Proton mobility in protonated amino acids and peptides

E LS EVI E R International Journal of Mass Spectrometry and Ion Processes 165/166 (1997) 339-347 and Ion Processes

Proton mobility in protonated amino acids and peptides

Alex G. Harrison*, Talat Yalcin’

Department of Chemistry, Universil?, of Toronto, 80 St. George Street, Toronto, Onturio MSS 3H6, Cunadu

Received 14 March 1997; accepted 23 June 1997

Abstract

The MD+ ions of a variety of amino acids and small peptides have been prepared using CD, and (CD&CO as chemical ionization reagents. Using tandem mass spectrometry the fragmentation reactions of these MD’ ions have been studied, both those occurring unimolecularly on the metastable ion time scale (CD, CI) and those occurring following collisional activation ((CD&CO CI). The results show that the added D+ has undergone extensive interchange (leading to H/D scrambling) with all labile hydrogens including carboxylic hydrogens, hydroxylic hydrogens, amidic hydrogens and amino hydrogens. The results indicate that the proton added to amino acids and simple peptides is very mobile and samples all positions bearing labile hydrogens prior to fragmentation of the protonated species. 0 1997 Elsevier Science B.V.

Keywords: Proton mobility; Protonated amino acids; Protonated peptides

1. Introduction

The major types of fragment ions produced on collisional activation of protonated peptides are well-documented [l-6]. Although high energy (keV) collisional activation often leads to charge- remote fragmentation reactions [7,8], low energy (eV) collisional activation most frequently leads to charge-directed fragmentation reactions [9-121.

* Corresponding author. Tel./fax: + 1 416 9783577. ‘Present address: Department of Chemistry, University of

Alberta, Edmonton, Alberta, Canada.

In many cases low energy CID leads to cleavage of an amide bond resulting in B,, ions if the charge remains on the N-terminus fragment and to Y,I” ions if the charge remains on the C- terminus fragment (Fig. 1). Recent ab initio and semiempirical molecular orbital calculations [ll-131 have shown that protonation at the amide nitrogen leads to considerable weakening of the amide bond and it is likely that this N-protonated species represents the reacting configuration for amide bond cleavage. (By contrast, protonation of the amide oxygen leads to strengthening of the amide bond [ll-131.1 However, it is clearly established that protonation at the amide nitrogen

016%1176/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOlhS-1176(97)00173-O

340 A.G. Hamion, T. Yalcin /International Journul of Muss Spectromefty and Ion Processes 165 / 166 (1997) 339-347

is not the thermodynamically favoured site of protonation of peptides [13-151. This has led many workers [8,10-12,161 to suggest that the proton added to the peptide does not remain fixed at the site of greatest proton affinity but is capable of migrating to positions of lower proton affinity prior to fragmentation of the protonated species. This has been called the ‘mobile proton model’ by Dongrk et al. [17]. Support for this model has come from deuterium labelling studies by Mueller et al. [18] and Johnson et al. [IY] which indicate extensive H/D mixing prior to collisionally activated dissociation of the MD’ ion of a number of small peptides, although it is not clear from the results presented that all labile hydrogens (those bonded to N, 0 or S> participate in the mixing process. In the present work we have studied the unimolecular (metastable ion) and collision-induced fragmentation of the MD+ ions of a variety of amino acids and small peptides to explore the extent of proton mobility prior to fragmentation. The MD’ ions were prepared by chemical ionization using either CD, or (CD,),CO as reagent gases. An early study 1201 of the D, and CD, chemical ionization of amino acids indicated extensive H/D mixing prior to fragmentation of the MD’ ions although the results were not amenable to quantitative interpretation since MH + ions also were observed in the mass spec- tra. Using tandem mass spectrometric techniques [21,22] we are able to make a more quantitative study of proton mobility in the present study.

2. Experimental

All experimental work was carried out using a ZAB-2FQ hybrid BEqQ mass spectrometer (VG Analytical Ltd., Wythenshawe, Manchester, UK) which has been described in detail previously [23]. Briefly, the instrument is a reversed geometry (BE) double-focusing mass spectrometer that is followed by a deceleration lens system, an rf-only quadrupole collision cell (9) and a quadrupole mass analyzer (Q>. The MD’ ions of the amino acids and peptides were prepared by chemical ionization (CI) using either CD, or (CD&CO as reagent gas. Because methane CI produces domi- nant CH_; and C,H: ions and PA(CH,) = 132

H2N COOH

R, YH-_FH-A,

&N-W- C;o,C+o + R,? A4

HjN-_CH-C-I$---COOH

e-2 II

y2

Fig. 1. Schematic of fragmentation of protonated peptides.

kcal mol.-’ [24] while PA(C2H,) = 163 kcal mol-’ [24], deuteronation of amino acids and peptides, whose proton affinities are greater than 200 kcal mol.-’ [25], is substantially exothermic and the MD’ ions are formed initially with coqiderable internal energy. To prepare MD’ ions of lower internal energy (CD,),CO (PA((CH,),CO) = 197 kcal mol-’ [24]) also was used as reagent gas with (CD,),COD+ as the major reactant ion. (ND, could not be used as reagent gas to give even gentler deuteronation because of H/D exchange between the deuteronated amino acid or peptide and deuteroammonia [26].) The EI/CI source was used in the CI mode at a source temperature of 200°C with an ionizing electron energy of 100 eV. The amino acid or peptide was introduced into the ion source either by a heated direct insertion probe or by a direct exposure probe.

In the metastable ion studies the MD+ ion of interest was mass-selected by the BE double- focusing mass spectrometer at 6 keV ion energy, decelerated to 20-40 eV energy and introduced into the rf-only collision cell q in the absence of collision gas. The ionic products of unimolecular fragmentation in the cell were measured by scan- ning the final quadrupole mass analyzer Q. Low energy collision-induced dissociation (CID) studies were carried out in the same fashion but with the addition of N, at an indicated pressure of 2-3 X lo-’ torr to the quadrupole collision cell. In both the metastable ion and CID studies 20-50 2-s scans were accumulated on a multi-channel analyer to improve signal-to-noise.

The amino acids used were obtained from

A.G. Ham’son, T. Yalcin /International Journal of Mass Spectrornetry and Ion Processes 165 /166 (1997) 339-347 341

Aldrich Chemical Co. while leucinamide and the peptides studied were obtained from Bachem Biosciences Inc.; all were used as received. The CD, and (CD,),CO reagents were obtained from CDN Isotopes, Vaudreuil, Quebec.

3. Results and discussion

3.1. Proton mobility in protonated amino acids

Table 1 presents the results obtained for fragmentation of the MD+ ions of a number of amino acids where the MD+ ion was obtained by CD, CI while Table 2 presents the results obtained when the MD+ ions were prepared by (CD,),CO CI. The CD, CI results refer to unimolecular metastable ion fragmentation (with CID results in brackets) while the results for the (CD,),CO CI systems refer to CID experiments since, in general, metastable ion signals were ex- tremely weak when perdeuteroacetone was used as the CI reagent. The calculated ratios included in Table 1 and Table 2 are based on complete mixing or scrambling of all labile hydrogens with the added D+; the method of calculation is out- lined in Appendix A.

The MH+ ions of the aliphatic amino acids alanine and leucine fragment primarily by elimination of H,O + CO [20,27-301, although metastable loss of CO from the MH+ ion of

Table 1 Unimolecular fragmentation of MD+ ions of amino acids”

alanine also is observed. The MD’ ions of these amino acids showed elimination of both [H,O + CO] and [HDO + CO] with the intensity ratios given in Table 1 and Table 2. These are essentially the same whether recording metastable ion fragmentation after CD, CI or CID following (CD&CO CI and are in agreement with the ratio calculated assuming complete scrambling of the labile H/D. Note that deuteronation of the amino group, the favoured site of protonation, followed by irreversible H+/D+ migration to the hydroxyl group of the carbonyl function would lead to - [H,O + CO]/ - [HDO + CO] = 2.0, neglecting isotope effects

Protonated proline also fragments primarily by loss of H,O + CO [27]. Metastable ion fragmentation of the MD + ion following CD, CI gave - [H,O + CO]/ - [HDO + CO] = 0.45, in reasonable agreement with the ratio of 0.50 expected for randomization of the labile H/D. The MD+ ion formed by (CD,),CO CI gave - [Hz0 + CO]/ - [HDO + CO] = 0.53 at low (5-10 eVj collision energies but the ratio decreased with in- creasing collision energy reaching a ratio of 0.35 at 45 eV collision energy. Clearly, there is a tendency for specific loss of the added deuterium as the collision energy is increased for reasons which are not clear.

The MH + ion of the hydroxylic amino acid serine fragments both by loss of H,O and by loss

Amino acid -H,O/-HDO - [H,O + CO]/ - [HDO + CO] -NH,/ - NHzD

Obs. Calc. Obs. Calc. Obs. Calc.

H-Ala-OH H-Leu-OH H-Pro-OH H-Ser-OH H-Asp-OH H-Asn-OH H-Tyr-OH H-Lys-OH H&x-NH,’

1.6 1.5 1.7 1.5

1.2 1.0 1.2 1.0 0.45 0.50 1.7 1.5 1.6 1.5 2.4 2.0 1.7(1.61b 1.5

1.1 1.0 0.73 0.67

‘MD + ions prepared by CD, CI. ‘CID data. ’ - [NH, + CO]/ -[NH,D + CO] ratio measured.

342 A.G. Hamion, T. Yalcin /International Journal of Mass Spectrometry and Ion Processes I65/ 166 (19971339-347

Table 2 Fragmentation of MD’ ions following collisional activationa

Amino acid -H,O/- HDO - [H,O + CO]/ - [HDO + CO]

Obs. Calc. Obs. Calc.

H-Ala-OH 1.1 1.0 H-Lcu-OH l.O(l.l)h 1.0 H-Pro-OH 0.53 -+ 0.35 0.50 H-Ser-OH 1.6 1.5 1.6 1.5 H-Asp-OH 1.6 1.5 1.4 1.5 H-Asn-OH 2.1 2.0 H-Tyr-OH 1.1 1.5(1.0) H-Lys-OH H-LeuNH,”

“MD’ ions prepared by (CD&CO CL “Number in brackets from metastable ion fragmentation. “Number in brackets calculated assuming phenolic hydrogen not involved in randomization.

- NH,/ - NH, D

Obs. Calc.

1.1 1.0 0.79 0.67

’ - [NH, + CO]/ - [NH2 D + CO] ratio measured. . -

of [H,O + CO] [27]. For the related amino acid threonine it has been shown [31] that loss of H,O involves the hydroxyl group of the side chain while loss of H,O + CO involves the carboxyl oxygens. In both metastable ion (CD, CI) and CID ((CD,),CO CI) studies we observed - H,O/ - HDO = 1.6 in agreement with the ratio of 1.5 calculated assuming complete mixing of the added deuterium with the hydrogens of the amino group, the hydrogen of the side-chain hydroxyl group and the hydrogen of the carboxylic acid group. Similarly, we observe -[H,O + CO]/ - [HDO + CO] = 1.7 (metastable ion, CD, CI> and 1.6 (CID, (CD&CO CI) also in reasonable agreement with the ratio of 1.5 calculated on the basis of randomization of all labile H/D. Note that if the carboxylic hydrogen was not involved in the scrambling preceding water loss a ratio of - H,O/ - HDO = 1.0 would be expected. Similarly, if the side-chain hydroxyl hydrogen was not involved in the mixing preceding water plus CO loss, a ratio -[HZ0 + CO]/ - [HDO + CO] = 1.0 would be expected. Clearly, all labile hydrogens are involved in the mixing process.

Aspartic acid contains a carboxylic acid group in the side chain and the MH+ ion fragments by loss of H,O and by loss of H,O + CO [27]. As the results in Table 1 and Table 2 show, the ratios - H,O/ - HDO and - [H,O + CO]/ - [HDO +

CO] observed in fragmentation of the MD’ ion formed by CD, CI or by (CD&CO CI are in agreement with the ratios calculated assuming scrambling of all labile H/D. The related amino acid asparagine contains an amide function in the side chain and the MH+ ion fragments primarily by loss of H,O + CO [27]. Fragmentation of the MD+ ion of asparagine showed -[HZ0 + CO]/ - [HDO + CO] = 2.4 when prepared by CD, CI and 2.1 when prepared by (CD&CO CI. Com- plete scrambling of all labile H/D, including the amide hydrogens, leads to a calculated ratio of 2.0 in reasonable agreement with the experimental data.

Protonated lysine fragments primarily by elimination of NH, and this has been shown to involve loss of the amino group of the side chain [27,32]. Fragmentation of the MD+ ion of lysine formed by either CD, CI or by (CD,),CO CI showed -NH,/- NH,D = 1.1, in good agreement with the ratio of 1.0 calculated assuming complete scrambling of all labile H/D. The interchange of the U- and w-hydrogens of protonated lysine is not unexpected since the high proton affinity of lysine (PA = 230 kcal mall’ 1251) has been at- tributed [33-351 to considerable hydrogen bond- ing between the two sites, similar to that observed for protonated cu,w-diamines 1361. The present results show that the carboxylic hydrogen also becomes involved in this exchange process.

A.G. Harrison, T. Yalcin /International Journal of Mass Spectrometry and Ion Processes 165 /I 66 (1997) 339-347 343

Protonated leucinamide fragments by loss of NH, + CO [30]. Fragmentation of the MD+ ion of leucinamide showed -[NH, + CO]/ - [NH,D + CO] = 0.73 (CD, CI) and 0.79 ((CD,),CO CD, compared to the ratio of 0.67 expected for complete scrambling of the labile H/D. Clearly, there is a slight preference for elimination of NH 3 + CO suggesting that H/D interchange has not led to complete scrambling.

Protonated tyrosine fragments by elimination of NH, and by elimination of H,O + CO [271. This was the one case examined where the results obtained depended on the method of formation of the MD+ ion. As the results in Table 1 show, the MD+ ion prepared by CD, CI showed - [H,O + CO]/ - [HDO + CO] = 1.7 in metastable ion fragmentation and 1.6 in CID fragmentation. These ratios are in good agreement with the ratio of 1.5 calculated assuming complete scrambling of all labile H/D, including the phenolic hydrogen. By contrast, following (CD&CO CI, CID of the MD+ ion resulted (Table 2) in a ratio -[HZ0 + CO]/ - [HDO + CO] = 1.1; this ratio is in agreement with the value of 1.0 calculated assuming that the phenolic hydrogen does not become involved in the H/D scrambling process. However, in the loss of ammonia, the MD’ ion formed by CD, CI fragmented to give -NH,/ - NH,D = 0.79 and the MD+ ion formed by (CD,),CO CI fragmented to give -NH,/ - NH,D = 0.62, both in moderate agreement with the ratio calculated assuming H/D scrambling which does involve the phenolic hydrogen. If the phenolic hydrogen is not involved in the scrambling process a ratio - NH,/ - NH,D = 0.33 would be expected. The rationalization of these rather conflicting results is not entirely clear. We do note, however, that exchange of the phenolic hydrogen of protonated tyrosine for deuterium on collision with ND, did not occur readily [26].

The results for the relatively simple amino acids studied thus indicate that, on the whole, there is complete equilibration or scrambling of all labile hydrogens prior to fragmentation on the metastable ion time scale or following collisional activation of stable ions. It is of interest to con- sider the mechanism(s) for this equilibration and

the energetics involved. There is general agreement that the thermochemically fdvoured site of protonation of simple amino acids is the a-amino group [14,33,37-391. Assuming initial protonation at this site, equilibration of the carboxyl hydrogen clearly involves reversible proton transfer to the carbonyl oxygen as shown in reaction A, Scheme 1. Theoretical estimates [37-391 indicate that the carbonyl proton affinity of glycine is 14-18 kcal mall’ less than the amino proton affinity; there- fore, an excitation energy of at least this magni- tude is required for exchange of the carboxyl hydrogen with the amino hydrogens. It should be noted that Hoppilliard and co-workers 129,371 have estimated that the critical energy for elimination of H,O + CO from protonated amino acids is of the order of 40-45 kcal mol-’ so that excitation energies up to this value can be present in the MH+/MD+ ion without fragmentation occurring. Similarly, interchange of the carboxylic hydrogen in the side chain of aspartic acid re- quires reversible proton transfer from the (Y- amino group to the carbonyl oxygen (reaction B, Fig. 1). Assuming that the remote carbonyl group has a proton affinity similar to that of propionic acid (PA = 192 kcal mol-’ [25] and using PA(aspartic acid) = 217 kcal mol-’ [25]) reaction B should be about 25 kcal mol- ’ endothermic but clearly possible for MH+/MD+ ions with excitation energies below the threshold for fragmentation.

Protonated serine also interchanges the hydroxylic hydrogen of the side chain with the other

PH OH R-CH-c, - R-CH-CC+ A

+tb$ cl AH, 'OH

0 CHz -d+3-!-,6 ‘C.-OH 9

CY 0 ’ ‘p-e-OH - Ii20

CHz F1 c/ \ HO y-c-m C

+b3

Scheme 1.

344 A.G. Hanison, T. Yalcin /International Journal of Mass ,Spectrometty and Ion Processes 165/166 (1997) 339-347

labile hydrogens (reaction C, Fig. 1). Assuming the proton affinity of the hydroxyl group is similar to that of n-propanol (PA = 191 kcal mol-’ [25]) and using PA(serine) = 217 kcal mall’ [25], reaction C is calculated to be about 26 kcal mall’ endothermic and interchange clearly is possible at internal energies of MH+/MD+ below the threshold for fragmentation.

The results for asparagine and leucinamide show that the hydrogens of the amide functions participate in the H/D exchange reactions. It is well-established [40-431 that amides preferen- tially protonate on the oxygen; consequently, the exchange involves H+/D+ transfer from the U- amino function to the less favoured amide nitrogen. Kinser et al. [44], from ab initio calculations, have concluded that the amide nitrogen in glycinamide has a proton affinity - 19 kcal mall ’ lower than the u-amino group. They also have calculated a critical energy for fragmentation of protonated glycinamide to CH, =NH,i + CO + NH, of - 46 kcal mall’ and a critical energy for transfer of a proton from the a-amino group to the amide nitrogen of - 21 kcal mol-‘. Although these energies will be somewhat different for protonated leucinamide, it appears very feasible that H/D interchange between the amine and the amide functions of leucinamide can occur at internal energies below the threshold for fragmentation.

The thermochemical data relevant to the asparagine system are less complete. The oxygen proton affinity of formamide has been reported as 201 kcal mall’ [42] while the oxygen proton affinity of acetamide is 206 kcal mall’ [25]. From ab initio calculations Lin et al. [45] have estimated that the nitrogen proton afhnity of formamide is - 14 kcal mall’ less than the oxygen proton aflinity. One can thus estimate that the proton affinity of the amide nitrogen in asparagine should be in the range 190-195 kcal mol-’ compared to a proton affinity of - 220 kcal mall’ for the amino group. Thus, the critical energy for H/D interchange between the amino and amide nitrogens is likely to be of the order of 30 kcal mall’ and interchange is expected at internal energies below the critical energy for fragmentation.

The interchange of the phenolic hydrogen of protonated tyrosine with other labile hydrogens, at least in some cases, is particularly intriguing. The proton affinity of phenol is 196 kcal mall’ [25] but this is known [46] to refer to protonation of the aromatic ring. DeFrees et al. [47] have estimated that the oxygen proton affinity of phenol is 13-20 kcal mol-’ less than the ring proton aftinity. Assuming that this lower proton affinity (176-183 kcal mall’) applies to the phenolic hydrogen of tyrosine and using PA(tyrosine) = 222 kcal mall’ [25], proton transfer from the amino position to the phenolic position, necessary for interchange to occur, should be 39-46 kcal mol-’ endothermic and, thus, require an excitation energy close to that required for fragmentation by loss of H,O + CO. The experimental results show that MD+ ions with energies at essentially the fragmentation threshold (metastable ions) interchange with the phenolic hydrogen but MD’ ions with lower internal energies which are activated by collision do not interchange with the phenolic hydrogen, possibly because the exchange rate is slow. It has been observed [26] that in the H/D exchange of protonated tyrosine with ND, the phenolic hydrogen does not participate.

3.2. Proton mobility in protonated peptides

The metastable ion fragmentation reactions (CD, CI) and CID reactions ((CD,),CO CI) of the MD+ ions of a few simple peptides also were examined. Protonated H-Leu-Gly-Gly-OH and protonated H-Gly-Leu-Gly-OH fragment on the metastable ion time scale exclusively by elimination of neutral glycine to form B, ions, nominally H-Leu-Gly and H-Gly-Leu; these also are major fragmentation channels following collisional activation. Fragmentation of the MD+ ion of the former tripeptide gave [B2 - H]/[B, - D] = 1.2 in metastable ion fragmentation and 1.1 following collisional activation, while fragmentation of the MD’ ion of H-Gly-Leu-Gly-OH gave [B, - H]/[B, - D] = 1.1 (metastable ion) and 1.0 (CID). (The symbolism B, - H and B, - D represent, respectively, B, ions containing only the H isotope and B, ions containing one D.) The ratios observed experimentally are in good agreement

A.G. Ham’son, T. Yalcin /International Journal of Mass Spedrome@ and Ion Processes 165/166 (19971339-347 345

with the ratio of 1.0 expected if the added D interchanges completely or randomizes with all labile hydrogens. Protonated H-Gly-Gly-Leu-OH fragments in both metastable and CID by elimination of water and by formation of protonated leucine (Y,). Fragmentation of the MD+ ion of the tripeptide gave - H,O/ - HDO = 2.2 in both metastable ion fragmentation and CID in agreement with the ratio of 2.0 calculated for complete scrambling of all labile H/D. The same peptide gave [H-Leu-OH.H+]/[H-Leu-OH.D+] = 0.56 (metastable ion) and 0.50 (CID) in agreement with the ratio 0.50 estimated on the basis of scrambling of all labile H/D; this estimation assumes that all the hydrogens transferred to the C-terminus amino acid during the formation of the Y, ion originate from labile positions with none originating from carbon-bonded hydrogens, in agreement with earlier labelling studies [18,48].

Protonated H-Ala-Ala-Ala-OH also fragments by loss of neutral alanine to form the B, ion. Fragmentation of the MD’ ion yielded [B, - HI/LB, - Dl = 1.1 (metastable ion) and 1.0 (CID) in good agreement with the calculated ratio of 1 .O. The protonated methyl ester H-Ala-Ala-Ala- 0Me.H’ fragments to form both the B, ion and the Y, ion, protonated alanine methyl ester. Frag- mentation of the MD+ ion gave [B2 - H]/[B, - D] = 0.63 (metastable ion) and 0.71 (CID), both in reasonable agreement with the calculated ratio of 0.67. The ratios [H-Ala-OMe.H+]/[H-Ala- OMe.D’l = 0.56 tmetastable ion) and 0.71 (CID) also were observed, in reasonable agreement with the ratio of 0.67 calculated on the basis of scrambling of the labile H/D. We also have examined the fragmentation reactions of the MD+ ions of the acetylated peptides Ac-Ala-Ala-Ala-OH and Ac-Ala-Ala-Ala-OMe. Both fragment to form the Yz ion and the B, ion AC-Ala-Ala+. In both cases the results showed that the Y, ion quantitatively incorporated the added deuterium as would be expected if only labile hydrogens are involved in the transfer that is necessary to form Y?. Frag- mentation of the tripeptide gave [B3 - H]/[B, - D] = 1.6 (me&stable ion) and 1.5 (CID) in agreement with the calculated ratio of 1.5. Fragmenta- tion of the methyl ester gave [B3 - H]/[B, - D] = 1.1 in both metastable ion and collisionally

activated dissociation, again in agreement with the ratio of 1.0 calculated on the basis of scrambling.

4. Conclusions

The present work extends and expands on earlier work [18-201 in showing a high mobility of the added proton in protonated amino acids and peptides. For those systems examined, the results show essentially complete scrambling of all labile H/D in MD’ ions fragmenting unimolecularly on the metastable ion time scale or following collisional activation. The results thus support fully the mobile proton model [17] which has been proposed to rationalize the fragmentation channels of protonated peptides. However, it should be noted that we were unable to prepare by CI the MD+ ions of arginine and histidine or of peptides containing these amino acids and it remains possible that in these systems the proton is less mobile. This could be particularly true for arginine-containing peptides, where the critical energy for H+/D’ migration may exceed the critical energy for fragmentation because of the high proton affinity of arginine (PA = 245 kcal mall’ [491X In this respect, we note that the lowest energy fragmentation reactions of protonated arginine (loss of NH,, loss of (H,N),C=NH and formation of (H,N),C+) [27] all centre around the guanidinyl function which is the site of protonation.

Acknowledgements

The authors are indebted to the Natural Sciences and Engineering Research Council (Canada) for financial support.

Appendix

If the labile H/D become completely ran- domized, the label distributions expected can be calculated from the number of combinations (M) of II things taken p at a time

M = n!/p!(n - p>! (1)

Thus, for loss of water from the MD’ ion of leucine one derives

346 A.G. Ham’son, T. Yalcin /International Journal of Mass Spectrometry and Ion Processes 165 /166 (1997) 339-347

M HZO=3!/2!1!=3

M uDO = M, x M, = (3!/1!2!) X (l!/l!O!) = 3

Thus one predicts - [H,O + CO]/ - [HDO + CO] = 1.0.

For loss of ammonia from the MD + ion of lysine one derives

M ,,=5!/3!2!=10

M NH,D = Mu, x M, = (5!/2!3!) x (l!/l!O!) = 10

Thus, one predicts -NH,/ - NH,D = 1.0.

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