negative-ion electrospray tandem mass spectrometry of peptides derivatized with...

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 33, 988È993 (1998)

Negative-ion Electrospray Tandem MassSpectrometry of Peptides Derivatized with4-Aminonaphthalenesulphonic Acid

Ingemar Lindh, Jan Sjo� vall, Tomas Bergman and William J. Griffiths*Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden

The value of derivatizing peptides at the C-terminus with 4-aminonaphthalenesulphonic acid (ansa) to aid in the denovo sequencing of peptides by mass spectrometry was assessed. Negative-ion electrospray of peptides is enhancedby derivatization with ansa. The derivatizing group also has the e†ect of increasing the mass of the peptides by 205Da, often shifting their deprotonated molecules to regions of the mass spectrum with reduced chemical noise.Collision-induced dissociation of naphthalenesulphonated peptide [M Ô H ]— ions results in abundant fragmentions formed by charge-remote fragmentations. The resulting fragmentation patterns are less complex than those ofthe underivatized analogues and are easier to interpret. Peptides were successfully derivatized at the low picomolelevel and sequenced on the hundred femtomole level using nano-electrospray tandem mass spectrometry. 1998(

John Wiley & Sons, Ltd.

KEYWORDS: electrospray ; tandem mass spectrometry ; derivatization ; peptides ; charge remote fragmentation

INTRODUCTION

In recent years there has been a revival of interest inderivatizing peptides for mass spectrometry.1 Watsonand co-workers2 derivatized peptides N-terminally withquaternary phosphonium salts via a novel syntheticmethod. The derivative formed is a quaternary phos-phonium cation which, when analysed by positive-ionfast atom bombardment (FAB) or matrix-assisted laserdesorption/ionisation (MALDI), gives intense[M ] H]` signals. The [M] H]` ions fragment inhigh-energy collision-induced dissociation (CID) reac-tions via charge-remote fragmentations (CRFs) with theresulting ions being N-terminal, making de novo aminoacid sequence determination by mass spectrometry(MS) relatively straightforward. Although the quaternaryphosphonium ion derivative was originally designed topromote CRF reactions upon high-energy CID, Linand Glish3 used this derivative in the analysis of pep-tides using a quadrupole ion trap mass spectrometer.Others4 have modiÐed peptides with the aim of pro-

* Correspondence to : W. J. Griffiths, Department of Medical Bio-chemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm,Sweden

E-mail : william.grifÐths=mbb.ki.seContract/grant sponsor : Swedish Medical Research Council ;

Contract/grant number : 03X-12551 ; Contract/grant number : 03X-219 ; Contract/grant number : 13X-10832.

Contract/grant sponsor : Karolinska Institutet.Contract/grant sponsor : Stiftelsen Lars Hiertas Minne.Contract/grant sponsor : Swedish Society for Medical Research.Contract/grant sponsor : Emil och Wera Cornells Stiftelsen.

moting charge-mediated fragmentation reactions, whichare prevalent at the low collision energies employedwith triple quadrupole instruments.

In our experience, a major problem for the low-levelanalysis of small molecules by positive-ion electrospray(ES) MS is the abundance of low-mass chemical noiseassociated with the ionization process. As far lesschemical noise is generated by negative-ion ES, smallacidic compounds may be more e†ectively analysed inthe negative-ion mode.5 This may be particularly sig-niÐcant for the application of nano-ES where analysis isoften attempted at the femtomole level.

In this laboratory, it has been shown that lipids deri-vatized at a carboxylic acid terminus with amino-sulphonic acids give more abundant [M [ H]~ ionsupon FAB ionization than their underivatized ana-logues.6 Furthermore, [M [ H]~ ions from lipids deri-vatized with aminosulphonic acids, give structurallyinformative product ions in high-energy CID reactions.6Peptides also contain a free carboxylic acid group andcan similarly be derivatized. We have previously report-ed the derivatization of tri-, tetra- and pentapeptideswith 4-aminonaphthalenesulphonic acid (ansa).7h9 Bothnegative-ion FAB7 and ES8,9 mass and CID spectrawere recorded. The CID spectra of the [M[ H]~ ionsshowed abundant C-terminal product ions formed byCRF reactions.

In the present work, we investigated the applicabilityof the derivatization method to an extended series ofpeptides. We also explored the possibility of carryingout the derivatization reaction on a picomole scale.Electrospray and nano-ES, mass and CID spectra wererecorded and their value in the de novo amino acidsequencing of the peptides was evaluated.

CCC 1076È5174/98/100988È06 $17.50 Received 21 April 1998( 1998 John Wiley & Sons, Ltd. Accepted 7 July 1998

MS/MS OF DERIVATIZED PEPTIDES 989

EXPERIMENTAL

Derivatization of peptides

Materials. 4-Aminonaphthalenesulphonic acid wasobtained from Fluka (Buchs, Switzerland) and peptidesH1ÈH6, M1ÈM10, bradykinin and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC) from Sigma (St Louis, MO, USA). Solvents wereof HPLC grade and distilled prior to use.

Methods. Peptides were acetylated before derivatizationwith ansa. A 200 ll volume of a 25% solution of aceticanhydride in methanol was added to a solution of thepeptide in 50 ll of water. After 3 h at room temperaturesolvents were removed under vacuum and the resultantacetylated peptides were used for derivatization withansa as described below.

The peptide derivatives listed in Table 1 were pre-pared by dissolving the peptide (1È100 nmol) in 50 ll ofcoupling bu†er. The coupling bu†er (pyridineÈHCl, pH5) was prepared by the addition of 5.3 ml of 32% HClto a solution of 8 ml of pyridine in 80 ml of water. TheÐnal volume was adjusted to 100 ml by the addition ofwater. A 50 ll volume of 1.0 M EDC and 400 ll of 250mM ansa, both of which were dissolved in the couplingbu†er, were added to the peptide solution. After 2 h at25 ¡C, the reaction was quenched by the addition of 50ll of acetic acid. After centrifugation, the solution wasinjected on to a reversed-phase high-performance(C18)liquid chromatographic (HPLC) column (5 lm,4.6] 250 mm; Vydac, Hesperia, CA, USA). Separationof the peptide derivatives from excess EDC, ansa andcoupling bu†er was achieved using a mobile phase ofacetonitrile (0È50% in 50 min) in aqueous 0.1% tri-Ñuoroacetic acid.

When derivatizing peptides at the picomole level theabove quantities were scaled down by a factor of 10.

Mass spectrometry

Negative-ion ES mass and CID spectra were recordedon an AutoSpec-OATOFFPD mass spectrometer10,11(Micromass, Manchester, UK). The instrument can beÐtted with either a conventional or nano-ES interface.For conventional ES, nitrogen was used as both thenebulizer and the bath gas, at Ñow-rates of 15 and 280 lh~1, respectively. The bath gas was heated to 80 ¡C.Sample can be introduced to the nano-ES interfacefrom either a gold-coated borosilicate capillary with a 5lm i.d. spraying oriÐce or by continuous infusion (10È1000 nl min~1) from a 20 lm i.d. fused-silica capillary.In general, peptide samples were sprayed from the boro-silicate capillaries, while calibrant samples were sprayedfrom the fused-silica capillary. With nano-ES, nebulizergas is not required and a reduced Ñow (10 l h~1) of bathgas is used. In studies using both interfaces the peptideswere sprayed from 50% aqueous methanol. For conven-tional ES the Ñow-rate was 5 ll min~1, whereas fornano-ES the borosilicate capillaries were loaded with1È2 ll of sample which was found to spray for 1È2 h.For the recording of mass spectra either a focal planedetector (FPD) or a fourth Ðeld-free region (FFR) o†-axis point detector was used.

The FPD is located between the magnet and thesecond electrostatic analyser on the AutoSpec massspectrometer. The FPD provides an improvement insensitivity of the order of 100-fold over the point detec-tor by simultaneously detecting ions over a wide massrange rather than detecting each mass sequentially ason the point detector. To do this, an additional electro-static analyser is Ðtted to the mass spectrometer afterthe magnet which will accept a range of masses from the

Table 1. Derivatized peptides studied

Internal

(I) or

No. of ÍMÉHËÉ m /z Error external (E)

Trivial name residues Sequence Theoretical Experimentalc (ppm) calibration

Ac-H3-ansa 3 Ac-TVL-ansa 577.2333 — — —

Ac-H1-ansaa 4 Ac-VGSE-ansa 618.1871 618.1871 0 E

Ac-H5-ansa 4 Ac-GGYR-ansa 697.2405 — — —

Ac-M1-ansa 5 Ac-IIGLM-ansa 791.3472 791.3476 0.5 E

Ac-H4-ansa 5 Ac-VHLTP-ansa 811.3449 — — —

Ac-H2-ansa 5 Ac-LPPSR-ansa 814.3558 814.3551 0.9 E

Ac-H6-ansa 5 Ac-FYGPV-ansa 827.3075 827.3114 4.7 E

Ac-M2-ansa 6 Ac-VGVAPG-ansa 744.3027 744.3023 0.5 E

Ac-M3-ansa 6 Ac-WHWLQL-ansa 1127.4773 — — —

Ac-M4-ansa 7 Ac-YPFPGPI-ansa 1035.4286 1035.4286 0 I

Ac2-M6-ansa 9 Ac-VGGYGYGA(AcK)-ansab 1158.4566 — — —

Ac-bradykinin-ansa 9 Ac-RPPGFSPFR-ansa 1305.5839 — — —

Ac3-M7-ansa 10 Ac-YGGFLR(AcK)YP(AcK)-ansab 1557.7200 1557.7230 1.9 I

Ac2-M8-ansa 11 Ac-I(AcK)NLQSLDPSH-ansab 1538.6950 1538.6953 0.2 I

Ac3-M9-ansa 12 Ac-PLSRTLSVAA(AcK)(AcK)-ansab 1599.8205 1599.8218 0.8 I

Ac3-M10-ansa 15 Ac-PLARTLSVAGLPG(AcK)(AcK)-ansab 1836.9682 — — —

a Dehydrated peptide.b AcK represents anN-acetylated lysine residue.c Accurate mass measurements were made on randomly selected peptides as indicated.

( 1998 John Wiley & Sons, Ltd. J. Mass Spectrom. 33, 988È993 (1998)

990 I. LINDH ET AL .

Figure 1. HPLC of products of naphthalenesulphonation ofAc-M2.

Figure 2. EC/CID spectra of ÍMÉHËÉ ion of Ac-M2-ansa (Ac-VGVAPG-ansa) (m /z 744.3). Spectra recorded with (a) conven-tional ES, 150 pmol injected, keV, He collision gas, (b)E

lab¼4

conventional ES, 150 pmol injected, eV, Xe collisionElab

¼400gas, (c) nano-ES, 10 pmol injected, eV, Xe collisionE

lab¼400

gas, (d) nano-ES, 1 pmol injected, eV, Xe collision gasElab

¼400and (e) nano-ES, 200 fmol injected, eV, Xe collisionE

lab¼400

gas.

magnetic sector and simultaneously focus them on tothe FPD. For the recording of mass spectra on theFPD the instrument was tuned to a resolution of 5000(full width at half maximum height (FWHM) deÐnition)and the FPD set to collect a mass range of 1.075 : 1 perexposure. Exposure times were 2 s.

For the recording of accurate mass spectra the instru-ment was tuned to resolutions in excess of 5000 (10%valley deÐnition) and ions were detected on the fourthFFR point detector. Voltage scans were performed overa mass range which was just great enough to includetwo calibrant ions, one on either side of the ion of inter-est. Scan rates were 15 s per scan. Mass calibration wasperformed by either bracketing sample spectra betweencalibrant spectra or by including calibration com-pounds in the sample solution.

The FPD was also used for the acquisition of B/Elinked-scan spectra. In this case the magnet was steppedacross the required mass range to give a series of arrayexposures which were “stitchedÏ together to give a spec-trum. The mass range was 1.05 : 1 per exposure, expo-sure times were 2 s and product ion resolution was 1000(FWHM deÐnition). In the B/E linked-scan experimentseither He or Ar collision gas was introduced into theÐrst FFR gas cell at a pressure sufficient to attenuatethe precursor ion beam intensity by 50%. The labor-atory frame collision energy was 4 keV.(Elab)CID spectra were also recorded using an orthogonalacceleration time-of-Ñight (OATOF) analyser. Mono-isotopic deprotonated molecules were selected by theEBE (E\ electric, B\ magnetic) sectors of the instru-ment and transmitted to the OATOF collision celllocated just after the Ðnal collector slit and containingXe at a pressure sufficient to attenuate the precursor ionbeam by about 80%. The laboratory frame collisionenergy was 400 eV. Undissociated precursor and(Elab)fragment ions were pulsed into the OATOF massanalyser. Precursor and fragment ion resolutions in theresulting spectra were in excess of 1000 (FWHMdeÐnition).

RESULTS

Derivatization

The acetylation reaction was found to be quantitative(yields [95%) in all cases. Both N-terminal and side-chain amino groups were N-acetylated. For some pep-tides methylation was found to occur at the carboxylgroups.

The acetylated peptides were quantitatively deriva-tized with ansa (yields [95%). An exception wasAc-H4, the yield of Ac-H4-ansa being only 75% as aresult of lactone formation. The above yields wereachieved when derivatization was carried out on thenanomole or picomole scale. Figure 1 shows an HPLCtrace of products of the naphthalenesulphonation ofAc-M2. For some of the other peptides several peakscorresponding to naphthalenesulphonated peptideswere observed on the chromatograms. For the peptidescontaining aspartic or glutamic acid residues, reaction



Figure 3. ES/CID spectra of the ÍMÉHËÉ ion of Ac-M4-ansa (Ac-YPFPGPI-ansa) (m /z 1035.4). Spectra recorded with (a) conventionalES, 150 pmol injected, eV, Xe collision gas and (b) nano-ES, 1 pmol injected, eV, Xe collision gas.E

lab¼400 E

lab¼400

products included mononapthalenesulphonated pep-tides or dehydrated and mononapthalenesulphonatedpeptides and doubly naphthalenesulphonated peptides.The presence of the above side products in the ES massspectra was indicative of peptides containing asparticand glutamic acid residues. ES mass spectra of the deri-vatized peptides showed strong [M[ H]~ ions.

Collision-induced dissociation of derivatized peptides

Conventional ES/CID spectra of the derivatized pep-tides listed in Table 1 were recorded on the FPD andwith the OATOF analyser. All of the small peptides(3È7 residues) derivatized with ansa gave structurallyinformative conventional ES/CID spectra, with theexception of Ac-M3-ansa. The spectra recorded at 400eV and 4 keV collision energies were essentially similar ;

however, high mass fragment ion peaks are moreintense in the 4 keV CID spectra [see Fig. 2(a) and (b)].Figures 2(a) and (b) and 3(a) are the conventionalES/CID spectra of Ac-M2-ansa and Ac-M4-ansa. (Thenomenclature used to describe the fragmentation isfrom Roepstor† and Fohlman;12 side-chain fragmenta-tions are as indicated in Scheme 1). All the CID spectracontain the sequence speciÐc backbone fragment ionsAX and/or @X, Y, AZ and/or @Z and the “high-energyÏside-chain fragmentation ions13,14 @V, @W, @a and @b.Larger peptides (9È15 residues) derivatized with ansaalso gave informative conventional ES/CID spectra,with the exception of However, for theAc3-M7-ansa.larger peptides greater amounts of sample were requiredto obtain interpretable spectra. The conventionalES/CID spectrum of the 12 residue peptide Ac3-M9-

is shown in Fig. 4(a).ansaDuring the course of the present study, a nano-ES

interface was installed in the AutoSpec-OATOFFPD

Scheme 1. Fragmentation pattern of deprotonated Ac-M2-ansa.

Figure 4. ES/CID spectra of the ÍMÉHËÉ ion of (Ac-PLSRTLSVAA(AcK)(AcK)-ansa) (m /z 1599.8). Spectra recorded withAc3-M9-ansa

(a) conventional ES, 600 pmol injected, eV, Xe collision gas and (b) nano-ES, 6 pmol injected, eV, Xe collision gas.Elab

¼400 Elab

¼400


992 I. LINDH ET AL .

mass spectrometer and it was decided to re-analyse arandom selection of peptides by nano-ES. As 1È2 ll ofsample tended to spray for 1È2 h, multiple experimentswere possible. From a 2 ll loading it was possible torecord (i) low-resolution (1000 resolution, 10% valleydeÐnition) mass spectra, (ii) accurate mass spectra (5000resolution, 10% valley) and (iii) 400 eV CID spectra.For the selected derivatized peptides 5 pmol were suffi-cient to carry out all of the above studies. The measuredaccurate masses are given in Table 1, and in each caseinterpretable CID spectra were obtained. It was foundthat high-quality nano-ES/CID spectra could beobtained from a few hundred femtomoles of sampleloaded into the capillary. Figure 2(c)È(e) show the nano-ES/CID spectra of the [M[ H]~ ions of Ac-M2-ansarecorded in a serial dilution experiment. It is clear thatan interpretable spectrum can be obtained from only afew hundred femtomoles of sample loaded. The beneÐtof the nano-ES interface is further illustrated in Figs 3and 4, which show a comparison of conventional andnano-ES/CID spectra.

DISCUSSION

With the ever increasing use of mass spectrometry byprotein sequencing laboratories there will be a growingnumber of peptide CID spectra which are not readilyinterpretable. At present many peptides are obtainedfrom trypsin digests and are analysed as doubly proto-nated molecules by positive-ion ES/CID. As such pep-tides have a basic C-terminus which binds one of theprotons, a series of C-terminal fragment ions are usuallyobserved. Only the mass of the peptide and a fewsequence ions are required for comparison with aprotein database from which the protein can be identi-Ðed and the peptide sequence postulated.15 However, apeptide derived from the C-terminal end of the protein(or one which is not obtained from a speciÐc enzymaticdigest) can have any of the 20 amino acids at its termini,making amino acid sequence determination more diffi-cult. This is especially important where recombinantproteins are concerned as these very often have raggedends. To aid sequencing of such peptides, derivatizationreactions can be carried out. By derivatizing peptideswith ansa, a stable negative charge site is introducedC-terminally into the peptide. CID of the deprotonatedmolecule results in only C-terminal fragment ions. Thisgreatly reduces the possibility of ambiguous sequenceions and facilitates de novo sequencing and databasesearching.

Accurate mass measurements have been used byorganic mass spectroscopists for many years16 in thedetermination of elemental compositions of small mol-ecules. It is feasible to use accurate mass measurementsto determine the elemental composition of molecules ofmass below 500 Da. Below this mass, unique elementalcompositions are separated by at least 1 ppm, and whencombined with complementary chemical informationsuch as valency rules, the required mass accuracydecreases to about 5 ppm. Conversely, it is not practicalto determine the unique elemental composition of largemolecules (1È5 kDa) as accuracies of 1 ppb would be

required.17,18 However, accurate mass measurementsdo have a practical utility in peptide analysis. Forinstance, the proposed amino acid sequence must Ðt themeasured accurate mass to within experimental error.Similarly, accurate mass measurements can be made useof in distinguishing between amino acid residues thatdi†er only slightly in mass, e.g. lysine and glutaminewhere only an 18 ppm accuracy is required for a peptideof mass 2000 Da. Clearly, accurate mass measurementsare also of considerable value in the area of databasesearching,19,20 greatly reducing the number of peptidemasses required to identify a protein.

In the present study, all 17 peptides were quantitat-ively derivatized. For peptides containing aspartate andglutamate residues, in addition to the mono-naphthalenesulphonated products, dehydrated and/ordinaphthalenesulphonated peptides may be formed. Theformation of these products is under further study andwill be discussed elsewhere.21 The ansa derivative ismost e†ective for the analysis of small peptides whichcan be sequenced by nano-ES/CID at the femtomolelevel. The AutoSpec double-focusing magnetic sectormass spectrometer, especially when Ðtted with anano-ES interface, allows the determination of depro-tonated molecule masses to high accuracy. In thepresent study, the average error in mass measurementwas 1 ppm, and only one peptide was mass measured togive an error in mass accuracy of greater than 2 ppm. Itwas found that the accuracy of mass determination wasindependent of whether an internal or bracketing cali-bration method was used.

CONCLUSION

Peptides can be quantitatively derivatized C-terminallywith 4-aminonaphthalenesulphonic acid and theresulting compounds give deprotonated molecules uponES ionization. The fragment ions formed by CID areonly C-terminal. Structurally informative spectra can beobtained for peptides ranging in size from 3 to 15 resi-dues, although the efficiency of ES/CID falls as the sizeof the peptide increases. For small peptide ansa deriv-atives (3È6 residues) de novo sequencing can be per-formed by nano-ES/CID from a few hundredfemtomoles of samples loaded. Using this methodology,peptides derived from the digestion of Syrian goldenhamster aldehyde dehydrogenase are now beingsequenced.22

Acknowledgements

This work was supported by the Swedish Medical Research Council(grant Nos 03X-12551, 03X-219 and 13X-10832), the research funds ofKarolinska Institutet, Stiftelsen Lars Hiertas Minne, the SwedishSociety for Medical Research and Emil och Wera Cornells Stiftelsen.I.L. thanks Stiftelsen Ragnhild och Einar Lundstro� ms Minne andSigurd och Elsa Goljes minne for Ðnancial support. Dr Y. Yang isthanked for her assistance in carrying out accurate mass measure-ments. The excellent work of the Micromass service engineers is grate-fully acknowledged.



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