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JOURNAL OF MASS SPECTROMETRY, VOL.

30, 339-347 (1995)

Proton Transfer Reactions of Multiply Charged


Peptide and Protein Cations and Anions

Rachel R. Ogorzalek Loot and Richard D. Smith*


Chemical Methods and Separations Group, Chemical Sciences Department, Pacific Northwest Laboratory, Richland,
Washington 99352, USA

Two types of gas-phase proton transfer reactions were examined with electrospray ionization-generated peptide and
protein ions; (i) bases reacting with multiply protonated molecules and (ii) acids reacting with multiply deprotonat-
ed molecules. For reactions of type (i) with bases spanning a range of proton affinities, the proton transfer reaction
rate was observed to increase with increasing proton affinity of the charge transfer reagent. Proton transfer was not
observed for the low proton affinity reagents (ethyl acetate, acetonitrile and water). These studies also qualitatively
measured for the first time the temperature dependences for reactions with multiply charged peptides and proteins.
Negative temperature dependences were observed for the weaker bases and positive dependences for the stronger
bases. A negative temperature dependence was also observed in the reaction of propionic acid with ( M - nH1"-
ions. Two hypotheses are proposed to explain the data. In the first, negative temperature dependences are attributed
to slightly exothermic reactions, while the positive dependences may reflect contributions from a competing clus-
tering pathway, a pathway which could be more dominant with the heavier reagents. Alternatively, the positive
temperature dependences may reflect the barrier in the reaction coordinate arising from the repulsion of like-
charged ions, while negative temperature dependences could reflect a cluster-mediated reaction in which charge
delocalization lowers the barrier to proton transfer. In the latter cases, clustering is invoked with the lower proton
affinity reagents because of the higher concentrations employed.

INTRODUCTION have pursued similar studies with quadrupole mass


Ikonomou and Kebarle" electro-
sprayed ions into a reduced-pressure reaction chamber
The development of electrospray ionization mass
containing tributylamine, while Feng and Konishi"'
spe~trometry'-~(ESI-MS) not only offers new capabil-
flooded an enclosed atmospheric pressure spray region
ities in molecular mass and structure determinations,
with ammonia. Lin and SunnerI8 examined positive-ion
but also opens up new classes of gas-phase ions for
charge distributions after exposure to gas-phase acids.
reaction studies. Ion-ion and ion-molecule reactions
Smith and c ~ - w o r k e r s ~used
~ - ~ the
~ 'Y-tube' inlet/
have been carried out previously with ESI-generated
reactor to expose ions to varying flows of reagent at
ion^.^-^' Studies of positive ion-negative ion reactions near atmospheric pressure. Based on such studies,
at our laboratory led to the development of a Y-shaped Winger et uLl3 argued that under more extreme inter-
capillary inlet-reactor which merges streams of
face conditions, ESI-generated cations can transfer
oppositely charged ions at near atmospheric pressures
protons to H 2 0 , and Ogorzalek Loo and co-
and transfers them to the mass ~pectrometer.~.'Ion-ion
w o r k e r ~ ~ ~have
, ' ~ , demonstrated
~~ that a disulfide-
experiments were pursued for which both reactant ions intact protein is more reactive to proton transfer than
were generated by ESI and in experiments where one an equally charged disulfide-reduced protein. Related
reactant was generated by ESI and the other was pro- ion-molecule reaction studies3' in an r.f.-only collision
duced in an electrical discharge in air. Proton transfer4 cell yielded several molecules of ammonia clustered to
and charge inversion5 reactions were studied with this the precursor ion and also demonstrated proton trans-
approach. fer.Iab Recently, Cassady et ul.lsc measured rates for
Experiments probing ion-molecule chemistry have proton transfer from ubiquitin.
been more common, including (i) proton trans- Gas-phase H-D exchange has been applied to multi-
fer,6-1 2.14-1 8 (ii) hydrogen-deuterium (H/D)
ply charged ions by McLafferty and c o - w ~ r k e r s 'and
~
exchange,' 3 * 1 9 (iii) collisional d i s s o c i a t i ~ n , ~(iv)
~ -col-
~~ by Winger et a l l 2 using a Fourier transform mass
lisional scattering,' 1,20*21 and (v) clustering30 reaction
spectrometer and a quadrupole mass spectrometer,
studies. McLuckey and c o - ~ o r k e r s ~were - ~ the first to respectively. The Cornell study" observed less H-D
demonstrate the dependence of reactivity on charge exchange for disulfide-linked proteins than for their
state for proton transfer of large multiply charged ions. reduced counterparts; both studies indicated that of the
Their quadrupole ion-trap experiments yielded rate
rapidly exchanged hydrogens in equally charged ions,
measurements for reactions with amines6 Other groups the disulfide-linked proteins exchanged H for D at
higher reaction rates than the disulfide-reduced pro-
* Paper prepared for Publication in Organic Mass Spectrometry. teins. These observations and the Fourier transform
t Present address: Protein and Carbohydrate Structure Facility,
2552 MSRB 11, University of Michigan, Ann Arbor, MI 48109-0674, mass spectrometric observations of differing exchange
USA rates and total exchanges for ions generated from differ-

CCC 1076-5174/95/020339-09 Received 7 July 1994


0 1995 by John Wiley & Sons, Ltd. Accepted 12 September I994
340 R. R. OGORZALEK LOO AND R. D. SMITH

ent solution conformers argue that elements of solution- inlet. Actual gas temperatures are expected to be lower
phase higher order structure are preserved in the gas than the inlet wall temperatures indicated owing to heat
phase." Recently, collisional scattering studies have transfer limitations. Additional desolvation was
supplied further evidence for gas-phase conformational achieved by collisional dissociation in the capillary
differences in proteins.".20.2' outlet-skimmer (0s)region at potential differences of
Numerous collisionally activated dissociation (CAD) 0-460 V.
studies of multiply charged ions have now been report- Ammonia (NH,), methylamine (MA), dimethylamine
ed,22p29ranging from demonstrations of facile cleavage (DMA) and trimethylamine (TMA), with proton affin-
at pro line^,^^ to differences in the cleavage between ities 871.5, 918.8, 953.1 and 973.6 kJ mol-', respec-
disulfide-intact and disulfide-reduced proteins,2hs27to t i ~ e l ywere
, ~ ~ delivered through a pressure regulator (set
CAD-MS comparisns of sequence homologies among for 1 atm delivery) and a needle valve. Water, acetonit-
proteins from different species.28 Collisional disso- rile and ethyl acetate, with proton affinities of 696.6,34
ciation of intact proteins as large as RMM 66 00028 789.133 and 849.4 kJ mol- 1 , 3 3 respectively, were intro-
and 15000OZ9has been reported. duced as mixtures with room air by employing a heated
In this paper, we explore proton transfer reactions of bubbler assembly upstream of shut-off and needle
multiply charged ions, determining the role of reagent valves. Diethylamine (DEA), triethylamine (TEA). and
proton affinity and reaction temperature. Reactions tributylamine (TBA), liquids at room temperature, were
with negatively charged proteins and gas-phase acids also introduced in this manner. Their proton affinities
are also considered. The role of proton affinity is have been determined to be 977.0, 1002.5 and 1017.5 kJ
worthy of examination in our inlet/reactor studies mol- ', r e ~ p e c t i v e l y .Addition
~~ of hexane-l,Bdiamine,
because of the reported reactivity of H 2 0 with multiply a solid at room temperature, (proton affinity estimated
charged proteins.' Our experimental assembly does to be 22.6 kJ mol-' higher than that of TEA)34 and
not insure that we probe thermodynamics; the results propionic acid, a liquid at room temperature, also
more likely reflect reaction kinetics. However, the employed the heated bubbler assembly. The gas lines
experiments are still of interest owing to the wide differ- were maintained at a higher temperature than the
ences observed in proton transfer rates for multiply bubbler assembly in order to prevent solidification in
charged ions,' in contrast to those usually observed for the gas lines. The mass flow for particular needle valve
singly charged ions. settings was determined by measuring the evacuation
rate of air-filled calibrated gas sampling bags and cor-
recting for the molecular mass of the reagent. For air-
EXPERIMENTAL reagent mixtures, the flow rate was corrected by the
average molecular mass of the mixture; reagent vapor
pressures were estimated using the Clausius-Clapeyron
Application of the inlet/reactor approach to proton equation and Trouton's rule.
transfer reactions of multiply charged ions has been One of the first tests performed with our inlet/reactor
described ' only a brief description of the was a confirmation that protein charge-state distribu-
apparatus is provided here. These experiments differ tions did not change on addition of air flowing through
from previous studies by quadrupole ion-traphp9 and the assembly at various needle valve settings The results
Fourier transform mass s p e ~ t r o m e t r y ' ~ ~ ~ ' ~ in a
(FTMS) of this test demonstrated that any shifts in protein
reaction occurs in the atmospheric pressure/vacuum charge-state distributions observed with amine reagents
interface, employing higher reagent gas flows. Reactions arose from reaction with the amine and were not arti-
in the high-pressure environment can rely on both bi- facts generated by the change in flow rate through the
and termolecular collisions. inlet/reactor.
The electrospray ionization atmosphere/vacuum All biochemical samples and diethylamine were
interface employed Y-shaped inlet/flow reactors con- obtained from Sigma Chemical (St Louis, MO, USA),
structed from three lengths of 0.16 cm od., 0.10 or 0.05 with the exception of bovine proinsulin, which was
cm i.d. stainless-steel tubing. Inlet/reactors were fabri- obtained from Novo BioLabs (Danbury, CT, USA) and
cated by silver soldering the tubing to a stainless-steel cr-galactosidase, obtained from Calbiochem (La Jolla,
post drilled with a 0.10 cm diameter Y-shaped CA, USA). Ethyl acetate, acetonitrile, methanol,
~ h a n n e l . ~ - ~ One
. ' ~ , 'arm
~ of the 'Y-tube' transported dimethylamine, trimethylamine, triethylamine, tribu-
ions while the other arm delivered the neutral proton tylamine, hexane- 1,6-diamine and propionic acid were
transfer reagent. Mixing of the two gas streams acquired from Aldrich Chemical (Milwaukee, WI,
occurred in the Y-shaped channel and the third arm USA). Ammonia and methylamine were purchased from
delivered the mixture to the mass spectrometer. An Matheson (East Rutherford, NJ, USA). Doubly distilled,
interaction time of a few ms is estimated for the protein deionized water was used for preparing protein solu-
ions with the reagent. Use of the 'Y-tube' apparatus in tions and as a protein transfer reagent.
ion-molecule reactions isolates the solution at the tip of
the ESI needle from the reagent, preventing elevation of RESULTS AND DISCUSSION
solution pH from exposure to amines.
The capillary was electrically heated with currents
from 11.0 to 14.5 A to drive the ESI process (e.g. desol- Reactions of a range of bases with cations
vate the droplet^).^"^^ Capillary temperatures were
determined by a chrome1 alumel thermocouple attached Proton transfer reactions of amines with multiply
to the outer wall of the atmospheric pressure side of the charged peptides and proteins shift charge-state dis-
PROTON TRANSFER REACTIONS 341

tributions very efficiently in the capillary inlet/reactor.


Figure 1 shows an ESI mass spectrum for melittin with 100
and without triethylamine ( < 1 x lo-' mol sec-') 80
where the highest charge state observed has decreased 60
by 2 charge states. On addition of more than
40
1.6 x mol s-' of dimethylamine to a-galactosidase
(RMM 85 OOO), the maximum charge observed shifted 20
+
from over 100 to approximately 65 . In addition to + -
less highly charged protein ions, protonated amines are v
s0
also observed as products in these experiments. They x
.-
c
40
are only observed when the electrospray is operating 30
and the amine reagent is flowing through the inlet/ a,
c
.-I= 20
reactor. These ions are attributed to proton transfer
from protonated solvent, protonated solvent clusters
a,
.=
>
-am
10 I ! I
and multiply protonated peptide and protein ions.
The role of proton affinity in shifting charge distribu- n 0 h li I\ i 7+

tions was examined by reacting horse heart cytochrome


c (RMM 12360), bovine ubiquitin (RMM 8565) and
4.0

3.0 (M+7H)7*
c l1
horse heart myoglobin (RMM 16591.5) with TEA,
TMA, MA and ammonia under the same conditions
(Table 1). Note that our observations for lower charge
states are limited by the 0-2000 m/z limit of our mass
spectrometer. Nevertheless, several conclusions can be
2.0

1 .o

0.0
6 1 1000 1400
8+
l i
li
1800
drawn from the data: (i) increasing the reagent flow m/z
increases the shift in the charge state distribution, i.e. Figure 2. Proton transfer from equine cytochrome c (RMM
yields a lower average charge for the protein; (ii) when 12360) to ammonia. NH, flow: (a) 0; (b) 6.4 x lo-'; mol sec-';
equal amounts of proton transfer reagents react with a (c) 2.4 x mol sec-'. The protein was electrosprayed from 5%
acetic acid-water with a methanol sheath, with a capillary inlet
protein, the reagent with the higher proton affinity shifts temperature of 100°C and 0s = +175 V. Dots in (a) indicate cyto-
the charge distribution to lower charge; and (iii) reac- chrome c dirner ions. A sulfate or phosphate adduct is observed in
tions are observed with NH, , despite its proton affinity (C).
being lower than that of the isolated amino acids (Fig. 2
illustrates the reactivity of NH, with cytochrome c).
The results for reactions with higher flows of TEA proton affinity reagent hexane-l$-diamine. Reactions
and hexane-1,6-diamine from our earlier with a under those conditions may be limited by the basicities
low-frequency quadrupole mass spectrometer (m/z 0- of the remaining arginines.
45000) are consistent with those in Table 1, although Owing to the short interaction time in the inlet/
little difference is observed between charge shifts reactor, these observations are likely to reflect the
obtained with TEA and those obtained with the higher kinetics of proton transfer from multiply charged ions.
They demonstrate that the reaction rates increase with
increasing proton affinity of the amine reagent, at least
for the low charge states to which our measurements
are most applicable. This result is consistent with Ikon-
1001 a (M+4H)4'
omou and Kebarle's suggestion" that the proton trans-
1 fer rates for multiply charged proteins might increase as
80 -
stronger bases were employed. They noted that for
60 5+ singly charged ions with exoergic deprotonation reac-
h

>
4 0 ~ 1 tions, rate constants are generally close to collision
3+ rates, while the slower deprotonation reactions of multi-
5
a
C,
c
.-I
20
0~
b
h l (I, I, 2+
ply charged cytochrome c with dimethylamine6 could
suggest that the reactions were endoergic. Recently,
a, ~..~ Cassady et aI.'sc also demonstrated that proton transfer
.?
c 80
-
m reaction rates for ubiquitin increased with the base's
$ 60- proton affinity.
- 1 40
Caution is necessary in extrapolating to the reactivity
of charge sites in a protein based on the proton afinities
20 of the isolated amino acids. Clearly the proton affinity

0
4: , !L 2+
of the isolated amino acid is influenced by its free amino
terminus. Moreover, intramolecular cyclizations,
2!0 500 800 1100 1400'
m/Z
increasing the proton affinity of free lysine, for example,
will have a different probability in the protein. Estimat-
Figure 1. ESI mass spectrum of melittin (RMM 2845) (a) ing the contributions from the entropy of reaction will
without and (b) with <1 x lo-' mol s-' triethylamine. The
peptide was electrosprayed from 5% acetic acid-water with a
be complicated in the protein. Despite all these caveats,
methanol sheath, with a capillary inlet temperature of 150 "C and the reactivity of NH, with multiply charged proteins is
an orifice-skimmer ( 0 s )bias = +250 V. still surprising and argues that electrostatic interactions
342 R. R. OGORZALEK LOO AND R. D. SMITH

Table 1. Cytochrome c, ubiquitin and myoglobin charge distributions after


proton transfer
Protein Reagenta Flow (mol sec-') Charging Maximum charge

Cytochrome c - 0 7+ to 20+ 16+


NH3 1.8 x 7+ to 14+ 11+
NH3 6.4 x l o - " 7+ to 12+ 9+, 10+
NH3 9.1 x 7+ to l o + 9+
NH3 3.7 x 10-5 7+ to lo+ a+, 9+
NH, 2.4 x 7+ to 8+ 7+
MA 4.8 x 7+ to 10+ 9+
MA 6.9 x 7+ to 9+ 7+
MA 2.8~10-~ 7+ 7+
TMA 9.7~ 10-7 7+ 7+
TMA 3.4 x 10-6 7+ 7+
TEA 4.5 K 10-7 7+ to 9+ 7+
TEA 6.5 x 7+ to 8+ 7+
TEA 2.6 x 7+ to 8+ 7+
Ubiquitin - 0 5+ to 12+ 1o+
NH3 6.4 x 5+ to 10+ 7+
NH3 9.1 X 54- t 0 7 + 7+
NH3 3.7 x 10-5 5+ to 7+ 7+
NH3 24 x 5+ to 6+ 6+
MA 4.8 x 5+ to 7+ 6+
MA 6.9 x 5+ to 6+ 5+
TMA 9.7 x 5+ to 6+ 5+
TEA 4.5~10-~ 5+ 5+
Myoglobin - 0 10+ to25+ 19+
NH, 6.4 x 10+ to 18+ 16+
NH3 9.1 x 10+ to 17+ 14+
NH3 3.7 x 10-5 9+ to i s + 12+, 13+
NH3 2.4~10-~ 9+toll+ 9+, 10+
MA 4.8 x 9+ to 14+ 12+
MA 6.9 x 9+ to 12+ 10+
TMA 9.7~10-7 g+toii+ 9+
TMA 3.4 x 10-6 9+ to 10+ 9+
TEA 4.5 x 10-7 9+ to l o + 9+
TEA 6.5 x 10-7 9+ 9+
a M A = methylamine; TMA-trimethylamine; TEA = triethylamine.

probably contribute substantially to proton transfer in The reactivity of water at flow rates ranging from 0 to
these systems. Proton transfer reactions with ammonia 8.3 x mol s p l was explored with horse heart cyto-
in a triple quadrupole mass spectrometer have also been chrome c. With stable H,O flow rates, shifts in the
reported recently.' 1cv18b protein charge-state distribution were not observed, in
In the light of the reactivity of ammonia, we won- contrast to an earlier report from our l a b ~ r a t o r y . ' ~
dered about the behavior of weaker bases. Reactions Recent FTMS studies' also failed to observe proton
with the lower proton affinity reagents ethyl acetate transfer to water from multiply protonated ubiquitins.
(839.7 kJ mol-I), acetonitrile, (788.3 kJ mol- I ) and The previous study was conducted under more extreme
H,O (696.6 kJ m 0 1 - l ) ~were
~ examined. No evidence of conditions (reaction time), which may account for the
charge-shifting reactions was observed for cytochrome different behavior observed. In this work, shifts in the
c, myoglobin and ubiquitin with ethyl acetate flows charge distribution were only observed for occasional
ranging from 0 to 1.6 x lo-' mol s - ' or acetonitrile scans and appeared to correlate with transmission of
flows ranging from 0 to 2.4 x lo-' mol s-'. Both water droplets through the reagent line. In those cases,
reagents showed adduct formation (e.g. protein-ethyl the resulting charge state distributions peaked at the
acetate complexes) for the high charge states of the [M + 10H]"+ charge state, independent of the vapor
three proteins (see Fig. 3). These adducts arise from flow rate, behavior inconsistent with a proton transfer
clustering in either the expansion or in the near- reaction. These results indicate that under the present
atmospheric pressure region of the capillary inlet, or conditions, observable proton transfer from multiply
from both regions. Regardless of where the adducts charged proteins requires bases stronger than ethyl
form, at modest capillary-skimmer biases they bear the acetate.
signature of an ion-molecule interaction-more adducts A previous study from our laboratory employed a
reside on high than on low charge-state protein ions. quadrupole mass filter with an m/z 45000 range to
Similar behavior has also been observed with the amine examine to extent of proton transfer from proteins to
reagents. TEA, TMA and hexane-1,6-diamine.I7 The lowest
PROTON TRANSFER REACTIONS 343

100 (M+1 OH)!O+ a 1 a


40 - (M+7H)7+
80
30 ~
I
3
w
60
40 v
-8 2 0 ~

.x
G 20 2
cn
1 0 ~
K c 5+
2 0 2
c 0~ I
C (M+1 0H)I O+ .-
I-

a, I 9 100~
(M+7H)'+ b
5
'
i ; 45 .-
c
-
a 2 8 0 ~ I
a,
?I 1o+
LT 30 60
I
40
15

0
6 I 1000 1400
d Z
1800
2o
0 ,!
?, ,I, 1 Jl
1

d Z
,I
Figure 3. Bovine ubiquitin (RMM 8565) in 1 : 1 methanol-5% Figure 4. Temperature dependence of proton transfer from
acetic acid-water electrosprayed with a methanol sheath with a bovine ubiquitin to 6.4 x mol s-' NH, with a capillary inlet
capillary temperature of 115°C and 0s = +135 V. (a) Without temperature of (a) 55 and (b) 150°C. The protein was electro-
ethyl acetate; (b) with 9.5 x mol s-' ethyl acetate. The sprayed from 1 : 1 methanol 5% acetic acid-water with a methanol
bimodal charge state distribution reflects contributions from two sheath and with 0s = +175 V.
conformer^.^'-^^

number of charges observed was close to the number of For these studies, a negative temperature dependence
arginine residues present in the proteins. Limited to an is defined as one in which the charge state distribution
m/z 2000 range for these experiments, we observed that shifts to higher charge with increasing temperature, i.e.
the abundance of the melittin [M 2HI2+ ion was + proton transfer reaction rates decrease with increasing
only weakly affected by increasing the flow of DMA, temperature. Similarly, for a positive temperature
implying that this ion is unreactive to DMA. Melittin dependence, proton transfer reaction rates increase with
has two arginines, three lysines and a free amino ter- increasing temperature and the charge-state distribution
+
minus; hence a shift to the 2 charge state is consistent shifts to lower charge. Temperature dependences were
with the remaining charges residing on the two argin- also measured without amines for acidic solutions of
ines. However, the melittin sequence has four potential myoglobin, cytochrome c and ubiquitin, to confirm that
charge sites in close proximity (residues 21-24; KRKR-), the charge distributions did not shift in the absence of a
and so another explanation for the stability of the dica- proton transfer reagent. Particular care must be taken
tion might be based on electrostatic (i.e., Coulombic)
considerations allowing only one charge site within resi-
dues 21-24 and another on the lysine at residue 7. The
N-terminal glycine is another potential charge site, but
its proton affinity is expected to be less than that of
DMA, while the values of the isolated amino acids
lysine and arginine are higher than that of
D M A . 3.3
~ 5,36
h
30
20 -
la 11+
(M+1 0H)I c+

I
9+

w
8
x 10-
Temperature dependences of proton transfer reactions .-
c
cn
c
s! 0 ~ - r h L J . L
Charge-state distributions for various peptides and pro- .-C b (M+9H)'
teins were examined with a nearly constant flow of $ 100-
.-
c
amine reagent as the capillary temperature was varied, 5
m 8 0 ~
as shown by examples given in Figs 4 and 5. (Because of n:
gas dynamics through the inlet, there is actually a small 60
decrease in flow with increasing temperature, but the
effect is small compared with the temperature depen-
dences observed.) Interface conditions other than the
capillary temperature were held constant. As is appar-
20
40
0
I 10+
I
1
j
IL
600 1000 1400 1800
ent from the measurements summarized in Table 2, d Z
weak bases display a negative temperature dependence
in their reactions with proteins, while stronger bases Figure 5. Temperature dependence of proton transfer from
equine myoglobin (RMM 16951.5) to 9.7 x lo-' mol s-' TMA
display a positive temperature dependence. The base at with a capillary inlet temperature of (a) 65 and (b) 160°C. The
which proteins change the direction of their tem- protein was electrosprayed from 5% acetic acid-water with a
perature dependence is protein dependent. methanol sheath and with 0s = +330 V.
344 R. R . OGORZALEK LOO AND R . D. SMITH

Table 2. Temperature dependences for proton transfer from proteins to amines


Most intense charge state
Reagent Proteina Lower temperatureb Higher temperatureb Temperature range (?C) Dependence"

NH, Cytochrome c 10+/11+ 12+ 55-1 80 (-)


Cytochrome c 9+/10+ 11+ 105-280 (-)
Melittin 3+ 4+ 160-290 (-1
Ubiquitind 7+ 8+/9+ 55-1 50 (-)
Myoglobin 15+/16+ 17+ 55-1 50 (-1
MA Ribonuclease A <7+ a+ 60-1 70 (-1
Cytochrome c 9+ 9+/1o+ 65-265 (-)
Ubiquitin 5+/6+ 6+/7+ 65-205 (-)
Myoglobin 12+ 12+ 67-1 70 (0)
DMA Cytochrome c 8+/9+ 7+ 145-270 (+I
D EA Albumin 46+' 40+' 120-240 (+?)
Albumin" 71 +' 63+' 145-270 (+?I
Lysozyme a+ 9+ 125-260 (-1
Lysozymee 1 o+ 12+ 130-260 (-1
Aprotinin 4+ 4+ (0)
125-235
Aprotinin' 5+ 5+ (-7)
125-235
Cytochrome c 9+ 7+ (+)
120-245
Cytochrome c9 9+' 8+' (+I
120-245
Ubiquiting 5+/6+ 6+/7+ (-1
80-1 80
Ubiquiting 6+/7+ 6+ (+?I
180-260
TMA Myoglobin 1 o+ <9+ (+I
65-160
Cytochrome c 9+' 7+' 60-1 20
(+)
Ubiquitin 5+/6+ 5+/6+ (0)
60-1 60
TEA Myoglobin 11+' lo+' (+I
75-1 20
Myoglobin 9+/10+ <9+ (+I
90-1 95
Cytochrome c 9+' 7+' (+I
60-1 55
Ubiquitin 6+' 5+' 75-1 55
(+)
a Proteins were electrosprayed from a 5% acetic acid-water solution with a methanol sheath, unless

indicated otherwise.
For a constant flow of amine reagent, the charge state of maximum intensity shifts from the first value
to the second across the range of temperatures given, unless indicated otherwise.
(-) indicates that the charge state increases for an increase in temperature, i.e. reaction rates decrease
for increasing temperature; (+) indicates that the charge state decreases for an increase in temperature,
i.e. reaction rates increase with increasing temperature; (0) indicates no apparent temperature depen-
dence.
Electrosprayed from 5% acetic acid-1 : 1 methanol-water with a methanol sheath.
Disulfide bonds reduced.
'Highest charge state observed.
Electrosprayed from water with a water sheath liquid flow.

with such measurements of positive temperature depen- charged data is not so simple; it does not explain why
dences in order to insure that contributions from ther- the temperature dependence changes sign for reactions
mally induced dissociation3 1,32 or thermal activation with more basic reagents, where the reaction is expected
followed by collisional dissociation in the interface to be more exergonic. It has been noted42 that the sign
(effects which also appear to shift the charge distribu- of AH for a reaction does not necessarily specify the
tion to lower charge states) are not misinterpreted as temperature dependence of the rate constant for systems
differences in proton transfer reaction rates. We based where the height of the central barrier in the reaction
our conclusions about positive temperature depen- path relative to the reactant channel is the determining
dences on observations obtained at temperatures and factor. In those cases, both exothermic and endothermic
interface conditions that did not yield such product directions may show a negative temperature coefficient
ions. It was confirmed that charge distributions did not for the reaction rate constant.42 Bursey and P e d e r ~ e n ~ ~
change with temperature in the absence of reagents for have specifically pointed out the problems in extracting
the temperature ranges employed. Temperature depen- thermochemical data from charge-transferring collisions
dences which are indicated by '? in Table 2 were rela- of multiply charged ions, problems due to the large
tively weak. Coulombic barrier arising from repulsion of two ions of
Observation of temperature dependences for these like charge in close proximity. It is clear that the pres-
systems is consistent with previous studies demonstrat- ence of a high barrier justifies positive temperature
ing that low collision efficiency reactions often proceed dependences for reactions which require thermal activa-
at temperature-dependent rates.40p42 Hence we might tion to overcome the barrier.
be inclined to rely heavily on information gleaned from Unfortunately, these insights do not appear to
ion-molecule reactions of singly charged ions, noting explain our data fully. If they are used to account for
that exergonic reactions with slow forward rate con- the positive temperature dependences observed with
stants often display negative temperature depen- strong bases, they do not then explain why the slower,
d e n c e ~ .However,
~~ the interpretation of our multiply less exothermic (or less nearly exothermic) reactions
P R O T O N TRANSFER REACTIONS 345

would have negative temperature dependences. The cal- erally adjust interface conditions downstream to lessen
culated repulsive potential based on their contributions to our spectra, but they could easily
contribute to the reaction pathway.
H,Nt CH2CH2CH,NH2 + HNR, +

We therefore offer two hypotheses to explain the tem-


-+ H,NfCH2CH,CH2NH2.-.HNR3+ perature dependences observed, both invoking clus-
tering. The first argues that competition from clustering
yielded values of +138.62 kJ mol-' for R = H and reduces proton transfer rates, the second argues that
+ '
144.43 kJ mol- for R = CH, .43 Based on these cal- clustering increases proton transfer rates by lowering
culations, it does not seem likely that an argument the barrier in the exit channel. The first hypothesis
could be made for an increase in barrier height for argues for enhanced clustering with the heavier bases
the reaction with TEA versus NH, that would be (despite their lower concentrations in the experiment),
large enough to alter the temperature dependence of the whereas the second argues enhanced clustering with the
reactions. lighter bases because of the much higher flows
It is interesting to speculate whether there may be employed. Future measurements of proton transfer
two different reaction mechanisms. Certainly the pres- rates with various bases may differentiate between these
ence of two mechanisms would easily explain why the mechanisms.
reactions of diethylamine with ubiquitin show negative These experiments did not investigate whether the
temperature dependences below 180 "C and positive temperature dependences varied with charge state. Such
temperature dependences above 180 "C. In one scenario, an investigation is potentially interesting. Clearly, the
the negative temperature dependences may reflect an amount and location of charge will affect the energetics
exergonic reaction, while the positive temperature of reactions, which could also explain why the base at
dependences may reflect competition from a clustering which proteins charged their charge dependence was
pathway, less favorable at higher temperatures. Clus- protein dependent. Steric effects may also modulate the
tering may be more of a factor with the heavier bases contributions of different channels.
because of the larger number of states in the complex. Yet another explanation supporting two different
McLuckey et d9 observed increased clustering for mechanisms for proton transfer might differentiate
hexane- 1,6-diamine in ion-trap experiments. between proton transfer to different amino acids,
An alternate scenario attributes the positive tem- arguing, for example, different behavior for transfer
perature dependences to a barrier in the reaction coor- from lysine versus histidine. That explanation appears
dinate, while the negative temperature dependences are to be invalid, particularly when one considers that cyto-
attributed to cluster-mediated proton transfer, a mecha- chrome c, which displays base-dependent temperature
nism which would be most favorable at low tem- dependences, has 19 Lys but only 2 Arg and 3 His. In
peratures, when cluster dissociation would be lessened. the cytochrome c charge-state population for which we
Should a charge-state neighbor many ammonia mol- probe temperature dependences, it seems likely that we
ecules, or primary or secondary amines (i.e. for heavily see transfers from lysines primarily. Moreover, because
clustered or 'solvated' species), it might be possible to our temperature dependence studies focus on the same
invoke charge delocalization at the transition in which charge-state populations for reaction with different
the amine cluster acquires a proton. Doing so could bases, it seems unlikely that different residues would
effectively increase the distance of the repulsive inter- react to yield the same product charge-state distribu-
action, decreasing the height of the barrier to proton tions depending on which base was employed.
transfer. Cluster formation leading to proton transfer
need not limited to the capillary inlet/reactor; it will
also arise in the supersonic expansion where lower tem- Proton transfer from propionic acid to anions
peratures arising from the cooling in the expansion
enhance cluster stability. Inlet temperatures still relate Reactions were also explored in which a gas-phase acid
to the amount of clustering occuring downstream in the (propionic acid) transfer protons to [M - nH]"-
supersonic expansion, as is evident from the fact that protein ions generated by ESI. Previous studies'
heated nozzles are used in molecular beam studies to employed an ion trap to explore proton transfer to
minimize van der Waal's c ~ m p l e x a t i o n ? The
~ possi- multiply deprotonated ions. A-chain insulin (oxidized
bility for reactions facilitated by cluster formation in the RMM 2532), equine myoglobin (RMM 16951.5) and
supersonic expansion would be unique to our inlet/ porcine pepsin (RMM 34584), selected on the basis of
reactor approach; thus, a different experimental our previous negative-ion ESI studies of polypeptides
approach may eliminate reactions with negative tem- and protein^:^ were electrosprayed from 3% ammonia-
perature dependences. We also point out that our water solutions with a methanol sheath. The results
experiment would be particularly susceptible to cluster from these reactions, summarized in Table 3, demon-
contributions for the weaker bases because the tem- strate that proton transfer from gas-phase acids to
perature dependences of the same charge-state popu- multiply deprotonated polypeptide and protein ions can
lations were examined with different proton transfer be studied with the inlet reactor. For myoglobin and
reagents. With weak bases, much higher reagent flows pepsin, the reactive groups should be the acidic residues
were required to shift the distributions to low charge Glu and Asp and the C-terminus. Oxidized A-chain
states, making reagent-protein complexes more likely. insulin has two Glu residues, a C-terminus and four
Amine adducts are readily observed in these experi- SO; groups as potential reaction sites.
ments at low temperatures, low interface voltages or The temperature dependence of the reaction of propi-
high reagent flows. At a given inlet temperature, we gen- onic acid with A-chain insulin (oxidized) was explored
346 R. R. OGORZALEK LOO AND R. D. SMITH

Table 3 A-chain insulin, pepin and myoglobin charge dis- CONCLUSION


tributions after proton transfer with propionic acid
Flow Maximum
These experiments demonstrate that our inlet/reactor
Protein (mol s-') Charging charge provides a suitable basis for studying proton transfer
A-chain insulin 0 3- to 6- 5-
from gas-phase bases to multiply protonated polypep-
(oxidized)
tides. These studies also extend previous proton transfer
1.8 x 3- to 6- 5- studies to gas-phase acids reacting with multiply depro-
3.9 x 10-5 3- to 4- 3- = 4- tonated molecules. In a comparison with bases span-
Pepsin 0 18- t o 3 6 - 28- ning a range of proton affinities, we find that the proton
3.9x 18- to 29- 25- transfer reaction rate increases with increasing proton
Myoglobin 0 9- to 1 7 - 13-~14- affinity of the charge-transfer reagent, consistent with
3 . 9 ~ 1 0 - ~ 9- t0 1 3 - 1 1- FTMS measurements."' Proton transfer was not
observed for the low proton affinity reagents ethyl
acetate, acetonitrile and water, although ethyl acetate
and acetonitrile preferentially formed adducts with the
for a propionic acid flow of 3.9 x mol SKI,as high charge states of the proteins examined.
shown by the example in Fig. 6 . At a capillary tem- These studies also measured the first temperature
perature of 220"C, the [M - 3HI3-and [M - 4HI4- dependences for reactions with multiply charged pep-
ions were observed, with the maximum intensity on the tides and proteins. Based on our examination of reac-
4- ion, but by 140°C, the charge state of maximum tions with ammonia, methylamine, dimethylamine,
intensity had shifted to the 3- ion, and by 110°C the diethylamine, trimethylamine and triethylamine, we find
4- ion had vanished and only the 3- ion was that the temperature dependences are negative for the
observed. The 110"C capillary temperature may have weaker bases and positive for the stronger bases, with
reduced the propionic acid flow through condensation the base at which the temperature dependence shifts
(it is lower than the boiling point of propionic acid), but from negative to positive being protein dependent.
such an action would have been observed as a positive Moreover, the reaction of diethylamine with ubiquitin
temperature dependence, and not the negative depen- displays a negative temperature dependence below
dence indicated. Hence we conclude that the reaction of 180"C and a positive temperature dependence above
oxidized A-chain insulin with propionic acid yields a 180°C. Based on these results, we suggest that two
negative temperature dependence for [M - 3HI3- and mechanisms for proton transfer operate in these experi-
[M - 4HI4- from 110 to 220°C. With data for only ments. Positive temperature dependences may reflect
one gas-phase acid, we find it premature to explain the the barrier to proton transfer arising from the nature of
cause of the negative temperature dependence. the transition state, where two like-charged ions in close
However, by analogy with the amine data, and in the proximity repel. A cluster-mediated proton transfer
light of the high propionic acid flows employed, contri- mechanism is proposed to explain the negative tem-
butions from clustering are plausible. perature dependence with weaker bases; the dependence
arises because clusters dissociate at higher temperatures.
It is suggested that charge delocalization possible with
the cluster mechanism could lower the barrier to proton
transfer by effectively increasing the distance between
a the like charges in the transition state. The inlet/reactor
100
approach encourages cluster formation by the high
(M-I 3 H p
80 reagent flow employed, particularly with weaker bases.
60 l i In addition, clusters can form readily in the supersonic
h

x
40 I. R expansion downstream of the inlet/reactor. Alternative-
ly, the negative temperature dependences may reflect an
c
zj 20 exergonic reaction, while the positive temperature
(I

2 0 dependences may reflect competition from a clustering


._ pathway, less favorable at higher temperatures. In this
a, b
.5
c
40
(M-lIH)I]-
case, the clustering could be more of a factor with the
m
- heavier bases because of a larger number of states in the
2 30
I complex. Negative temperature dependences were also
I observed in reactions of multiply deprotonated poly-
20
i I peptides and proteins with propionic acid.
10

0
Acknowledgements
9
We thank Scott McLuckey for proposing alternative explanations for
the temperature dependences. Joseph Loo, Harold Udseth, John
Figure 6. Equine myoglobin in 3% ammonia-water electro- Fulton, Bill Robins, and Philip Andrews also provided helpful dis-
sprayed with a methanol sheath and 0 s = -375 V and a capillary cussions. We acknowledge support of the US Department of Energy
temperature of 180°C. (a) Without propionic acid and (b) with through the Oflice of Health and Environmental Research and
3.9 x rnol s-' of propionic acid. Charge states are labeled for through internal Exploratory Research of the Molecular Science
the apomyoglobin ions. Dots indicate heme-attached myoglobin Research Center (Contract DE-AC06-76RLO 1830). Pacific North-
ions. west Laboratory is operated by Battelle Memorial Institute.
PROTON TRANSFER REACTIONS 347

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