Comparison between Enhanced MALDI In-source Decay by

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A comparison between enhanced MALDI in-source decay by ammonium persulfate and N- or C-terminal derivatization methods for detailed peptide structure determination Anita Horvati#, Ivana Dodig, Tomislav Vuleti#, Dubravko Pavokovi#, Zdenko Hamersak, Ana Butorac, and Mario Cindri# Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac303436n • Publication Date (Web): 12 Mar 2013 Downloaded from http://pubs.acs.org on March 20, 2013

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Analytical Chemistry

A comparison between enhanced MALDI in-source decay by ammonium persulfate and N- or C-terminal derivatization methods for detailed peptide structure determination

Anita Horvatić†, Ivana Dodig†, Tomislav Vuletić‡, Dubravko Pavoković+, Zdenko Hameršak†, Ana Butorac§, Mario Cindrić*,†

†

Ruñer Bošković Institute, Bijenička cesta 54, Zagreb, Croatia

‡

Institute of Physics, Zagreb, Croatia

+

Faculty of Science, University of Zagreb, Croatia

§

Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia

* To whom correspondence should be addressed. E-mail: [email protected], Fax: +38514561010

ABSTRACT

Amino acid sequencing and more detailed structure elucidation analysis of peptides and small proteins is a very difficult task even if the state-of-the-art mass spectrometry is employed. To make this task easier chemical derivatization methods of N-terminus with 4-sulfophenyl-isothiocyanate (SPITC) or C-terminus with 2methoxy-4,5-dihydro-1H-imidazole (Lys-tag) can enhance peptide fragmentation or fragment ionizability, via proton mobility/localization mechanisms making MS2 spectrum more informative and less demanding for structural interpretation. Observed disadvantages related to both derivatization methods are sample- and

ACS Paragon Plus Environment

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time-consuming procedures and the increased number of reaction by-products. A novel, sulfate radical in-source formation method of MALDI mass spectrometry based on chemically enhanced in-source decay (ISD) can be accomplished by simple addition of ammonium persulfate (APS) in matrix solution. This method enables effective decomposition of peptide ions already in the first stage of MS analysis where a large number of fragment ions are produced. The resultant MALDI-ISD mass spectra (MS after APS MALDI-ISD) are almost equivalent to conventional, CID MS2 spectra. These fragment ions are further subjected to the second stage of the MS and consequently MS3 spectra are produced, which makes the sequence analysis more informative and complete (CID MS2 is thus equivalent to CID MS3). Multiply-stage MS after APS addition showed enhanced sensitivity, resolution and mass accuracy compared to peptide derivatization (SPITC and Lys-tag) or conventional MS and MS2 analysis and offered more detailed insight into peptide structure.

Key words: MALDI-TOF/TOF • mobile and localized proton • MALDI-in source decay• SPITC-4-sulfophenyl-isothiocyanate • Lys-tag-2-methoxy-4,5-dihydro-1Himidazole • ammonium persulfate (APS)


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INTRODUCTION

Complete determination of protein/peptide primary structure should be the final result of amino acid sequencing.1,2 Edman degradation has proven to be a powerful and practical strategy for the amino acid sequence analysis.1,3 However, Edman degradation is time-consuming method that requires at least couple of picomole protein/peptide quantities limited to the analysis of only 20 naturally-occurring amino acids (posttranslational modifications and position of disulfide bonds cannot be deduced by Edman sequencing). Furthermore, proteins/peptides that have blocked N-termini are inaccessible to the Edman degradation reaction. On the other hand, in the past 20 years protein and peptide sequencing has been advanced by mass spectrometry based methods due to their unmatched sensitivity and repeatability, ability of posttranslational modification analysis and ability of quantitative and highthroughput analyses.3-5 Yet, posttranslational modifications and unfavorable charge distribution in proteins/peptides challenges the unambiguous amino acid sequence analysis by mass spectrometry, sometimes making the obtained sequence incomplete.6-9 Even some, relatively small peptides (up to 5000 Da) remain only partially sequenced by MS or MS2 analyses. Therefore, in recent years, multistage mass spectrometry (MS3 to MSn) has been introduced in order to increase confidence in peptide structure elucidation, even though rearrangement reactions and decrease of the ion yields, characteristic for MS to MS2 to MS3 transition experiments,

hinder

the

analysis.10,11

Prerequisite

for

primary

structure

protein/peptide sequence analysis by mass spectrometry is the generation of a complete series of fragment ions of one of the types, or a series of fragment ions combined from several of the types , i.e. a-, b-, c- and x-, y-, z-type ions.12,13,14


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Beside technical improvements in mass spectrometry instrumentation, chemically-enhanced ionization and fragmentation emerged as significant means for peptide sequencing.15 The use of chemical tagging of N- or C-terminus in MS-based protein/peptide

sequencing

exploits

mobile

and

localized

proton

models

framework.16,17 Both models are important to fully understand the peptide dissociation in the gas phase and can be very helpful in the selection process of a precursor ion. Two derivatization reagents, 4-sulfophenyl-isothiocyanate (SPITC, initiates activation of an extra mobile proton)18 and 2-methoxy-4,5-dihydro-1H-imidazole (Lys-tag, acts as a strong non-ionic base that localizes the proton)19,20 are frequently used to tag Nterminal amine or C-terminal lysine, respectively (Figure 1). While SPITC increases the fragmentation rate of peptides and directs their fragmentation into a predictable pattern, Lys-tag improves the peptide ionizability (MS) and y-series ions signal intensity (MS2).21,22 SPITC, in analogy to the Edman reagent cannot be applied if the N-terminus of the peptide is blocked, but it does not change masses of MS2 fragments and simplifies the interpretation of the resulting spectra. Lys-tag can be applied if the N-terminus is blocked, however it changes the masses of the derivatized peptide and MS2 fragments, making the interpretation of the resulting spectra more difficult. Another disadvantage related to both derivatization methods is the increased number of reaction by-products hindering the interpretation of MS spectra. In this work we present a new concept of MALDI enhanced in-source decay (ISD) initiated with a chemical reagent added to the matrix solution (ammonium persulfate, APS). We include the sequence analysis of two model peptides Daptomycin and Enfuvirtide by MALDI-MS based on this concept. These two, a cyclic peptide and a peptide with blocked N-terminus, are an appropriate choice to enable


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comparison of three different approaches to amino acid sequencing: conventional Nand C-terminal derivatization based methods and the novel, in-source sulfate radical formation method: MALDI-ISD followed by MS2 (Figure 1). APS addition enables effective decomposition of peptide ions already in the first stage of MS analysis where a large number of fragment ions are produced. The resultant MS spectra (MS after APS MALDI-ISD) are almost equivalent to conventional CID MS2 spectra. These fragment ions are further subjected to the second stage of the MS making the sequence analysis more informative and complete. These MS2 spectra (MS2 after APS MS3) are equivalent to conventional CID MS3. In MALDI, radical induced fragmentation of the peptide analyte can be easily achieved in-source with several different matrices. ISD enhanced by matrix that features hydrogen abstraction mechanism (5-nitro salicylic acid or 2,5-bis(2hydroxyethoxy)-7,7,8,8-tetracyanoquinodimethane) was utilized as a tool for amino acid sequencing of peptides and/or proteins.23 These exhibited prominent formation of a-series ions, explained as end-products of hydrogen-deficient peptide radicals formation. On the other hand, ISD based on hydrogen radicals produced after 2,5dihydroxybenzoic acid or 1,5-diaminonaphthalene matrix ionization at elevated laser fluence leads to c-, z-, w-, and d-series ion formation.24 Hydrogen-deficient peptide radicals or hydrogen radicals formation do not lead to the CID-like fragmentation (formation of b-, and y-series ions and internal fragments) obtained after APS addition to the CHCA matrix. CID-like fragments subjected to the second stage of the MS (MS2) would produce another b- and y-series of ions that can be easily assigned. Therefore, APS addition to the CHCA matrix and sulfate radical formation effect on the peptide fragmentation in the gas phase could be considered as conventional CID


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enhancement, as opposed to hydrogen-deficient peptide radicals or hydrogen radicals formation.

MATERIALS AND METHODS

Preparation of intact samples. Peptide samples, Daptomycin (Chengdu Kaijie Bio-Phamaceuticals Co., Ltd. China) and Enfuvirtide (Cubist Pharmaceuticals, USA) each were dissolved in deionized water to concentration of 0.05 mg/mL. Volume of 10 µL of each sample solution was purified using ZipTip C4 pipette tips (Millipore, Bedford, USA) and dried, then dissolved in 10 µL of CHCA matrix (αCyano-4-hydroxycinnamic acid, 5 mg/mL, Sigma–Aldrich, St. Louis, USA) and dried onto the MALDI plate (volume of 1 µL). At the end, 0.05 µg of peptide was deposited onto the MALDI plate. Enfuvirtide tryptic digest. Trypsinolysis of Enfuvirtide was performed by mixing 60 µL of intact sample (0.05 mg/mL), 40 µL of 100 mM NH4HCO3 (pH 7.8) and 1 µL of trypsin (1 mg/mL, Merck, Darmstadt, Germany) and incubation for 18 h at 37 °C. After digestion, 10 µL of digestion mixture were purified using ZipTip and dried, then mixed with 5 µL of CHCA matrix. Volume of 1 µL was deposited onto the MALDI plate and dried. Preparation of derivatized samples (SPITC and Lys-tag). Daptomycin derivatization was carried out by using SPITC (4-sulfophenyl-isothiocyanate; Sigma– Aldrich, St. Louis, USA). Enfuvirtide derivatization was carried out by using Lys-tag (2-methoxy-4,5-dihydro-1H-imidazole;

C/D/N

Isotopes,

Pointe-Claire,

Quebec,

Canada). Derivatized Daptomycin was prepared by addition of 8.5 µL of SPITC reagent (10 mg/mL SPITC in 20 mM NaHCO3; pH 9) to the purified (10 µL, ZipTip)


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and dried sample. After heating the mixture for 30 minutes at 56 °C the reaction was quenched by addition of 10 µL of 5% trifluoroacetic acid (TFA). Dried Enfuvirtide tryptic peptide mixture was diluted with 50 µL of 0.1% TFA, and derivatized by 50 µL of 0.8 M Lys-tag according to the procedures described in literature.19,20 The mixture was incubated for 1 h at 55 °C. After the derivatization reactions, 10 µL of derivatized peptide samples were purified using ZipTip, mixed with 5 µL of CHCA matrix and spotted onto the MALDI plate (volume of 1 µL). Preparation of intact sample with addition of APS. Peptide samples were prepared under the same procedure described for the intact peptides. Volume of 0.5 µL of 30 mM APS aqueous solution (ammonium persulfate; Sigma–Aldrich, St. Louis, USA) was spotted onto the formed droplet of 1 µL peptide sample that was previously purified, dried and mixed with CHCA matrix. Prior to analysis, volume of 1.5 µL of peptide/ CHCA / APS matrix solution was left to dry onto the MALDI plate. MALDI TOF/TOF-MS. For direct profiling and MS2 fragmentation study of intact and derivatized peptides a 4800 Plus MALDI TOF/TOF analyzer (Applied Biosystems Inc., Foster City, CA, USA) equipped with a 200 Hz, 355 nm Nd:YAG laser was used. Acquisition was performed in positive ion reflector mode. Instrument parameters were set using the 4000 Series Explorer software version (V 3.5.3, Applied Biosystems Inc., Foster City, CA, USA). Mass spectra were obtained by averaging 1000 laser shots covering the mass range between m/z 9 and 4500. MS2 was achieved by 1 or 2 kV collision induced dissociation (CID) in positive ion mode. For both MALDI-TOF MS and MS2 analysis, CHCA matrix was used. Dried purified samples (both intact and derivatized) were diluted in 5 µL of matrix, 1 µL of mixture was spotted onto the MALDI plate and allowed to dry.


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RESULTS

Daptomycin MS2 and MS3 experiments with addition of ammonium persulfate. Simple addition of ammonium persulfate in the matrix solution dramatically changed the appearance of the Daptomycin MS spectrum turning it into a MS2-like spectrum (MALDI-ISD MS). This spectrum, in comparison to the classical CID MS2 spectrum, exhibited enhanced resolution, mass accuracy and signal-tonoise ratio, which facilitated the identification of amino acid sequence through more than 50 potential precursor ions (Figure 2a and 2b). MALDI-ISD MS spectrum fragmentation pattern in the mass range between m/z 570 and 1620 appears as a mirror reflection to the CID MS2 spectrum of the precursor ion at m/z 1620.7. Since CID MS2 spectrum did not provide enough supportive data for informative structural and sub-structural sequence analysis (Figure 2a and 3), Daptomycin SPITC derivatization or MALDI-ISD followed by MS2 analysis were required (Figure 2b, 2c and 3). MALDI-ISD MS fragments generated in-source by addition of ammonium persulfate are further dissociated in MS2 experiment, allowing a rare opportunity to choose precursor ion in the first stage of the MS experiment for the MS3. MS2 analysis of only one internal fragment at m/z 1280.4 selected in the group of 50 MALDI-ISD MS potential precursor ions (described in details in Supporting Information Figures S-1, and -2 and Table S-1) filled data gaps obtained after CID MS2 analysis (missing fragments b5, b6, b9, b11 and b12) necessary for aforementioned structural analysis. Thus, determination of the amino acid sequence along with chemical modifications and prosthetics groups is possible without demanding N- or C-terminal derivatization procedure (Figure 2c and 3) or


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implementation of an MS3 instrument with the decrease in sensitivity inherent to the multistage MS3 procedure. A similar method for enhanced MALDI in-source decay based on saturated ammonium sulfate addition in the matrix solution has recently been published as an efficient in-source fragmentation procedure.25,26 The authors

explained the

phenomenon of enhanced in-source peptide decay in details. When MALDI spot is formed, hydrated sulfate anions exclude the peptide by removing protective coat of water. Higher local concentration of the peptide and a lack of the protective coat of water facilitate desorption and fragmentation. Concentration of ammonium sulfate used in this experiment was 0.5 M to 3 M, which is approximately two orders of magnitude higher than ammonium persulfate concentrations used in the procedure presented here. Although ammonium sulfate and persulfate are structurally similar compounds, the effect they have on ionization and fragmentation is not entirely comparable. The addition of ammonium sulfate in the matrix solution does enhance ionization and fragmentation and decreases the number and intensity of adducts (Na+, K+, NH4+), however only when a relatively high quantity is added. Conversely, even a small amount of ammonium persulfate shows a more pronounced effect on fragmentation, while retaining the effect on ionization. Presumably, the effect on fragmentation is due to persulfate propensity for sulfate radical formation. Sulfate radical formation is routinely used to catalyze the polymerization of acrylamide and it is explained in details and well known from the literature.27 We remind that ammonium persulfate is a strong oxidizing agent and radical initiator. Oxidizing effect of ammonium persulfate on proteins/peptides was not observed in enhanced MALDI in-source decay experiments (e.g. oxidation of amino acids prone to react with an oxidant Tyr, Met, Cys, Trp and His). The radical formation mechanism is initiated in


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MALDI experiments

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when photodissociation and thermal decomposition of

ammonium persulfate occur. Eventually, persulfate radicals formed by laser irradiation cleave peptide bonds resulting in a large number of internal cleavages that occur already in-source.

A mobile proton: MS2 analysis of derivatized (SPITC) cyclic lipopetide Daptomycin. The SPITC-derivatized cyclic peptide underwent facile fragmentation, predominantly resulting in b-series ions in the MS2 spectra (Figure 2c). After SPITC derivatization of L-kynurenine amine (Figure 3) and activation of an extra mobile proton, distant peptide bonds in Daptomycin ion were dissociated making MS2 sequencing complete. However, migration of a mobile proton is obtained up to Lthreonine peptide bond (b5 ion), facilitating cleavage of only 9 peptide bonds, out of 13. The applied derivatization method and the following MS2 analysis are appropriate for relatively small cyclic peptides approx. up to m/z 2000. Larger cyclic peptide would face limitations of proton migration, while larger Lys-containing cyclic peptides should be derivatized additionally by separate lysine-protecting procedure (e.g. Lystag derivatization followed by purification and SPITC derivatization, again followed by another purification procedure).

A localized proton: MS2 analysis of derivatized (Lys-tag) tryptic fragments and of intact Enfuvirtide. Enfuvirtide contains acetylated N-terminus and amidated C-terminus that change MS2 characteristics of the peptide precursor and product ions.28 Both these synthetic modifications should not hinder the overall formation of b- and y-series of ions by localization of the proton on amide C-terminus or by neutralization of N-terminus basicity, concurrently making the fragmentation


10

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process less energetically demanding. As shown in previous example, Lys-tag derivatization method applied on cyclic lipopeptide without appropriate amine reactive centre in the molecule was not feasible. Without a reactive centre in the molecule derivatization methods are useless. SPITC derivatization method could not be applied on Enfuvirtide due to the N-terminal acetylation, thus different analytical approaches were considered. Top-down approach i.e. conventional MS2 analysis (precursor ion at m/z 4490.2) did not lead to the determination of all amino acids and chemical modifications in the sequence. That made sequence analysis of the intact molecule incomplete (data not shown) even though in theory both termini chemical modifications would not suppress formation of y- and b-ions, necessary for primary structure determination. We note that Enfuvirtide peptide ion can be concisely described in basic group notation; CH3CONH

- YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF -

(N-amide+) -

(peptide bonds+)

-

CONH2 (C-amide+)

where: (+) represents moderate proton affinity, (++) high proton affinity and (+++) extremely high proton affinity. Consequently, bottom-up analysis based on trypsin digestion was opted for as an intermediate step towards the sequence structure determination. Fragments produced after trypsinolysis altered the formerly equalized charge distribution in the Enfuvirtide peptide ion into:


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T1 fragment

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CH3CONH -

YTSLIHSLIEESQNQQEK

-

COOH

(N-amide+) -

(peptide bonds+)

-

(C-Lys++)

NEQELLELDK

-

COOH

(peptide bonds+)

-

(C-Lys ++)

WASLWNWF

-

CONH2

(peptide bonds+)

-

(C-amide+)

T2 fragment

NH2 (N-amine++) -

T3 fragment

NH2 (N-amine++) -

Although all three tryptic peptides with their charge distributions and proton affinity sites followed standard bx-yz fragmentation pathway by mobilization of the added proton localized at N-terminal amino group (T3) or Lys side chain (T1 and T2), derivatization of T2 fragment was still needed for informative structural and substructural sequence analysis. In the MS2 analysis of intact T2 fragment, bx-yz ion pairs formation were gradually decreased to signal-to-noise (S/N) lower than 5, as ladder sequence grew in number. Finally, b8-y8 signals were barely detectable after the accumulation of 10 or more mass spectra, and the last, b9-y9 pair of signals in the ladder sequence was below reliable detection limits (Figure 4a). After lysine derivatization of the T2 tryptic fragment, the proton affinity distribution in the peptide was changed from T2 (++)-(+)(++) to T2-Lys-tag (++)-(+)-(+++), yielding the improvement of y-ions ionizability and amino acid sequence determination exclusively through y-series of ions (Figure 4b). Difference between the proton affinities of N- and C-terminus is directly proportional to ln(bx/yz) value.14 Inadequate difference in proton affinity between termini can be easily changed by introduction of a strong basic group with extremely high proton affinity, preferably on C-terminal side chain (e.g. Lys-tag imidazole


12

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group). Singly protonated tryptic peptide that contains strong basic group at the Cterminus requires elevated energy for mobilization of the extra proton to reach energetically less favored protonation sites. Dissociation energy requirements for the mobile proton activation depend on the basicity of the side-chain or N-terminus. Cterminal side-chain amino acids can be sorted in accordance to the decreasing proton affinity: Lys-imidazole-containing > Homoarg-containing > Arg-containing > Lys-containing > Non-basic peptides. Additionally, intact tryptic fragments T1 and T3 were effortlessly analyzed by conventional MS2 (Figure 4c and 4d). Thus, chemical modifications on T1 Lys-terminated and T3 Phe-terminated tryptic peptide fragments were not required (n.b. T3 tryptic peptide cannot be derivatized by the applied reaction). The derivatization of the peptide mixture under the reaction conditions described in the literature (cf. time and reagent quantities in Materials and methods)19,20 resulted in incomplete labeling of T1-lysine and derivatization of other tryptic peptides shown on Figure 5, hence hampering MS spectra elucidation. At the same time derivatized and non-derivatized, miscleaved and cleaved Enfuvirtide tryptic fragments were present in MS spectrum - a cumulative number of fragments arising from a relatively small protein was very high (5 non-derivatized and 4 derivatized fragments). Time-consuming and demanding analysis of derivatized/nonderivatized MS fragments were completed after MS2 analysis of derivatized T2 tryptic peptide, where the fragment masses were recalculated to Lys-tag mass increment of m/z 68 (see Supporting information Figure S-3 for recalculated masses details). However, the applied method allowed for straightforward Enfuvirtide sequence analysis in a highly reproducible manner.


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Enfuvirtide MALDI-ISD MS and MS2 experiments with addition of ammonium persulfate. The analysis of Enfuvirtide tryptic digest, especially derivatized T2-Lys-tag tryptic peptide required several experiments making the analysis of the peptide sequence time-consuming and laborious. Mass spectrometry data processing was hampered due to the increment in the fragment masses of the y-series ions after addition of the tag. Additionally, sequence analysis of tryptic peptides has required elevated sample consumption due to the formation of miscleaved fragments, and due to laborious reaction condition optimization. In short, bottom-up principle was successfully applied on the amino acid sequence analysis of a chemically modified small protein without losing the important structural data but a one-step (instead of a three-step) sample preparation procedure of the analyzed peptide would be more appreciated.7,8,29 The mass range of topdown mass spectrometry has been extended to proteins as large as 229 kDa,30 but the data collected by this approach is often incomplete or of high complexity, thus hindering detailed structural protein analysis. However, a fast, unambiguous and complete MS analysis is required in many fields of biological research and pharmaceutical science.31,32 MALDI-ISD MS and MS2 experiments based on the addition of ammonium persulfate allow medium top-down type of experiment without the reduction of sequence information quality (17 out of 64 in-source fragments were subjected to MS2 experiment). Quite the contrary, data reliability and validity were increased under these conditions (increased resolution, mass accuracy and signal-tonoise ratio relative to conventional MS2 and MS3 experiments). Enfuvirtide MS spectra with 27 in-source fragments of low intensity recorded before and 64 in-source fragments of high intensity detected after addition of ammonium persulfate are presented in Figure 6. Neither of 27 in-source fragments of low intensity obtained by


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conventional MALDI experiment was intensive enough for informative MS2 analysis, on the contrary, complete and reliable Enfuvertide structural analysis of the 17 most intense fragments was performed after APS addition in MS2 experiment (MALDI-ISD MS analysis of 64 in-source generated fragments and MS2 analysis of m/z 2060.9 and 2448.2 precursor ions can be found in Supporting Information Figures S-4-S-6 and Table S-2). MS2 fragmentation of only two in-source precursor ions m/z 2060.9 and 2448.2 produced after APS addition were sufficient for Enfuvertide primary structure determination. The applied method simplified and accelerated the sequence analysis up to a point that enzymatic digestion and derivatization were not needed. Obtained data validity and reproducibility were probed by multiple MS, MALDI-ISD MS and MS2 experiments without a significant decrease of the ion yield characteristic for MS to MS2 to MS3 transition experiments. On the other hand, MALDI-ISD MS followed by MS2 experiments greatly increase the amount of structural information obtainable for a given peptide sequence.

CONCLUSIONS

Top-down mass spectrometry is in focus of instrumental development for more than twenty years, but is not yet a high-throughput method.29 We presume that the routine implementation of the multistage mass spectrometry would be the next logical step in the top-down approach development.33,34 On the other side, chemically assisted fragmentation and ionization based on mobile and localized proton models (SPITC and Lys-tag, respectively) facilitate protein sequence determination in bottom-up experiments.15,21 However, incomplete derivatization, unpredictable and unwanted side-reactions (posttranslational modifications, reactive cysteines and


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methionines, miscleaved lysines etc.) could add to the complexity of the peptide mixture making the analysis more difficult or unfeasible. Combining the advantage of MALDI technique to predominantly generate singly charged ions with enhanced insource fragmentation (due to ammonium persulfate addition in the matrix) and the power of multistage MS, we have developed a new technology platform for complete peptide primary sequence determination that avoids laborious sample preparation procedures. The sequence can be unambiguously determined by assignations of b-, y- and internal fragment-ions produced in the MS, that is in MALDI-ISD MS. Collected data could be further validated through MS2 analysis. Each step of enhanced pseudo multistage analysis, in contrast to the conventional multistage analysis, retained the resolution, mass accuracy and ion yield of the previous step. Even though the technology platform was developed on pharmaceutical peptides, the implementation of the proposed technology in the high-throughput peptide and protein structural analysis should be feasible due to the complementarity between existing software platforms and enhanced pseudo MS multistage analysis.34,35


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ACKNOWLEDGMENTS

This work is supported by the project of Croatian Ministry of Science, Education and Sports 098-0000000-3454. Participation of Tomislav Vuletić in this work was supported by Croatian UKF Grant 22/08. A. Horvatić and I. Dodig contributed equally to this work.

REFERENCES

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FIGURES

Figure 1. Enhanced MS2 fragmentation accomplished through the activation of an extra mobile proton by N-terminal labeling of peptide with 4-sulfophenylisothiocyanate (SPITC) or proton localization of Lys-containing peptide by C-terminal labeling with 2-methoxy-4,5-dihydro-1H-imidazole (Lys-tag). Unlike SPITC and Lystag covalent derivatization, ammonium persulfate enhances in-source decay accomplished through radical formation in the gas phase (MALDI ammonium persulfate thermal and photochemical decomposition).


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Figure 2. MALDI-TOF mass spectra of Daptomycin cyclic lipopeptide: (a) MS/MS of non-derivatized precursor ion at m/z 1620.7 and (b) MALDI-ISD MS, that is MS after APS addition (mirror image) (c) MS2 of SPITC-derivatized precursor ion at m/z 1835.7 with denoted b-series ions in the mass range between m/z 135 and 1660. Question mark denotes missing b-series signals.


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Figure 3. Theoretical monoisotopic fragment masses of Daptomycin cyclic lipopeptide fragmentation sequence (b-series and decanoic acid amide denoted as c1 ion). Masses are calculated after lactone ring gas-phase MS/MS dissociation. Calculated masses were not altered by SPITC derivatization of L-kynurenine amine. Sequence signal interruptions observed in CID MS2 are marked as ‡‡.


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Figure 4. MALDI-TOF MS2 of T2, T2-Lys-tag, T1, and T3 tryptic fragments. T2-Lystag, T1, and T3 tryptic fragments designate the complete Enfuvirtide sequence, while T2 fragment provides only partial sequence information. Precursor ions were (a) T2 at m/z 1230.59 with missing fragment pairs b8, b9 and y8, y9 indicated with question


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marks, (b) T2-Lys-tag at m/z 1298.63 (marked with an asterisk), (c) T1 at m/z 1108.54 and (d) T3 at m/z 2189.03.


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Figure 5. MALDI-TOF MS of Enfuvirtide tryptic peptide fragments. T1, T2 and T3, as well as miscleaved T1-2 and T2-3 represent non-derivatized peptide tryptic fragments. T1*, T2* and miscleaved T1-2*, T2-3* denote peptide tryptic fragments derivatized at lysine (Lys-tag) with the theoretical mass increment of m/z 68.037 relative to non-derivatized peptides.


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Figure 6. MALDI-TOF mass spectrum of Enfuvirtide, with designated fragment ions, (a) without addition of APS (b) MALDI-ISD MS spectrum with 64 peptide fragments of high intensity obtained after APS addition. Fragment ions obtained in MALDI-ISD MS spectrum were subjected to multistage mass spectrometry (MS2). Detailed comparison of in-source fragment ions intensity and sequence coverage before and after addition of APS can be found in Supporting Information.


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“ For TOC only ”:

TOC 1

TOC 2

TOC 3


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Comparison between Enhanced MALDI In-source Decay by

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