Enhanced Ionization of Phosphopeptide Using Ammonium Phosphate as Matrix Additive by MALDI-MS

Chinese Journal of Chemistry, 2008, 26, 1863—1869 Full Paper

* E-mail: [email protected]; Tel.: 0086-021-54237416; Fax: 0086-021-54237961 Received November 9, 2007; revised January 18, 2008; accepted June 17, 2008. Project supported by the National Science and Technology Key Project (Nos. 2007CB914100, 2009CB825607 and 2006AA02Z134), Shanghai

Leading Academic Discipline B109, the National Natural Science Foundation of China (Nos. 20405003, 30672394 and 30530040), Program for New Century Excellent Talents in University (NCET-06-0360) and Shanghai Rising-Star Program (No. 06QA14004).

© 2008 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Enhanced Ionization of Phosphopeptide Using Ammonium Phosphate as Matrix Additive by MALDI-MS

ZHANG, Ying(张莹) LU, Hao-Jie*(陆豪杰) YANG, Peng-Yuan(杨芃原)

Department of Chemistry and Institute of Biomedical Sciences, Fudan University, Shanghai 200433, China

A new method based upon adding ammonium phosphate as a matrix additive to enhance the ionization effi-ciency of phosphopeptide in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was de-scribed. Furthermore, influences of different phosphate salts at various concentrations on the ionization efficiency of phosphopeptide were investigated systematically, finding that the signal intensity for phosphopeptide 48FQ[pS]EEQQQTEDELQDK63 digested from β-casein were 5 to 8 times increased in an optimized condition with 10 mmol/L ammonium monobasic phosphate or 3 to 4 times increased with 10 mmol/L ammonium dibasic phosphate as additive to matrix 2,5-dihydroxybenzoic acid, respectively. Compared with the most optimized matrix system that was currently reported for special ionization of phosphopeptides, the signal intensity of this phos-phopeptide was also enhanced by twice when introducing 5 mmol/L ammonium dibasic phosphate into matrix 2,4,6-trihydroxyacetophenone. In addition, the mechanism was also discussed, assuming that the cooperation func-tion of ammonium cation and phosphate anion was of great importance in enhancing the ionization efficiency of phosphopeptide in MALDI-MS.

Keywords phosphopeptide, ionization, MALDI, mass spectrometry

Introduction

Protein phosphorylation has increasingly become the focus of attention in cell biology and biomedical sci-ences because it is intimately involved in many impor-tant physiological functions.1-3 Phosphorylation is in-volved in the storage of phosphate in proteins and bind-ing of metal ions4 and plays a critical role in transmit-ting extracellular signals to the nucleus5,6 and regulating protein activity.7 Thus, determination of the phospho-protein and its sites has been an essential step in the analysis of the bio-function of many biological systems. Recently, numerous mass spectrometry-based tech-niques have been developed in analysis of phosphopro-teins,8-16 given their potential for rapid and highly sensi-tive identification of protein. Especially, peptide map-ping with MALDI-MS serves as an attractive method for fingerprinting phosphopeptides.12,13,17

However, during the decades, determination of phospho-related posttranslational modifications in pro-teins still presents a number of challenges to mass spec-trometry although tremendous effort has been put into the development of methodology.18-20 As we know, firstly, in complex organism tissues or cellulars, phos-phoprotein is usually present at substoichiometric levels, moreover, the low degree of phosphopeptides generated from finite phosphoprotein always suffers the suppres-sive effect from non-phosphorylated peptide.3 Secondly,

in an MALDI-MS positive mode, phosphorylated pep-tides are often detected with low content or not at all due to their intrinsic properties that influence their ioni-zation behavior.18,19 Phosphopeptides often contain one or more high density negatively charged phosphate groups and can carry a substantial accumulated charge in solution, which not only increased the difficulty of sample preparation but also impeded the ionization process. As a result, the desorption and ionization effi-ciency of phosphopeptides has been often reduced sub-stantially when compared with their non-phosphorylated counterparts in MALDI, furthermore, signal suppression usually tends to be serious in the presence of other pep-tides.1,2,4 It has been reported that desorption/ionization efficiency for phosphopeptides in MALDI-MS is ap-proximately an order of magnitude lower than that of nonphosphorylated forms.21

Nowadays, the identification of phosphoproteins and phosphopeptides with mass spectrometry is possible by many direct and indirect means. Generally speaking, approaches to improve phosphopeptide detection with MALDI-MS included selectively enriching phos-phopeptides such as immobilized metal affinity chro-matography (IMAC)10,22-24 or antibody25 and enhancing the ionization of phosphopeptides.21,26-28 Use of matrix additives to enhance phosphopeptide response in MALDI-MS is a very appealing approach, which in a

1864 Chin. J. Chem., 2008, Vol. 26, No. 10 ZHANG, LU & YANG


large extent minimizes sample handling and is compati-ble with the enrichment method. In the past years, addi-tives such as phosphate acid (H3PO4)

25 and ammonium salts of ammonium citrate [(NH4)3Cit] and ammonium acetate (CH3COONH4) have been warranted success-fully in enhancing the signal of phosphopeptides in MALDI-MS analysis.26 Yet, to the best of our knowl-edge, no report has appeared on the use of phosphate salts (e.g., ammonium phosphate) as matrix additives for enhancing the ionization efficiency of phosphopep-tides in MALDI-MS.

Herein, we have performed a systemic investigation on the influence of phosphate salt additives on MALDI- MS analysis of phosphopeptides, showing that ammo-nium phosphate of proper concentration added in matrix 2,5-dihydroxybenzoic acid (DHB) significantly en-hanced the ionization efficiency of phosphopeptides. S/N values were 5 to 8 times increased at an optimized condition with 10 mmol/L ammonium dibasic phos-phate [(NH4)2HPO4] or 3 to 4 times increased with 10 mmol/L ammonium monobasic phosphate (NH4H2PO4)

as DHB additives respectively for phosphopeptide 48FQ[pS]EEQQQTEDELQDK63 digested from β-casein. Furthermore, when 5 mmol/L (NH4)2HPO4 as a matrix additive added into matrix 2,4,6-trihydroxyacetophenone (THAP), the ionization efficiency of phosphopeptides was enhanced by twice compared with the most optimized matrix system currently reported.

Experimental

Materials

The matrixes of DHB, THAP and standard protein β-casein were purchased from Sigma (St. Louis, MO, USA). Acetonitrile (ACN) was purchased from Merck (Darmstadt, Germany). Trifluoroacetic acid (TFA) was purchased from TEDIA (Fairfield, OH, USA). Modified porcine trypsin was purchased from Promega, Madison (WI, USA). The reagents of ammonium bicarbonate (NH4HCO3), phosphoric acid (H3PO4), sodium phos-phate (Na3PO4), sodium monobasic phosphate (NaH2PO4), sodium dibasic phosphate (Na2HPO4), po-tassium monobasic phosphate (KH2PO4), potassium dibasic phosphate (K2HPO4), (NH4)2HPO4 and (NH4)H2PO4 were purchased from Shanghai (the 4th Reagent Factory of Shanghai, China). All used water was purified from Milli-Q organic-free system (Milli-pore, Bedford, MA, USA).

Phosphoprotein digestion

Digestion of bovine β-casein was performed with modified porcine trypsin. Experimental procedure and condition consulted to the method previously reported,25 in short, proteolytic digestion of stock solutions of β-casein (approximately 1 mg•mL－1) was performed using trypsin (modified/sequencing grade) at an en-zyme-to-substrate ratio of 1∶40 in 25 mmol/L ammo-nium bicarbonate at 37 ℃ for 12 h and stored at －20

℃ until further analysis. Tryptic digestion of bovine β-casein generated the monophosphorylated peptide 48FQ[pS]EEQQQ-TEDELQDK63 (Mw 2059.82 Da) and the tetraphosphorylated peptide 16RELEELNVP- GEIVE[pS]L[pS][pS][pS]EESITR40 (Mw 3121.26 Da), and the monophosphopeptide was considered in the fol-lowing discussion.

Sample Preparation for MALDI MS

Two individual matrix systems were 7.5 mg•mL－1

DHB and 10 mg•mL－1 THAP in 50% acetonitrile con-taining 0.1% trifluoroacetic acid (TFA) and used as control. Different types of phosphate salts were added into the DHB matrix system, respectively, resulting in final concentration of phosphate salts ranging from 1 to 20 mmol/L. H3PO4 was added with a final concentration of 1.0%. Similarly with the DHB matrix system, differ-ent types of phosphate salts and phosphoric acid with concentration grads were also added into the THAP ma-trix system respectively. The THAP/(NH4)3Cit matrix system was 10 mg•mL－1 THAP containing 0.5 mg• mL－1 (NH4)3Cit. Subsequently, 0.5 µL of the sample solution was mixed with a 1∶1 (V∶V) ratio of the ma-trix solution on the MALDI target using the dried drop-let method.

MALDI MS spectrometry

All the mass spectra were taken from a 4700 Pro-teomics Analyzer of MALDI-TOF/TOF-MS (Applied Biosystems, Framingham, MA, USA). The instrument was operated at an accelerating voltage of 20 kV. A 200 Hz pulsed Nd:YAG laser (355 nm) was used for MALDI. Proteolytic peptides of standard Myoglobin with known molecular masses were used for calibration. All spectra were taken from signal averaging of 1000 laser shots. The laser intensity was kept properly con-stant for all of the samples.

Results and discussion

Optimization of matrix system

Phosphate salts were added into a DHB matrix sys-tem with concentrations of 1, 5, 10, 15 and 20 mmol/L, respectively, in order to investigate the effects of dif-ferent types and concentrations of phosphate salts on the phosphopeptide ionization behavior. Figures 1a—1f showed mass spectra of 60 fmol•µL－1 β-casein diges-tion with 10 mmol/L six different phosphate salts of Na3PO4, Na2HPO4, NaH2PO4, K2HPO4, (NH4)2HPO4 and NH4H2PO4 as matrix additives, respectively. Figure 2 showed the spectrum of 120 fmol•µL－1

β-casein di-gestion in 7.5 mg•mL－1 DHB matrix without additional additives as control. Signal at m/z 2060.82 Da assigned to the monophosphopeptide was barely observed (Fig-ures 1a—1d), corresponding to Na3PO4, Na2HPO4, NaH2PO4, and K2HPO4, no matter different concentra-tions of the salt additive were used (data not shown). These results indicated that these salts severely ruined

Phosphopeptide Chin. J. Chem., 2008 Vol. 26 No. 10 1865


Figure 1 Mass spectra of 60 fmol•µL－1 β-casein digestion in DHB matrix system with 10 mmol/L different phosphate salts. (a) Na3PO4; (b) Na2HPO4; (c) NaH2PO4; (d) K2HPO4; (e) (NH4)2HPO4; (f) NH4H2PO4.



Figure 2 Mass spectrum of 120 fmol•µL－1 β-casein digestion in DHB matrix without additional additive.

the spectra instead of signal improvement. However, DHB/(NH4)2HPO4 or DHB/NH4H2PO4 matrix system showed a great ability to enhance the ionization effi-ciency of phosphopeptide as shown in Figures 1e and 1f. Take a comparison of Figures 1e, 1f with Figure 2, the S/N got from 60 fmol•µL－ 1 phosphopeptide in the DHB/(NH4)2HPO4 matrix system was equal to those got from 400—500 fmol•µL－1 phosphopeptide with the DHB matrix, and S/N got from 60 fmol•µL－1 phos-phopeptide in the DHB/NH4H2PO4 matrix system was equal to those got from 200—300 fmol•µL－1 in the DHB matrix system. To sum up, the S/N of the phos-phopeptide at m/z 2060.82 Da under the optimized con-ditions was 5—8 times enhanced in DHB/(NH4)2HPO4 and 3—4 times enhanced in DHB/NH4H2PO4 than using DHB matrix system without additional additives.

The effect of concentrations of (NH4)2HPO4 and NH4H2PO4 on the ionization efficiency of phosphopep-tide was investigated (as shown in Figure 3). The two ammonium phosphates performed the similar changing trend between phosphopeptide ionization efficiency and salt concentration. The S/N of phosphopeptide increased with increasing the salts concentration and reached the maximum signal intensity when the salt concentration

Figure 3 Effect of concentration of (NH4)2HPO4 and NH4H2PO4 on the ionization efficiency of phosphopeptide.

was 10 mmol/L, afterward, with increasing the salts concentration, no notable improvement of S/N was ob-served. To sum up, an optimized concentration 10 mmol/L of (NH4)2HPO4 or NH4H2PO4 as matrix addi-tives gave the highest S/N for the phosphopeptide.

Comparison between DHB matrix systems with ad-ditives of (NH4)2HPO4, NH4H2PO4 and H3PO4

The capability of enhancing ionization of phos-phopeptide with three additives (NH4)2HPO4, NH4H2- PO4 and H3PO4 was investigated. Figure 4 showed the

Figure 4 Average S/N of the (a) phosphopeptide (m/z 2061 Da) and (b) nonphosphopeptide (m/z 1383 Da) obtained from 10 sam-ple spots with 10 mmol•L－1 (NH4)2HPO4, 10 mmol•L－1 NH4- H2PO4 and 1% H3PO4 as additives, respectively.



S/N of the phosphopeptide (a) and nonphosphopeptide (b) in 7.5 mg•mL－1 DHB matrix system containing 10 mmol•L－1 (NH4)2HPO4, 10 mmol•L－1 NH4H2PO4, 1% H3PO4, respectively. Each column represented the av-erage S/N obtained from ten different sample spots. On every spot 0.5 µL of sample with a concentration of 60 fmol•µL－1 was firstly dropped, followed by adding 0.5 µL of matrix solution. It can be clearly seen that the S/N of phosphopeptide (m/z 2060.82 Da) gave the most no-table increase when (NH4)2HPO4 was used as an addi-tive. Taking a comparison, when (NH4)2HPO4 was used as an additive, the average S/N of phosphopeptide was about twice higher than those of the two other additives. According to these results mentioned above, the capa-bility of enhancing ionization of phosphopeptide can be ranked in the order of (NH4)2HPO4, NH4H2PO4, H3PO4 at their own optimized concentration. Another interest-

ing phenomenon was that the S/N of nonphosphopeptide was improved in the DHB/(NH4)2HPO4 matrix system in the mean time (shown in Figure 4b). When (NH4)2HPO4 was used as an additive, the average S/N of nonphosphopeptide (m/z 1383.28 Da) got from these 10 different spots was nearly twice higher than those of the two others.

(NH4)2HPO4, NH4H2PO4 and H3PO4 as additives in matrix THAP

(NH4)2HPO4 also showed its compatibility with the matrix THAP that was reported for special ionization of phosphopeptides.20 (NH4)2HPO4 was added into 10 mg•mL－1 THAP matrix solution with the final salt con-centration ranging from 1 to 20 mmol/L. 10 mg•mL－1 THAP containing 0.5 mg•mL－1 (NH4)3Cit was used as control. Figure 5 showed two typical mass spectra of

Figure 5 Spectra of 40 fmol•µL－1 β-casein digestion with (a) THAP/(NH4)2HPO4, (b) THAP/(NH4)3Cit and (c) average S/N of phos-phopeptide (m/z 2061 Da) obtained from 10 different sample spots with THAP/(NH4)2HPO4 and THAP/(NH4)3Cit matrix systems respec-tively.



40 fmol•µL－1 β-casein digestion with (a) THAP/(NH4)2- HPO4 and (b) THAP/(NH4)3Cit matrix systems respec-tively. (NH4)2HPO4 as a matrix additive gave better signal intensity than (NH4)3Cit, usually, twice higher S/N can be achieved as shown in Figure 5c. The best S/N of phosphopeptide (m/z 2060.82 Da) was got with 5 mmol/L (NH4)2HPO4 in the THAP matrix. NH4H2PO4

and H3PO4 as additives were also tested but giving no signal improvement (data not shown).

Hereto, it can be assumed that both ammonium cation and phosphate anion have great contribution to enhance the ionization of phosphopeptide in the MS positive mode and their corporation worked much better than these two types of ions that were separated. Fur-thermore, it also demonstrated that the ion of 4NH＋ gave much contribution to ionization of phosphopeptide than the counter ion. The existence of ammonium can substitute Na＋/K＋ by H＋ (from 4NH＋ ) thereby reduc-ing the matrix clusters and leading to a better desorption and ionization process.28 Secondly, the ammonium can neutralize or partially neutralize phosphate groups of phosphopeptide by forming ( 4NH＋ )3n

34(PO )n－ , which

makes them easily complex with H＋. Thus the ioniza-tion efficiency of phosphopeptide could be enhanced in the positive mode.27 Thirdly, the incorporation of am-monium salts has changed the crystal shape of the DHB matrix, and large needle like crystal turned into a small one,29 which was benefitial for ionization and also could improve the reproducibility. As for the phosphate anion, according to these previous references, phosphopeptides as a charged ion formed a crystal lattice with matrix after solvent was evaporated.25,29 The matrix system containing 3

4PO − would reach a higher ionic intensity with the evaporation of solvent, which could make more phosphopeptide ions incorporate into the matrix crystal. In our experiment, the high charge density of (NH4)2HPO4 made the ionic strength of sample and ma-trix mixture reach a high level during the evaporation of solvent.25 More protonated phosphopeptide was forced to incorporate into the matrix crystal during the forma-tion of the matrix crystal. The ionic strength of NH4H2PO4 was higher than that of H3PO4, therefore, the ionization efficiency was better with NH4H2PO4 as a matrix additive than with H3PO4.

In short, (NH4)2HPO4 and NH4H2PO4 as additives in the matrix assisted the ionization of phosphopeptide by forming an ion pair between 4NH＋ and phosphopep-tide, as well as forcing the crystallizations. It was proved that (NH4)2HPO4 was a more favorable matrix additive in analysis of phosphoprotein digestion since this salt could not only enhance the ionization of phosphopeptide but also nonphosphopeptide.

Conclusion

In this paper, we have systematically investigated the influence of different types of phosphate salts on the ionization efficiency of phosphopeptides, phosphate ammonium can significantly enhance the ionization ef-

ficiency, especially the (NH4)2HPO4 at an appropriate concentration demonstrates a prominent contribution. 7.5 mg•mL － 1 DHB matrix solution containing 10 mmol/L (NH4)2HPO4 gave a 5—8 times signal en-hancement and 7.5 mg•mL－1 DHB matrix solution con-taining 10 mmol/L NH4H2PO4 gives a 3—5 times signal enhancement of phosphopeptide (m/z 2060.82 Da) than those of the DHB matrix systems without additional additives. Moreover, as for (NH4)2HPO4 in the other matrix of THAP, the S/N of phosphopeptide is twice higher than using (NH4)3Cit as matrix additive. There-fore, the incorporation of ammonium phosphate can enhance the detection of phosphopeptide by MALDI- MS at the positive mode to a large extent. This method in some sense could simply and efficiently identify phosphoprotein.

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(E0711093 ZHAO, X. J.)

Enhanced Ionization of Phosphopeptide Using Ammonium Phosphate as Matrix Additive by MALDI-MS

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