JOURNAL OF MASS SPECTROMETRY, VOL. 31, 1188-1190 (1996)

JMS Letters

Dear Sir,

Differentiation Between the Isomeric Amino Acids Leucine and lsoleucine Using Low-Energy Collision-Induced Dissociation Tandem Mass Spectroscopy

In the field of peptide sequencing, the use of low-energy collision-induced dissociation (CID) tandem mass spectrom- etry (MS/MS) using quadrupole instruments has emerged as a potent technique. Hunt et al.' pioneered in this field and have brought the technology to the point of approaching in utility MS/MS carried out on more sophisticated instruments e.g. four-sector magnetic double-focusing instruments applying high-energy CID. High-energy CID spectra are generally con- sidered to be important in establishing amino acid sequences unambiguously, as in the case of the assignment of leucine us. isoleucine, the isomeric pair of amino acids. In the literature' and recent books on mass spe~t rometry ,~ ,~ it is postulated that low-energy CID spectra obtained with triple-quadrupole MS cannot differentiate leucine from isoleucine.

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Using fast atom bombardment (FAB) MS in early studies on octapeptides and decapeptides, it was reported that the immonium ions R-CH=NH2+ formed by loss of CH,O, from [MH] + are characteristic of the presence of correspond- ing amino acids.5 In the case of leucine or isoleucine, m/z 86 represents the immonjum ion C,H,-CH=NH2+. In 1985, Aubagnac et d6 using FAB MS/MS selected this ion to dis- tinguish between the two amino acids leucine and isoleucine. The difference was based on the distinct relative abundances in the resulting CID spectra. The noticeable abundant forma- tion of m/z 44 and 69 ions agreed with the structures of the two m/z 86 ions derived from leucine and isoleucine, respec- tively. The m/z 44 ion is formed by the McLafferty rearrange- ment with loss of propene whereas the m/z 69 ion is formed by loss of NH,. This method was validated by Heerma and Bathelt' also using FAB MS/MS for various derivatized and underivatized peptides containing leucine and isoleucine at different positions. In a recent study, Heerma and co-worker? used model oligopeptides containing both residues with leucine and isoleucine at the N-terminus. They showed that

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Figure 1. ES mass spectrum of peptide RFEMFRELNEALELKDAOAGKE(NH,) (concentration 0.02 mg ml-') at cone voltages of (a) 50 and (b) 75 V. Eluent, H,O-CH,CN (1 : 1 ) ; flow rate. 10 11 min-' ; source temperature, 80 "C.

CCC 1076--5174/96/101188-03 0 1996 by John Wiley & Sons, Ltd.

Received 1 July 1996 Accepted 15 July 1996

JMS LETTERS 1189

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Figure 2. Product ion spectra of m/z 86 (irnrnonium ion of leucine/isoleucine) of (a) the peptide RFEMFRELNEALELKDAQAGKE(NH,), (b) L-leucine and (c) L-isoleucine. Conditions: cone voltage, 75 V; collision energy, 12 eV; argon pressure, 0.5 Pa; source temperature, 80 "C.

the characteristics of the spectrum of the amino acid present in the peptide at the N-terminus predominates in the mixed tandem mass spectrum. It was nicely demonstrated for two 21-residue polypeptides that one of the polypeptides, the spec- trum of which was very similar to that of pure isoleucine, con- tained solely isoleucine and that the other, which gave a

mixed spectrum, indicated the presence of both leucine and isoleucine. Moreover, the isoleucine dominating in the spec- trum was probably at the N-terminal position.

In February 1996, during the execution of an international interlaboratory exercise to identify unknown peptides with molecular masses ranging from - 500 to 4OO0, we used in con-

1190 JMS LETTERS

trast to the above-mentioned examples a low-energy quadru- pole MS/MS instrument to distinguish leucine from isoleucine. One of the peptides to be identified during this exercise was RFEMFRELNEALELKDAQAGKE-NH, with an average molecular mass of 2623.97. In the electrospray (ES) spectrum recorded with a VG Quattro I1 mass spectrometer, the formation of the immonium ion (m/z 86) proved to be strongly dependent on the applied cone voltage, as shown in Fig. 1. The formation of the immonium ion is evident on increasing the cone voltage up to at least 75-100 V. The resulting product ion spectra from the m/z 86 precursor ion show different relative abundances and characteristic ions at m/z 57, 58 (isoleucine) and 43 (leucine), allowing one to dis- tinguish between the two amino acids as shown in Fig. 2. In the figure the product ion spectrum of the above-mentioned peptide is compared with the product ion spectra of leucine and isoleucine, indicating the presence of solely leucine in the peptide. With additional results obtained after tryptic digestion the complete sequence of the peptide was elucidated with the unambiguous assignment of leucine us. isoleucine. In the same way, using the precursor ion at m/z 86 from the ES spectrum of the intact peptide VEHYDNIEQKIDDID- HEIADLQAKITRLVQQHPRIDE with an average molecu- lar mass of 4438.88, it could be derived that we were dealing with a mixture of isoleucine and leucine. In spite of the ratio of six isoleucine residues to two leucines in the peptide, the characteristic ion at m/z 43 of leucine is still present in the spectrum with an abundance of -25%. These results indicate that comparable structural information can be obtained as reported by Heerma and co-workerss on intact peptides using FAB MS/MS based on high-energy CID on sector instru- ments.

During the culmination of our work, Dookeran ef aL9 reported in May 1996 the distinction of leucine and isoleucine using a hybrid BEqQ MS instrument. In this case, the sub- stantial abundance of the immonium ion was again obtained by FAB MS; however, discrimination was made by low- energy CID, resulting in similar results to those obtained by high-energy CID. This supports our view that differentiation between leucine and isoleucine can be made using low-energy CID provided that the m/z 86 immonium ion is abundant.

In conclusion, the use of high cone voltage (75-100 V) frag- mentation and low-energy CID MS/MS supplies suflicient information to distinguish between leucine and isoleucine isomers. In the case of peptides containing both leucine and isoleucine, a tryptic digest can be carried out, combined with digestion with endopeptidase,'O which hydrolyses the N atom of leucine or isoleucine resulting in peptides having leucine or isoleucine as their N-terminal residues.

The following experimental conditions were applied: 10 p1 flow injection; eluent, H,O/CH,CN (1:l); flow rate, 10 11 min-'. The following MS conditions were applied: cone voltage, 75 V [with the exception of the cone voltage of SO V in Fig. l(a)]; collision energy, 12 eV; argon pressure, 0.5 Pa; and source temperature, 80 "C.

Acknowledgements

The authors thank Jim Hancock, Defence Research Establishment, Suffield, Canada, for organizing the international interlaboratory exercise on the elucidation of unknown peptides and Eric Wils of the TNO Prim Maurits Laboratory for editorial assistance and valuable comments.

Yours, ALBERT G. HULST and CHARLES E. KIENTZ TNO Prins Maurits Laboratory, P.O. Box 45, 2280 AA Rijswijk, The Nctherlands

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