m/z 2189.2). Increasing the concentration of acetic anhydride at pH 6.0 resulted in the acetylation of other sites, and 1 M acetic anhydride eventually resulted in the acetylation of all the three amino groups of dynorphin A. Decreasing the pH to 3.3 led to a lower dependency of the concentration of acetic anhydride on Nα selectivity; acetylation at pH 3.3 gave more than an 80% Nα selectivity over a wide range of acetic anhydride concentrations (0.2–1 M) and the highest Nα selectivity (90%) with 0.5 M acetic anhydride (Fig. 2). These results demonstrate that Nα-selectivity depends on not only the pH of the solvent but also the concentration of acetic anhydride used. The above method could be used to identify an N-blocked peptide in a peptide mixture. Cytochrome c, an N-terminally blocked (acetylated) protein, was subjected to tryptic digestion without protecting the ε-amino groups of its lysine residues. The digest (50 pmol) was dissolved in 0.1% aqueous acetic acid and treated with a 1 : 1 mixture of 0.5 M d 0-acetic anhydride and d6-acetic anhydride in acetonitrile for 5 min on ice, which allowed for the peptides modified with the reagents to be differentiated from the intact ones by MS. The ion peaks observed as doublet peaks with 3 Da spacing corresponded to the internal peptides, while the peak at m/z 589.2, which showed a natural isotopic distribution, could be assigned to the N-terminal peptide (Ac-GDVEK) of cytochrome c (Figs. 3, 4). The Nα selective acetylation procedure described here is an easy-to-use protocol and is useful for the facile identification of an N-blocked peptide(s), even when Lys residues are involved, in a peptide mixture or a Page 2 of 3

N α SeLectIve AcetYL atIon oF PePtIDes

Vol. 1 (2012), A0010

REFERENCES 1) 2)

3)

Fig. 4.

ESI-MS/MS spectrum from MH+ at m/z 589.2 in Fig. 3. The backbone fragment ions proved the site of acetylation as well as the sequence.

4)

5)

protein digest when carried out in combination with stableisotope labeling.

CONCLUSION Nα Selective acetylation with acetic anhydride is a simple and rapid method, which does not require any pre-treatment or purification prior to mass measurement. It could be useful in a variety of protein analyses such as amino acid sequencing and quantitative proteomics.

© 2012 The Mass Spectrometry Society of Japan

6)

7)

8)

F. E. Regnier, S. Julka. Primary amine coding as a path to comparative proteomics. Proteomics 6: 3968–3979, 2006. P. Liu, F. E. Regnier. An isotope coding strategy for proteomics involving both amine and carboxyl group labeling. J. Proteome Res. 1: 443–450, 2002. C. Lemmel, S. Weik, U. Eberle, J. Dengjel, T. Kratt, H. D. Becker, H. G. Rammensee, S. Stevanovic. Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling. Nat. Biotechnol. 22: 450–454, 2004. M. J. Noga, J. J. Lewandowski, P. Suder, J. Silberring. An enhanced method for peptides sequencing by N-terminal derivatization and MS. Proteomics 5: 4367–4375, 2005. M. J. Noga, A. Asperger, J. Silberring. N-Terminal H3/D3acetylation for improved high-throughput peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer. Rapid Commun. Mass Spectrom. 20: 1823–1827, 2006. T. Keough, R. S. Youngquist, M. P. Lacey. A method for highsensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 96: 7131–7136, 1999. T. Mikami, K. Yanagi, H. Nakazawa. Proceedings of the 48th ASMS Conference on Mass Spectrometry and Allied Topics, Long Beach, CA, 2000. T. Mikami, K. Yanagi, H. Nakazawa. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, 2001.

Page 3 of 3

N (α) Selective Acetylation of Peptides.

A chemical tag at the peptide N-terminus, in combination with MS, can be useful for quantitative analysis, N-terminal peptide identification, or pepti...
570KB Sizes 0 Downloads 0 Views