Hydrogen/Deuterium Exchange

8/13/2019 Hydrogen/Deuterium Exchange

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Research Topic task started on Thu Feb 5, 2004 at 2:36 PM

2 Research Topic candidates were identified in CAPLUS.

using the phrase "electrospray mass spectrometry"

Selected 1 of 2 candidate topics.11692 references were found containing the concept "electrospray mass spectrometry".

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150 references were found when refined using the phrase "deuterium exchange"

Copyrights:

CAPLUS: Copyright 2003 ACS (The UK patent material in this product/service is UK Crown copyright and is made

available with permission. (C) Crown Copyright. The French (FR) patent

material in this product/service is made available from Institut National de

la Propriete Industrielle (INPI).)REGISTRY: Copyright 2003 ACS (Some records contain information from GenBank(R). See also: Benson D.A.,

Karsch-Mizrachi I., Lipman D.J., Ostell J., Rapp B.A., Wheeler D.L.

Genbank. Nucl. Acids Res. 28(1):15-18 (2000). Property values tagged

with IC are from the ZIC/VINITI data file provided by InfoChem.)

CASREACT: Copyright 2003 ACS (Some records from 1974 to 1991 are derived from the ZIC/VINITI data file

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from the period prior to 1986.)

CHEMLIST, CHEMCATS: Copyright 2003 ACS

Bibliographic Information

Dynamics and Ligand-Induced Solvent Accessibility Changes in Human Retinoid X Receptor Homodimer

Determined by Hydrogen Deuterium Exchange and Mass Spectrometry. Yan, Xuguang; Broderick, David;

Leid, Mark E.; Schimerlik, Michael I.; Deinzer, Max L. Departments of Chemistry, Biochemistry and Biophysics

and Pharmaceutical Science, Oregon State University, Corvallis, OR, USA. Biochemistry (2004), 43(4), 909-

917. CODEN: BICHAW ISSN: 0006-2960. Journal written in English. AN 2004:31167 CAPLUS (Copyright

2004 ACS on SciFinder (R))

Abstract

Receptors for retinoic acid act as ligand activated transcription factors. The three-dimensional structure of the

retinoid X receptor (RXR) ligand binding domain has been detd., but little information is available concerning the

properties of the protein in soln. Hydrogen/deuterium exchange followed by electrospray ionization mass

spectrometry was used to probe the soln. conformation of the recombinant human RXR homodimer ligand bindingdomain in the presence and absence of 9-cis-retinoic acid (9-cis-RA). Within the exptl. time domain (0.25-180 min),about 20 amide hydrogens showed decreased exchange rates in the presence of satg. concns. of 9-cis-RA as

compared to those found for the homodimer in the absence of ligand. Most of the amides were located in peptides

derived from regions of the protein shown by the X-ray structure to interact with the bound ligand: the amino

termini of helixes 3 and 9, the two sheets, helix 8, the H8-H9 loop, and the carboxyl terminus of helix 11.Unexpectedly, protection was also obsd. in peptides derived from helixes 7, 10, 11, and the H7-H8 and H10-H11

loops, regions that are not directly in contact with bound 9-cis-RA. These results suggest that the binding of ligand

results in addnl. effects on the conformation or dynamics of the homodimer in soln. as compared to those obsd. for

the X-ray structure. Overall, the change in deuterium exchange induced by the binding of 9-cis-RA correlated


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reasonably well with changes in hydrogen bonding, residue depth, and/or solvent accessibility predicted from the

crystal structure.


Monitoring of high pressure protein denaturation by hydrogen/deuterium exchange and electrospray

ionization mass spectrometry. Petry, Inga; Szewczuk, Zbigniew. Faculty of Chemistry, University of Wroclaw,Pol. Editor(s): Benedetti, Ettore; Pedone, Carlo. Peptides 2002, Proceedings of the European Peptide Symposium,

27th, Sorrento, Italy, Aug. 31-Sept. 6, 2002 (2002), 854-855. Publisher: Edizioni Ziino, Castellammare di Stabia,

Italy CODEN: 69EYXG Conference written in English. AN 2004:28713 CAPLUS (Copyright 2004 ACS on

SciFinder (R))


Elucidation of the fragmentation pathways of azaspiracids, using electrospray ionisation, hydrogen/deuterium

exchange, and multiple-stage mass spectrometry. Sierra, Monica Diaz; Furey, Ambrose; Hamilton, Brett;

Lehane, Mary; James, Kevin J. PROTEOBIO, Mass Spectrometry Centre for Proteomics and Biotoxin Research,

Department of Chemistry, Cork Institute of Technology, Cork, Ire. Journal of Mass Spectrometry (2003),

38(11), 1178-1186. CODEN: JMSPFJ ISSN: 1076-5174. Journal written in English. AN 2003:975783

CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

Collision-induced dissocn. (CID) mass spectra were generated for azaspiracids using electrospray ionisation (ESI),

and hydrogen/deuterium (H/D) exchange was used to ascertain the no. and type of replaceable hydrogens in the three

predominant azaspiracid toxins. H/D exchange was conveniently achieved using deuterated solvents for liq.

chromatog. (LC). Using ion-trap mass spectrometry, multiple-stage CID expts. (MSn) on the protonated and fully

exchanged ions were performed to decipher characteristic fragmentation pathways. The precursor and product ions

from azaspiracids lost up to five water mols. from different regions during MSn expts. and it was possible to

distinguish between the water losses from different mol. regions. These studies confirmed that the first waterloss ion

in the spectra of azaspiracids resulted from dehydration at the vicinal diol at C20-C21. Five MS dissocn. pathways

were identified that resulted from fragmentation of the carbon skeleton of azaspiracids producing nitrogen-contg.

ions. Two pathways, involving cleavage of the E-ring and C27-C28, gave ions that were found in all azaspiracids.Three pathways, A-ring, C-ring and C19-C20 cleavages, were useful for distinguishing between azaspiracid analogs.

The same product ions from backbone fragmentation were also obsd. using hybrid quadrupole time-of-flight mass

spectrometry (QqTOFMS). The fragmentation of the A-ring was the most facile and was exploited in the

development of LC/MSn methods for the anal. of azaspiracids.


Probing protein folding and binding by electrospray ionization Fourier transform mass spectrometry. Eyles,

S. J.; Gumerov, D. R.; Gierasch, L. M.; Kaltashov, I. A. Departments of Chemistry, Polymer Science and

Engineering, University of Massachusetts, Amherst, MA, USA. Advances in Mass Spectrometry (2001), 15

499-500. CODEN: AMSPAH ISSN: 0568-000X. Journal written in English. AN 2003:974607 CAPLUS

(Copyright 2004 ACS on SciFinder (R))

Abstract

The dynamics and ligand-binding mechanism of cellular retinoic acid binding protein I (CRABP I) were

investigated. An amide H/D exchange was used to probe protein conformational stability. Mildly denaturing

conditions were employed to increase the population of intermediate states. Structural anal. of folding intermediates

was carried out by inducing dissocn. of protein ions in the gas phase and measuring the deuterium content of each

fragment ion as a function of the H/D exchange time in soln. Preliminary data indicate that under mildly denaturing

conditions, presence of RA leads to significant stabilization of the protein conformation.


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Liquid chromatography-multiple tandem mass spectrometry for the determination of ten azaspiracids,

including hydroxyl analogues in shellfish. Lehane, Mary; Saez, Maria Jose Fidalgo; Magdalena, Ana Brana;

Canas, Isabel Ruppen; Sierra, Monica Diaz; Hamilton, Brett; Furey, Ambrose; James, Kevin J. Department of

Chemistry, Mass Spectrometry Centre for Proteomics and Biotoxin Research, PROTEOBIO, Cork Institute ofTechnology, Cork, Ire. Journal of Chromatography, A (2004), 1024(1-2), 63-70. CODEN: JCRAEY ISSN:

0021-9673. Journal written in English. AN 2003:967554 CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

Azaspiracids (AZAs) are a group of polyether toxins that cause food poisoning in humans. These toxins, produced

by marine dinoflagellates, accumulate in filter-feeding shellfish, esp. mussels. Sensitive liq. chromatog.-electrospray

ionisation mass spectrometry (LC-ESI-MSn) methods have been developed for the detn. of the major AZAs and their

hydroxyl analogs. These methods, utilizing both chromatog. and mass resoln., were applied for the detn. of 10 AZAs

in mussels (Mytilus edulis). An optimized isocratic reversed phase method (3 m Luna-2 C18 column) sepd. 10azaspiracids using acetonitrile/water (46:54, vol./vol.) contg. 0.05% trifluoroacetic acid (TFA) and 0.004%

ammonium acetate in 55 min. Analyte detn. using MS3 involved trapping and fragmentation of the [M+H]+ and

[M+H-H2O]+ ions with detection of the [M + H-2H2O]+ ion for each AZA. Linear calibrations were obtained forAZA1, using spiked shellfish exts., in the range 0.05-1.00 g/mL (r2 = 0.997) with a detection limit of 5 pg(signal:noise = 3). The major fragmentation pathways in hydroxylated azaspiracids were elucidated using

hydrogen/deuterium (H/D) exchange expts. An LC-MS3 method was developed using unique parent ions and

product ions, [M + H-H2O-C9H10O2R1R3]+, that involved fragmentation of the A-ring. This facilitated the

discrimination between 10 azapiracids, AZA1-10. Thus, this rapid LC-MS3 method did not require complete

chromatog. resoln. and the run-time of 7 min had detection limits better than 20 pg for each toxin.


Atmospheric Pressure Gas-Phase H/D Exchange of Serine Octamers. Takats, Zoltan; Nanita, Sergio C.;

Schlosser, Gitta; Vekey, Karoly; Cooks, R. Graham. Department of Chemistry, Purdue University, West Lafayette,

IN, USA. Analytical Chemistry (2003), 75(22), 6147-6154. CODEN: ANCHAM ISSN: 0003-2700. Journal

written in English. CAN 139:347298 AN 2003:801957 CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

The recently discovered homochiral serine octamer has been a focus of interest because of its possible implications

for the origin of homochirality in living systems. Electrospray ionization (ESI) and sonic spray ionization (SSI)

tandem mass spectrometry have been used to generate this unusually stable magic no. cluster. Several structures

have been suggested for the serine octamer, based on tandem mass spectrometry, ion mobility measurements, and

quantum mech. calcns. We now report exptl. hydrogen/deuterium (H/D) exchange data, which demonstrate the

existence of two different structures for the serine octamer. These forms undergo exchange at significantly different

rates. One form may correspond to soln.-phase assembled clusters and the other to octamers formed during the

ionization process. Expts. done at higher resoln. confirm that the exptl. observations made here apply to the serine

octamer without interference from metaclusters, namely, higher order clusters (Ser16 + 2H)+2, etc., the 12C isotopesof which have mass-to-charge ratios identical to the protonated octamers. H/D exchange of racemic serine shows

predominantly the extensively exchanged ion population, as well as providing evidence that racemic serine generates

abundant metaclusters. The evidence presented here shows that one type of serine octamer is responsible for the

strong chiral effects assocd. with the formation of these magic no. clusters. These slowly exchanging more fragile

clusters are the octamers that might have played a role in homochirogenesis.


Applying a new algorithm for obtaining site specific rate constants for H/D exchange of the gas phase proton-


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bound arginine dimer. Reuben, Bryan G.; Ritov, Ya'acov; Geller, Orit; McFarland, Melinda A.; Marshall, Alan

G.; Lifshitz, Chava. School of Applied Science, South Bank University, London, UK. Chemical Physics Letters

(2003), 380(1,2), 88-94. CODEN: CHPLBC ISSN: 0009-2614. Journal written in English. CAN 139:396147

AN 2003:783726 CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

A new algorithm has been developed for extg. site-specific rate consts. for hydrogen/deuterium (H/D) exchange in

gas phase protonated amino acids, their clusters and peptides. The algorithm minimizes the mutual entropy or the

Kullback-Leibler information divergence between the obsd. concns. and the model. Electrospray ionization-mass

spectrometry (ESI-MS) results from fast flow tube and Fourier transform ion cyclotron resonance (FT-ICR) expts.,

resp., were modeled. The results for protonated glycine were in excellent agreement with previous literature data.

Four rate consts. were found, three of them identical corresponding to the three equiv. hydrogen atoms of the

protonated amine group and a fourth higher one corresponding to the single carboxyl hydrogen. New results for the

proton-bound dimer of arginine demonstrated a single high site-specific rate const. and fourteen low ones. These

results are in agreement with the ion-zwitterion structure of (arginine)2H+ that has a single carboxyl hydrogen atom.


Electrospray-ionization mass spectrometry for protein conformational studies. Grandori, Rita. Institute ofChemistry, Johannes Kepler University, Linz, Austria. Current Organic Chemistry (2003), 7(15), 1589-1603.

CODEN: CORCFE ISSN: 1385-2728. Journal written in English. AN 2003:752233 CAPLUS (Copyright

2004 ACS on SciFinder (R))

Abstract

The possibility to study large mols. and their non-covalent interactions by mass spectrometry (MS) has opened novel

ways to investigate protein folding and binding reactions. MS can be applied to protein conformational studies in

two conceptually different ways. One approach uses MS to monitor mass changes produced by conformation-

sensitive reactions, such as hydrogen/deuterium (H/D) exchange, alkylation and radiolysis. The second approach

directly exploits the conformation dependence of the charge-state distributions (CSDs) of the multiply charged

protein ions produced by electrospray-ionization (ESI). This review focuses on the information that has been

provided by the latter kind of studies. An attempt is made to summarize and discuss the available evidence about themechanism underlying this technique and its possible applications. The results of the studies described here include

equil. and kinetic characterization of protein folding transitions and detection of folding intermediates. The case

studies of myoglobin (Mb) and cytochrome c (cyt c) are discussed in particular detail. The unprecedented

advantages offered by MS in the anal. of heterogeneous samples can now be applied to the study of dynamic systems

involving different conformational states.


Method for characterizing metabolites using hydrogen/deuterium exchange. Lam, Wing Wah; Ramanathan,

Ragulan. (Warner-Lambert Company LLC, USA). Eur. Pat. Appl. (2003), 42 pp. CODEN: EPXXDW EP

1345028 A1 20030917 Designated States R: AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LI, LU, NL, SE, MC, PT,

IE, SI, LT, LV, FI, RO, MK, CY, AL, TR, BG, CZ, EE, HU, SK. Patent written in English. Application: EP 2003-

4825 20030305. Priority: US 2002-364373 20020314. CAN 139:240320 AN 2003:734673 CAPLUS


Patent Family Information

Patent No. Kind Date Application No. Date

EP 1345028 A1 20030917 EP 2003-4825 20030305

R: AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LI, LU, NL, SE, MC, PT, IE,

SI, LT, LV, FI, RO, MK, CY, AL, TR, BG, CZ, EE, HU, SK


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WO 2003076930 A1 20030918 WO 2003-IB883 20030303

W: AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN,

CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,

GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS,

LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, OM, PH,

PL, PT, RO, RU, SD, SE, SG, SK, SL, TJ, TM, TN, TR, TT, TZ, UA,

UG, US, UZ, VN, YU, ZA, ZM, ZW, AM, AZ, BY, KG, KZ, MD, RU,

TJ, TM

RW: GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW, AT, BE, BG,

CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HU, IE, IT, LU, MC,

NL, PT, RO, SE, SI, SK, TR, BF, BJ, CF, CG, CI, CM, GA, GN, GQ,

GW, ML, MR, NE, SN, TD, TG

US 2003175979 A1 20030918 US 2003-387613 20030313

JP 2004028993 A2 20040129 JP 2003-68691 20030313

Priority Application

US 2002-364373P P 20020314

Abstract

A system and method for performing hydrogen/deuterium (H/D) exchange in an electrospray ionization (ESI) source

is described. The system includes a liq. chromatograph-mass spectrometer (LC-MS), which is equipped with an ESI

source that provides for introduction of a sheath liq. The resulting system employs deuterated solvent, such as

deuterium oxide, as the sheath liq., which allows H/D exchange expts. to be performed online. This directly provides

information for detg. the no. and position of exchangeable hydrogens, aiding in the elucidation of the structures of

drug metabolites. To demonstrate the usefulness of the invention, the hydrogen/deuterium exchange in the

metabolites of PD 0200126 was examd.


Gas-phase hydrogen/deuterium exchange of adenine nucleotides. Crestoni, Maria Elisa; Fornarini, Simonetta.

Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita di Roma "LaSapienza," Piazzale A. Moro 5, Rome, Italy. Journal of Mass Spectrometry (2003), 38(8), 854-861. CODEN:

JMSPFJ ISSN: 1076-5174. Journal written in English. AN 2003:710107 CAPLUS (Copyright 2004 ACS on

SciFinder (R))

Abstract

Gas-phase hydrogen/deuterium exchange reactions of (de)protonated (sodiated) adenosine-5'-mono-, di- and

triphosphate ions with CD3OD, CD3CO2D and ND3 were achieved using a combination of electrospray ionization

and Fourier transform ion cyclotron resonance mass spectrometry. The reaction kinetics are dependent on factors

such as the charge state, the phosphate chain length, the properties of the exchange reactants and the sodium content.

The results indicate that the overall H/D exchange may involve specific sites even if endowed with high energetic

barriers. The enhanced reactivity exhibited by adenosine polyphosphate ions compared with adenosine-5'-

monophosphate suggests a crit. role of the polyphosphate chain in rendering conformationally accessible remote H-

donor sites. Low-energy collision-induced dissocn. of (sodiated) adenine nucleotides anions supports the aptitude of

the (poly)phosphate chain in probing distant sites via the intermediacy of a cyclic structure.


Dissecting interdomain communication within cAPK regulatory subunit type IIusing enhanced amidehydrogen/deuterium exchange mass spectrometry (DXMS). Zawadzki, Kerri M.; Hamuro, Yoshitomo; Kim,

Jack S.; Garrod, Siv; Stranz, David D.; Taylor, Susan S.; Woods, Virgil L., Jr. Department of Chemistry and


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Biochemistry, University of California, La Jolla, CA, USA. Protein Science (2003), 12(9), 1980-1990.

CODEN: PRCIEI ISSN: 0961-8368. Journal written in English. CAN 140:37918 AN 2003:683213 CAPLUS


Abstract

CAMP-dependent protein kinase (cAPK) is a heterotetramer contg. a regulatory (R) subunit dimer bound to twocatalytic (C) subunits and is involved in numerous cell signaling pathways. The C-subunit is activated allosterically

when two cAMP mols. bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B,

resp.). Each cAMP-binding domain contains a conserved Arg residue that is crit. for high-affinity cAMP binding.

Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and

cAPK activation. To better understand the local and long-range effects that the Arg-to-Lys mutation has on the

dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RIIsubunit was probed byelectrospray mass spectrometry. Mutant proteins contg. the Arg-to-Lys substitution in either cAMP-binding domain

were deuterated for various times and then, prior to mass spectrometry anal., subjected to pepsin digestion to localize

the deuterium incorporation. Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen

exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is

indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB-B) leads to increased

exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B

domain and is believed to be functionally linked to the cAB-B domain. This interdomain communication appears tobe a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain.


Investigation of apparent mass deviations in electrospray ionization tandem mass spectrometry of a

benzophenone-labeled peptide. Leite, John F.; Dougherty, Dennis A.; Lester, Henry A.; Shahgholi, Mona.

Division of Biology, California Institute of Technology, Pasadena, CA, USA. Rapid Communications in Mass

Spectrometry (2003), 17(15), 1677-1684. CODEN: RCMSEF ISSN: 0951-4198. Journal written in English.

CAN 140:59921 AN 2003:590146 CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

In a previous study utilizing benzophenone-based topol. probes to study conformationally dependent changes inmouse muscle nicotinic acetylcholine receptor (nAChR) topol., electrospray ionization tandem mass spectrometric

(ESI-MS/MS) anal. led to a consistent -2.0 Da mass deviation from expected values. In the present study a synthetic

peptide, corresponding to nAChR 1 subunit residues 130-139, was photolabeled. MS/MS anal. of this peptide

using an ion trap confirmed the previously obsd. mass deviation, assocd. only with fragment ions that contain the

incorporated benzophenone moiety. Anal. of peak profiles for the photolabeled ions does not indicate the typical

"peak fronting" that produces a mass shift when labile ions are prematurely ejected from the ion trap. Rather,

hydrogen/deuterium (H/D) exchange expts. support the hypothesis that a chem. rearrangement involving Ph

migration and ketone formation has formed an unexpected oxidized peptide, with mol. mass 2 Da less than that

expected, that is isolated for collision-induced dissocn. in the ion trap together with the predicted precursor due to the

broad ion isolation window specified.


Hydrogen/deuterium exchange of electrosprayed ions in the atmospheric interface of a commercial triple-

quadrupole mass spectrometer. Takats, Zoltan; Schlosser, Gitta; Vekey, Karoly. Institute of Chemistry, Mass

Spectrometry Department, Chemical Research Center of the Hungarian Academy of Sciences, Budapest, Hung.

International Journal of Mass Spectrometry (2003), 228(2-3), 729-741. CODEN: IMSPF8 ISSN: 1387-3806.

Journal written in English. AN 2003:581546 CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract


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A novel H/D exchange technique capable of the deuteration of electrosprayed ions has been developed. H/D

exchange was carried out by introducing deuterating agent (e.g., d-MeOH) into the curtain gas flow of a com. triple

quadrupole mass spectrometer. In contrast to the widely used H/D exchange techniques, the ions are not trapped in

this case. The main advantages of this technique are the ease of use and applicability on most com. mass

spectrometers, including quadrupole-type instruments.Effect of various instrumental parameters was investigated in

detail, including spray voltage, spray position, partial pressure of the deuterating agent in the curtain gas, gas flow

rates, the orifice-to-skimmer potential and the source temp. Among these only the partial pressure of the deuterating

agent in curtain gas, orifice-to-skimmer potential and source temp. influenced the efficiency of H/D exchange.

These suggest that the H/D exchange is likely to occur in the fore-vacuum region of the atm. interface. Anal.

capabilities of the technique were demonstrated by differentiation of lysine and glutamine protonated mol. ions.

Selective quantitation of lysine and glutamine mixts. was achieved, with a lower limit of detection of 1.5% for

glutamine and 0.2% for lysine. H/D exchange of multiply charged macromol. ions can also be carried out using this

technique, which was demonstrated using cytochrome c.


Hydrogen Exchange-Mass Spectrometry Analysis of -Amyloid Peptide Structure. Wang, Steven S.-S.;Tobler, Scott A.; Good, Theresa A.; Fernandez, Erik J. Department of Chemical Engineering, Texas A&M

University, College Station, TX, USA. Biochemistry (2003), 42(31), 9507-9514. CODEN: BICHAW ISSN:

0006-2960. Journal written in English. CAN 139:175511 AN 2003:557199 CAPLUS (Copyright 2004 ACSon SciFinder (R))

Abstract

-Amyloid peptide (A) is the primary protein component of senile plaques in Alzheimer's disease and is believed tobe responsible for the neurodegeneration assocd. with the disease. Ahas proven to be toxic only when aggregated;however, the structure of the aggregated species assocd. with toxicity is unknown. In the present study, we use

hydrogen-deuterium isotope exchange (HX)-electrospray ionization mass spectrometry (MS) along with enzymic

digestion as a tool to examine at near residue level, the changes in Astructure assocd. with aggregation to a fibrilform. Our results show that the structure of Aintermediate species formed early in the course of fibrillogenesis isdependent upon solvent conditions. Addnl., the HX-MS data of peptic Afragments suggest that the C-terminalsegment of the peptide is approx. 35% protected from exchange in fibril-contg. samples, relative to monomeric Aspecies prepd. in DMSO/H2O. The N-terminus (residues 1-4) is completely unprotected from exchange, and thefragment contg. residues 5-19 is over 50% protected from exchange in the fibril-contg. samples. This work

contributes to our understanding of Astructure assocd. with aggregation and toxicity and further application of thisapproach may aid in the design of agents that intervene in the Aaggregation processes assocd. with neurotoxicity.Bibliographic Information

Conformational dynamics of partially denatured myoglobin studied by time-resolved electrospray mass

spectrometry with online hydrogen-deuterium exchange. [Erratum to document cited in CA139:32723].

Simmons, Douglas A.; Dunn, Stanley D.; Konermann, Lars. Departments of Chemistry and Biochemistry,

University of Western Ontario, London, ON, Can. Biochemistry (2003), 42(30), 9248. CODEN: BICHAW

ISSN: 0006-2960. Journal; Errata written in English. AN 2003:519782 CAPLUS (Copyright 2004 ACS on

SciFinder (R))

Abstract

An erratum.


Conformational dynamics of partially denatured myoglobin studied by time-resolved electrospray mass


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spectrometry with online hydrogen-deuterium exchange. Simmons, Douglas A.; Dunn, Stanley D.;

Konermann, Lars. Departments of Chemistry and Biochemistry, University of Western Ontario, London, ON,

Can. Biochemistry (2003), 42(19), 5896-5905. CODEN: BICHAW ISSN: 0006-2960. Journal written in

English. CAN 139:32723 AN 2003:316127 CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

This study demonstrates the use of electrospray mass spectrometry in conjunction with rapid online mixing ("time-

resolved" ESI-MS) for monitoring protein conformational dynamics under equil. conditions. The

hydrogen/deuterium exchange (HDX) kinetics of mildly denatured myoglobin (Mb) at pD 9.3, in the presence of

27% acetonitrile, were studied with millisecond time resoln. Anal. ultracentrifugation indicates that the av. protein

compactness under these solvent conditions is similar to that of native holomyoglobin (hMb). The mass spectrum

shows protein ions in a wide array of charge and heme binding states, indicating the presence of multiple coexisting

conformations. The exptl. approach used allows the HDX kinetics of all of these species to be monitored sep. A

combination of EX1 and EX2 behavior was obsd. for hMb ions in charge states 7+ to 9+, which predominantly

represent nativelike hMb in soln. The EX1 kinetics are biphasic, indicating the presence of two protein populations

that undergo conformational opening events with different rate consts. The EX2 kinetics obsd. for nativelike hMb

are biphasic as well. All other charge and heme binding states represent non-native protein conformations that are

involved in rapid interconversion processes, thus leading to monoexponential EX2 kinetics with a common rate

const. Burst phase labeling for these non-native proteins occurs at 125 sites. In contrast, the nativelike proteinconformation shows burst phase labeling only for 88 sites. A kinetic model is developed which is based on the

assumption of three distinct (un)folding units in Mb. The model implies that the free energy landscape of the protein

exhibits a major barrier. The crossing of this barrier is most likely assocd. with slow, cooperative opening/closing

events of the heme binding pocket. Rapid conformational fluctuations on either side of the barrier give rise to the

obsd. EX2 kinetics. Simulated HDX kinetics based on this model are in excellent agreement with the exptl. data.


Improving hydrogen/deuterium exchange mass spectrometry by reduction of the back-exchange effect.

Kipping, Marc; Schierhorn, Angelika. Max Planck Research Unit for Enzymology of Protein Folding, Halle/Saale,

Germany. Journal of Mass Spectrometry (2003), 38(3), 271-276. CODEN: JMSPFJ ISSN: 1076-5174. Journal

written in English. CAN 139:361072 AN 2003:281269 CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

The measurement of deuterium incorporation kinetics using hydrogen/deuterium (H/D) exchange expts. is a valuable

tool for the investigation of the conformational dynamics of biomols. in soln. Expts. consist of two parts when using

H/D exchange mass spectrometry to analyze the deuterium incorporation. After deuterium incorporation at high

D2O concn., it is necessary to decrease the D2O concn. before the mass anal. to avoid deuterium incorporation under

artificial conditions of mass spectrometric prepn. and measurement. A low D2O concn., however, leads to back-

exchange of incorporated deuterons during mass anal. This back-exchange is one of the major problems in H/D

exchange mass spectrometry and must be reduced as much as possible. In the past, techniques using electrospray

ionization (ESI) had the lowest back-exchange values possible in H/D exchange mass spectrometry. Methods for the

measurement of H/D exchange by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) that

have been developed since 1998 have some significant advantages, but they could not achieve the back-exchange

min. of ESI methods. Here, we present a protocol for H/D exchange MALDI-MS which allows for greaterminimization of back-exchange compared with H/D exchange ESI-MS under similar conditions.


Dynamics of cAPK Type IIActivation Revealed by Enhanced Amide H/2H Exchange Mass Spectrometry(DXMS). Hamuro, Yoshimoto; Zawadzki, Kerri M.; Kim, Jack S.; Stranz, David D.; Taylor, Susan S.; Woods,

Virgil L. Department of Medicine, University of California, La Jolla, CA, USA. Journal of Molecular Biology

(2003), 327(5), 1065-1076. CODEN: JMOBAK ISSN: 0022-2836. Journal written in English. CAN


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139:113593 AN 2003:231341 CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

CAMP-dependent protein kinase (cAPK) is a key component in numerous cell signaling pathways. The cAPK

regulatory (R) subunit maintains the kinase in an inactive state until cAMP satn. of the R-subunit leads to activation

of the enzyme. To delineate the conformational changes assocd. with cAPK activation, the amidehydrogen/deuterium exchange in the cAPK type IIR-subunit was probed by electrospray mass spectrometry. Threestates of the R-subunit, cAMP-bound, catalytic (C)-subunit bound, and apo, were incubated in deuterated water for

various lengths of time and then, prior to mass spectrometry anal., subjected to digestion by pepsin to localize the

deuterium incorporation. High sequence coverage (>99%) by the pepsin-digested fragments enables us to monitor

the dynamics of the whole protein. The effects of cAMP binding on RIIamide hydrogen exchange are restricted tothe cAMP-binding pockets, while the effects of C-subunit binding are evident across both cAMP-binding domains

and the linker region. The decreased amide hydrogen exchange for residues 253-268 within cAMP binding domain

A and for residues 102-115, which include the pseudosubstrate inhibitory site, support the prediction that these two

regions represent the conserved primary and peripheral C-subunit binding sites. An increase in amide hydrogen

exchange for a broad area within cAMP-binding domain B and a narrow area within cAMP-binding domain A

(residues 222-232) suggest that C-subunit binding transmits long-distance conformational changes throughout the

protein.


Identification of the degradation product of ezlopitant, a non-peptidic substance p antagonist receptor, by

hydrogen deuterium exchange, electrospray ionization tandem mass spectrometry (ESI/MS/MS) and nuclear

magnetic resonance (NMR) spectroscopy. Kamel, Amin M.; Zandi, Kathleen S.; Massefski, Walter W.

Department of Pharmacokinetics, Dynamics and Drug Metabolism, Groton Laboratories, Pfizer Global Research

and Development, Groton, CT, USA. Journal of Pharmaceutical and Biomedical Analysis (2003), 31(6), 1211-

1222. CODEN: JPBADA ISSN: 0731-7085. Journal written in English. CAN 139:250395 AN 2003:229374

CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

The degrdn. product of ezlopitant was isolated from low specific activity material and identified by soln. phase

hydrogen/deuterium (H/D) exchange and electrospray ionization tandem mass spectrometry (ESI/MS/MS) to be an

iso-Pr peroxide analog of ezlopitant. The structure of the degradant was further confirmed by NMR (NMR)

spectroscopy utilizing complete 1H and 13C assignments. Studies were also performed to identify the factors

responsible for the oxidative degrdn. of ezlopitant, which included salt form, storage conditions and salt formation

solvent. Of all the variable studies over a 3 wk period, only a change in the salt form prevented this oxidative

degrdn.


Downsizing improves sensitivity 100-fold for hydrogen exchange-mass spectrometry. Wang, Lintao; Smith,

David L. Department of Chemistry, University of Nebraska, Lincoln, NE, USA. Analytical Biochemistry

(2003), 314(1), 46-53. CODEN: ANBCA2 ISSN: 0003-2697. Journal written in English. CAN 139:347613AN 2003:190730 CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

This paper describes a method that substantially improves the sensitivity of high-performance liq. chromatog.

hydrogen exchange-mass spectrometry (HPLC HX MS). The success of this method relies on using a capillary

HPLC column (0.1 mm ID 5 cm L) to increase the sensitivity of electrospray ionization, while keeping anal. timesshort to minimize hydrogen/deuterium (H/D) exchange. A small, immobilized pepsin column and a capillary C18


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trap were included in the capillary HPLC MS system to provide rapid digestion, peptide concn., and desalting while

maintaining slow H/D exchange conditions. To minimize the anal. time, dead vols. and capacities of all components

were optimized. Fully deuterated cytochrome c and its fully deuterated peptic peptides were used to evaluate

deuterium recovery at amide linkages. The deuterium recovery measured at low flow rates using this system

spanned a range of 66-77% (av. of 71%), which was similar to the range measured for a much larger system (67-

80%, av. 75%). Signal levels of most peptides for the down-sized system increased by about 100-fold compared

with the signal for the larger system. These results greatly strengthen the HPLC HX MS technique for studies where

the quantity of protein is small.


Fragmentation reactions of protonated peptides containing glutamine or glutamic acid. Harrison, Alex G.

Department of Chemistry, University of Toronto, Toronto, ON, Can. Journal of Mass Spectrometry (2003),

38(2), 174-187. CODEN: JMSPFJ ISSN: 1076-5174. Journal written in English. CAN 139:117670 AN

2003:155585 CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

A variety of protonated dipeptides and tripeptides contg. glutamic acid or glutamine were prepd. by electrospray

ionization or by fast atom bombardment ionization and their fragmentation pathways elucidated using metastable ionstudies, energy-resolved mass spectrometry and triple-stage mass spectrometry (MS3) expts. Addnl. mechanistic

information was obtained by exchanging the labile hydrogens for deuterium. Protonated H-Gln-Gly-OH fragments

by loss of NH3 and loss of H2O in metastable ion fragmentation; under collision-induced dissocn. (CID) conditions

loss of H-Gly-OH + CO from the [MH - NH3]+ ion forms the base peak C4H6NO+ (m/z 84). Protonated dipeptides

with an -linkage, H-Glu-Xxx-OH, are characterized by elimination of H2O and by elimination of H-Xxx-OH plus

CO to form the glutamic acid immonium ion of m/z 102. By contrast, protonated dipeptides with a -linkage, H-Glu(Xxx-OH)-OH, do not show elimination of H2O or formation of m/z 102 but rather show elimination of NH3,

particularly in metastable ion fragmentation, and elimination of H-Xxx-OH to form m/z 130. Both the - and -dipeptides show formation of [H-Xxx-OH]H+, with this reaction channel increasing in importance as the proton

affinity (PA) of H-Xxx-OH increases. The characteristic loss of H2O and formation of m/z 102 are obsd. for the

protonated -tripeptide H-Glu-Gly-Phe-OH whereas the protonated -tripeptide H-Glu(Gly-Gly-OH)-OH shows lossof NH3 and formation of m/z 130 as obsd. for dipeptides with the -linkage. Both tripeptides show abundantformation of the y''2 ion under CID conditions, presumably because a stable anhydride neutral structure can be

formed. Under metastable ion conditions protonated dipeptides of structure H-Xxx-Glu-OH show abundant

elimination of H2O whereas those of structure H-Xxx-Gln-OH show abundant elimination of NH3. The importance

of these reaction channels is much reduced under CID conditions, the major fragmentation mode being cleavage of

the amide bond to form either the a1 ion or the y''1 ion.

Particularly when Xxx = Gly, under CID conditions the initial loss of NH3 from the glutamine contg. dipeptide is

followed by elimination of a second NH3 while the initial loss of H2O from the glutamic acid dipeptide is followed

by elimination of NH3. Isotopic labeling shows that predominantly labile hydrogens are lost in both steps. Although

both [H-Gly-Glu-Gly-OH]H+ and [H-Gly-Gln-Gly-OH]H+ fragment mainly to form b2 and a2 ions, the latter also

shows elimination of NH3 plus a glycine residue and formation of protonated glycinamide. Isotopic labeling shows

extensive mixing of labile and carbon-bonded hydrogens in the formation of protonated glycinamide.


Charge State Distribution and Hydrogen/Deuterium Exchange of -Lactalbumin and -LactoglobulinPreparations by Electrospray Ionization Mass Spectrometry. Alomirah, Husam; Alli, Inteaz; Konishi, Yasuo.

Department of Food Science and Agricultural Chemistry, McGill University, Ste. Anne de Bellevue, QC, Can.

Journal of Agricultural and Food Chemistry (2003), 51(7), 2049-2057. CODEN: JAFCAU ISSN: 0021-8561.

Journal written in English. CAN 138:319977 AN 2003:139870 CAPLUS (Copyright 2004 ACS on SciFinder

(R))

Abstract


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Charge state distribution (CSD) and hydrogen/deuterium (H/D) exchange of prepns. of -lactalbumin (-Lac) and -lactoglobulin (-Lg) were investigated using electrospray ionization mass spectrometry (ESI-MS). Storage of -Lacat pH 3 resulted in substantial changes in its CSD, with the emergence of new ion species and shifts toward higher

charge state, indicating less stable conformation. ESI spectra of -Lac kept at pH 5.5 for 4 days showed stable

conformation; however, extending the storage period resulted in substantial changes in CSD and a decrease in the

stability of holo--Lac (Ca2+-bound form). In comparison to apo--Lac, the relative intensity of holo--Lac washigher at pH 6.8 but lower at pH 8 during the storage period. -Lg showed stable CSD at pH 3, substantial changesat pH 5.5, and minor changes at pH 6.8 and 8 during storage. The H/D exchange results demonstrate that the

conformation of holo--Lac was more stable than that of apo--Lac and that the conformation of -Lg variant Bwas more stable than that of the -Lg variant A. Kinetics of H/D exchange indicated that -Lac and -Lg fractionsobtained from whey protein prepns. have the same or improved conformational stabilities compared to those of -

Lac and -Lg stds. The presence of four or more hexose residues in -Lac enhanced its conformational stability; thepresence of two hexose residues in -Lg resulted in a less stable conformation.Bibliographic Information

Conformations of Gas-Phase Lysozyme Ions Produced from Two Different Solution Conformations. Mao,

Dunmin; Babu, Kodali Ravindra; Chen, Yu-Luan; Douglas, D. J. Department of Chemistry, University of BritishColumbia, Vancouver, BC, Can. Analytical Chemistry (2003), 75(6), 1325-1330. CODEN: ANCHAM ISSN:

0003-2700. Journal written in English. CAN 138:233897 AN 2003:130892 CAPLUS (Copyright 2004 ACS

on SciFinder (R))

Abstract

Near pH 2.0, lysozyme in water is in its native conformation, and in water/methanol (2/8) it adopts a helical

denatured conformation (Kamatari et al. Protein Sci. 1998, 7, 681-688). Hydrogen/deuterium (H/D) exchange of

lysozyme in soln. confirms that it is partially unfolded at pH 2.0 in water/methanol (vol./vol. = 2/8). With

electrospray ionization (ESI) mass spectrometry (MS), lysozyme in water produces ions with charges +7 to +12, with

the greatest intensity at +10, whereas lysozyme in water/methanol (2/8) produces ions with charges +6 to +12 with

the greatest intensity at +7. Thus, lysozyme is an exception to the rule that a protein denatured in soln. forms higher

charge states than the same protein in its folded native conformations in soln. Because the same charge states areproduced from these two soln. conformations, a direct comparison of the properties of the gas-phase ions produced

from two very different soln. conformations is possible. The conformations of lysozyme ions in the gas phase were

studied using cross section measurements and gas-phase H/D exchange. Similar cross sections and H/D exchange

levels were obsd. for same-charge states of lysozyme ions formed from the native and helical denatured

conformations in soln. Cross sections show that the ions have compact structures. Thus, disulfide-intact gaseous

lysozyme ions generated from the denatured state in water/methanol (2/8) refold into compact structures in the gas

phase on a time scale of milliseconds or less.


Hydrogen exchange studies on Alzheimer's amyloid-peptides by mass spectrometry using matrix-assistedlaser desorption/ionization and electrospray ionization. Kraus, Mario; Bienert, Michael; Krause, Eberhard.

Forschungsinstitut fur Molekulare Pharmakologie, Berlin, Germany. Rapid Communications in Mass

Spectrometry (2003), 17(3), 222-228. CODEN: RCMSEF ISSN: 0951-4198. Journal written in English. CAN


Abstract

The conformation and aggregation behavior of synthetic Alzheimer's amyloid peptides (A) has been investigatedusing hydrogen-deuterium exchange measured by electrospray ionization mass spectrometry and matrix-assisted


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laser desorption/ionization mass spectrometry. Mass spectrometric fragmentation of deuterated Apeptides wascarried out by collision-induced dissocn., inlet fragmentation, and post-source decay. In contrast to the C-terminally

truncated peptides A(1-40) and A(1-36) showing full hydrogen-deuterium exchange, A(1-42) and thepyroglutamyl peptide Pyr3-A(3-42) produced more complex signal patterns resulting from the formation of -sheet-structured oligomers having 18-20 strongly protected protons. Using mass spectrometric fragmentation the

results show that the reduced isotope exchange of A(1-42) can be attributed to the central part of the chaincomprising residues 8-23. This confirms involvement of the hydrophobic binding domain LVFFA in the course ofAaggregation and demonstrates that hydrogen-deuterium exchange in combination with mass spectrometry is wellsuited for structural anal. of monomeric and reversibly assocd. amyloid peptides using picomole quantities of

material.


A mass spectrometry investigation of the conformational changes in adrenocorticotropic hormones. Lin,

Hui; Dass, Chhabil. Department of Chemistry, The University of Memphis, Memphis, TN, USA. European

Journal of Mass Spectrometry (2002), 8(5), 381-387. CODEN: EJMSCL ISSN: 1469-0667. Journal written in


Abstract

Electrospray ionization-mass spectrometry (ESI-MS) was employed to study methanol-induced conformational

changes in adrenocorticotrophic hormone (ACTH). ACTH, a 39-residue peptide, is a member of the

proopiomelanocortin family of peptides. Charge-state distribution (CSD) and hydrogen-deuterium (H/D) exchange

were used to monitor the conformational changes as a function of methanol concn. The latter expts. were conducted

via time-resolved ESI-MS in a continuous-flow app. The CSD and the H/D exchange exptl. data both reveal that

ACTH exists, presumably in a random coil open structure in aq. media, but assumes a more compact helical

conformation with increased concn. of methanol. The H/D exchange expts. also reveal that 79% of ACTH is present

as -helix in mixed water-methanol solvent media.


Hydrogen/deuterium exchange of hydrophobic peptides in model membranes by electrospray ionization massspectrometry. Hansen, Raino K.; Broadhurst, R. William; Skelton, Paul C.; Arkin, Isaiah T. Department of

Biochemistry, University of Cambridge, Cambridge Centre for Molecular Recognition, Cambridge, UK. Journal

of the American Society for Mass Spectrometry (2002), 13(12), 1376-1387. CODEN: JAMSEF ISSN: 1044-0305.


(R))

Abstract

We demonstrate here that the hydrogen/deuterium solvent exchange (HDX) properties of the transmembrane

fragment of the M2 protein of Influenza A (M2-TM) incorporated into lipid vesicles or detergent micelles can be

studied with straightforward electrospray (ESI) and nanospray mass spectrometry (MS) configurations provided that

key factors, including sample prepn. techniques, are optimized. Small unilamellar vesicle prepns. were obtained by

solubilizing dimyristoyl phosphatidylcholine (DMPC) and the M2-TM peptide in aq. soln. with n-octyl--D-glycopyranoside, followed by dialysis to remove the detergent. Electron microscopy expts. revealed that subsequent

concn. by centrifugation introduced large multilamellar aggregates that were not compatible with ESI-MS. By

contrast, a lyophilization-based concn. procedure, followed by thawing above the liq. crystal transition temp. of the

lipid component, maintained the liposome size profile and yielded excellent ion fluxes in both ESI-MS and nano-

ESI-MS. Using these methods the global HDX profile of M2-TM in aq. DMPC vesicles was compared with that in

methanol, demonstrating that several amide sites were protected from exchange by the lipid membrane. We also

show that hydrophobic peptides can be detected by ESI-MS in the presence of a large molar excess of the detergent

Triton X-100. The rate of HDX of M2-TM in Triton X-100 micelles was faster than that in DMPC vesicles but


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slower than when the peptide had been denatured in methanol. These results indicate that the accessibility of

backbone amide sites to the solvent can be profoundly affected by membrane protein structure and dynamics, as well

as the properties of model bilayer systems.


An electrospray ionization-flow tube study of H/D exchange in protonated serine. Ustyuzhanin, Pavel;Ustyuzhanin, Juliya; Lifshitz, Chava. Department for Physical Chemistry and The Farkas Center for Light Induced

Processes, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel. International Journal of Mass

Spectrometry (2003), 223-224(1-3), 491-498. CODEN: IMSPF8 ISSN: 1387-3806. Journal written in English.


Abstract

An electrospray ionization-fast flow technique has been employed to study the H/D exchange reactions of protonated

serine with ND3 and CH3OD. A flow rate of 2.51017 mols. s-1 ND3 at an overall pressure of 0.22 Torr and areaction time of 21.4 ms is sufficient to exchange up to five labile hydrogens. With deuterated methanol raising the

flow rate to 1.81018 mols. s-1 enabled observation of four exchanges and traces of a fifth exchange. Relativeabundances for the various cations vs. neutral flow rates were measured. Optimum apparent and site-specific rate

consts. were deduced by simulated fits based on solns. of simultaneous first-order differential equations. Four site-

specific rate consts. deduced for the reaction of protonated serine with CH3OD, 4.110-11, 1.910-12, 1.910-12,and 1.910-12 cm3 per mol. s-1 are very similar to the ones deduced for protonated glycine with CH3OD.Bibliographic Information

Gas phase hydrogen/deuterium exchange of proteins in an ion trap mass spectrometer. Evans, Sarah E.;

Lueck, Nathan; Marzluff, Elaine M. Department of Chemistry, Grinnell College, Grinnell, IA, USA.

International Journal of Mass Spectrometry (2003), 222(1-3), 175-187. CODEN: IMSPF8 ISSN: 1387-3806.


(R))

Abstract

Electrospray ionization ion trap mass spectrometry (ESI-ITMS) coupled with gas phase hydrogen/deuterium (H/D)

exchange is demonstrated to be a useful tool to investigate the gas phase conformations of proteins when coupled

with a mechanistic understanding of exchange. We have investigated the H/D exchange of multiple charge states of

lysozyme, cytochrome c, ubiquitin, insulin, thioredoxin and melittin zwith deuterated methanol in the ion trap. This

allows a direct comparison of exchange of these well studied proteins under identical conditions. For all proteins

except lysozyme, exchange results in some peak broadening but no evidence of distinct conformers is obsd. Qual.,

trends in exchange levels are consistent with prior Fourier transform ion cyclotron resonance mass spectrometry (FT-

ICR) expts. Consistent with mechanistic studies which have shown that amine hydrogens in peptides exchange

rapidly, a correlation between the no. of amine hydrogens and exchange level is obsd. Highest levels of exchange

are obsd. for proteins without disulfide bonds, and for proteins which are protonated on sites other than arginine.

Both of these observations are explained by the "relay" mechanism of exchange. These results indicate that a further

understanding of both the dynamics of gas phase mols. and mechanisms of exchange are necessary to relate gasphase H/D exchange data more directly to protein conformation.


Mapping of protein:protein contact surfaces by hydrogen/deuterium exchange, followed by on-line high-

performance liquid chromatography-electrospray ionization fourier-transform ion-cyclotron-resonance mass

analysis. Lam, TuKiet T.; Lanman, Jason K.; Emmett, Mark R.; Hendrickson, Christopher L.; Marshall, Alan G.;

Prevelige, Peter E. Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, USA.


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Journal of Chromatography, A (2002), 982(1), 85-95. CODEN: JCRAEY ISSN: 0021-9673. Journal written in


Abstract

For protein complexes too large, uncrystallizable/insol., or low concn. for conventional x-ray diffraction or NMR

anal., the contact surface(s) may be mapped by comparing H/2H exchange rate (and thus solvent accessibility) ofbackbone amide hydrogens in free vs. complexed protein(s). The protein is first exposed to 2H2O, allowed to

exchange for each of several reaction periods, and then digested with pepsin. The extent and rate of H/2H exchange

is detd. by measuring the increase in mass with H/2H exchange period for each of the peptides. Here, we present an

exptl. protocol that combines rapid (to minimize back-exchange) HPLC front-end sepn. with ultrahigh-resoln. mass

anal. (needed to distinguish the isotopic distributions of dozens of peptides simultaneously). The method is used to

study the assembled human immunodeficiency virus type capsid protein (CA) and its sol. form.


Gas-phase behavior of negative ions produced from thiazidic diuretics under electrospray conditions.

Garcia, Patrice; Popot, Marie Agnes; Fournier, Francoise; Bonnaire, Yves; Tabet, Jean Claude. L.A.B./F.N.C.F.,

Chatenay-Malabry, Fr. Journal of Mass Spectrometry (2002), 37(9), 940-953. CODEN: JMSPFJ ISSN: 1076-

5174. Journal written in English. CAN 138:243414 AN 2002:779660 CAPLUS (Copyright 2004 ACS onSciFinder (R))

Abstract

A systematic mass spectrometric study of 10 thiazidic diuretics and related compds. was undertaken by mass

spectrometry (MS) with electrospray ionization in the neg. ion mode. Collisional dissocn. "in-source" (CID-MS) and

in a low-pressure collision cell (CID-MS/MS) were compared in both excitation regions. Spectra obtained by CID-

MS and by CID-MS/MS were matched. Using the two methods, loss of HCl and consecutive dissocns. from 2HCl

losses were exhibited from compds. such as methyclothiazide and trichlormethiazide but not from other thiazidic

diuretics that contain chlorine substituents in the arom. moiety. However, deprotonated dichlorphenamide gave rise

to loss of HCl by CID-MS and CID-MS/MS. For other diuretics such as hydroflumethiazide and

hydrochlorothiazide, the loss of HCN and [HCN + SO2] was relevant. Reaction mechanisms were checked by

means of deuterium-hydrogen exchange, which showed that deprotonation took place regioselectively on theheterocyclic moiety. The cleavage pathways require mol. isomerization forming ion-dipole complexes prior to

decompns., allowing long-distance proton transfer for neutral elimination. Identifications of the most specific

fragmentations presented in this paper were applied to the screening and unambiguous identification of diuretics for

horse doping control.


Hydrogen/deuterium exchange and mass spectrometric analysis of a protein containing multiple disulfide

bonds: solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSF). Yan,Xuguang; Zhang, Heidi; Watson, Jeffrey; Schimerlik, Michael I.; Deinzer, Max L. Department of Chemistry,

Oregon State University, Corvallis, OR, USA. Protein Science (2002), 11(9), 2113-2124. CODEN: PRCIEI

ISSN: 0961-8368. Journal written in English. CAN 137:363256 AN 2002:674090 CAPLUS (Copyright 2004

ACS on SciFinder (R))

Abstract

Studies with the homodimeric recombinant human macrophage colony-stimulating factor beta (rhM-CSF), showfor the first time that a large no. (9) of disulfide linkages can be reduced after amide hydrogen/deuterium (H/D)

exchange, and the protein digested and analyzed successfully for the isotopic compn. by electrospray mass

spectrometry. Anal. of amide H/D after exchange-in shows that in soln. the conserved four-helix bundle of (rhM-


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CSF) has fast and moderately fast exchangeable sections of amide hydrogens in the A helix, and mostly slowexchanging sections of amide hydrogens in the B, C, and D helixes. Most of the amide hydrogens in the loop

between the 1 and 4 sheets exhibited fast or moderately fast exchange, whereas in the amino acid 63-67 loop,located at the interface of the two subunits, the exchange was slow. Solvent accessibility as measured by H/D

exchange showed a better correlation with the av. depth of amide residues calcd. from reported x-ray crystallog. data

for rhM-CSF than with the av. B-factor. The rates of H/D exchange in rhM-CSFappear to correlate well with theexposed surface calcd. for each amino acid residue in the crystal structure except for the D helix. Fast hydrogenisotope exchange throughout the segment amino acids 150-221 present in rhM-CSF, but not rhM-CSF, providesevidence that the C-terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the D

helix is due to interaction of the C-terminal tail with this helical segment.


Noncovalent dimerization of paclitaxel in solution: evidence from electrospray ionization mass spectrometry.

Lorenz, Sarah A.; Bigwarfe, Paul M., Jr.; Balasubramanian, Sathyamangalam V.; Fetterly, Gerald J.; Straubinger,

Robert M.; Wood, Troy D. Department of Chemistry, State University of New York at Buffalo, Buffalo, NY,

USA. Journal of Pharmaceutical Sciences (2002), 91(9), 2057-2066. CODEN: JPMSAE ISSN: 0022-3549.


(R))

Abstract

Paclitaxel, a unique antimitotic chemotherapy agent that inhibits cell division by binding to microtubules and

prevents them from "depolymg.," has received widespread interest because of its efficacy in fighting certain types of

cancer, including breast and ovarian cancer. Paclitaxel undergoes aggregation at millimolar concns. in both aq.

media and solvents of low polarity (mimicking hydrophobic environments). Its aggregation may have impact on its

aq. stability and its ability to stabilize microtubules. Here, we investigated the dimerization phenomenon of

paclitaxel by electrospray ionization mass spectrometry (ESI-MS). Paclitaxel dimers were stable in solns. of

acetonitrile/aq. ammonium acetate (80/20) and aq. sodium acetate/acetonitrile (92/8 or 95/5) at various pH values.

Addnl. expts. using soln.-phase hydrogen/deuterium exchange were employed to ascertain whether or not the obsd.

dimers were formed in soln. or as an artifact of the ESI process by ion-mol. reaction. The evidence supports

formation of the dimer in soln., and the approach used can be extended to investigation of other types of drug-druginteractions.


Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray

ionization time-of-flight mass spectrometry. Van Den Bremer, Ewald T. J.; Jiskoot, Wim; James, Richard;

Moore, Geoffrey R.; Kleanthous, Colin; Heck, Albert J. R.; Maier, Claudia S. Department of Biomolecular Mass

Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht

University, Utrecht, Neth. Protein Science (2002), 11(7), 1738-1752. CODEN: PRCIEI ISSN: 0961-8368.


(R))

Abstract

Nano-electrospray ionization time-of-flight mass spectrometry (ESI-MS) was used to study the conformational

consequences of metal ion binding to the colicin E9 endonuclease (E9 DNase) by taking advantage of the unique

capability of ESI-MS to allow simultaneous assessment of conformational heterogeneity and metal ion binding.

Alterations of charge state distributions on metal ion binding/release were correlated with spectral changes obsd. in

far- and near-UV CD and intrinsic tryptophan fluorescence. In addn., hydrogen/deuterium (H/D) exchange expts.

were used to probe structural integrity. The present study shows that ESI-MS is sensitive to changes of the

thermodn. stability of E9 DNase as a result of metal ion binding/release in a manner consistent with that deduced


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from proteolysis and calorimetric expts. Interestingly, acid-induced release of the metal ion from the E9 DNase

causes dramatic conformational instability assocd. with a loss of fixed tertiary structure, but secondary structure is

retained. Furthermore, ESI-MS enabled the direct observation of the noncovalent protein complex of E9 DNase

bound to its cognate immunity protein Im9 in the presence and absence of Zn2+. Gas-phase dissocn. expts. of the

deuterium-labeled binary and ternary complexes revealed that metal ion binding, not Im9, results in a dramatic

exchange protection of E9 DNase in the complex. In addn., our metal ion binding studies and gas-phase dissocn.

expts. of the ternary E9 DNase-Zn2+-Im9 complex have provided further evidence that electrostatic interactions

govern the gas phase ion stability.


Melittin-diacylphosphatidylcholine interaction examined by electrospray ionization fourier transform ion

cyclotron resonance mass spectrometry. Akashi, Satoko; Takio, Koji. Division of Biomolecular

Characterization, RIKEN (The Institute of Physical and Chemical Research), Saitama, Japan. Journal of the Mass

Spectrometry Society of Japan (2002), 50(2), 67-71. CODEN: JMSJEY ISSN: 1340-8097. Journal written in


Abstract

The structure of melittin in the presence of dioleoylphosphatidylcholine (DOPC) was investigated using hydrogen-deuterium (H/D) exchange in conjunction with collision induced dissocn. (CID) in an rf-only hexapole ion guide

with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). The

deuterium incorporation into backbone amide hydrogens of melittin in the presence of DOPC was analyzed at

different time points examg. the mass of each fragment ion produced by hexapole-CID. The percentage of deuterium

incorporation into the fragments of melittin was less than 45% at initial 10 s of isotope exchange. It increased

rapidly as the exchange period was prolonged. After 300 s of incubation in D2O about 85% of amide hydrogens

were exchanged with deuterium. When melittin was incubated in D2O in the presence of dodecylphosphocholine

(DPC), the rate of isotope exchange was reduced at every time point. In the case of melittin alone, more than 80%

amide hydrogens were exchanged with deuterium within 10 s. By comparing these time courses, it seems that the

contact with DOPC did not induce melittin to change its conformation. DOPC possibly just shielded the melittin

mol. while DPC induced melittin to form some stable conformation, such as helical structure. It was revealed that

H/D exchange and MS anal. enabled to study such structural changes of a peptide brought about by the interaction

with two types of phospholipid even in the presence of 50-fold amt. of lipid.


Characterizing the fragmentation of 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime and selected

metabolites using ion trap mass spectrometry. Zhou, Lian; Voyksner, Robert D.; Thakker, Dhiren R.; Stephens,

Chad E.; Anbazhagan, Mariappan; Boykin, David W.; Hall, James E.; Tidwell, Richard R. Division of Medicinal

Chemistry and Natural Products, School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill,

NC, USA. Rapid Communications in Mass Spectrometry (2002), 16(11), 1078-1085. CODEN: RCMSEF

ISSN: 0951-4198. Journal written in English. CAN 137:379622 AN 2002:431606 CAPLUS (Copyright 2004

ACS on SciFinder (R))

Abstract

A novel prodrug [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289)] of the promising antimicrobial

agent, 2,5-bis(4-amidinophenyl)furan (DB75), has excellent oral activity. It is currently undergoing phase II clin.

evaluation as an orally administered drug candidate against African trypanosomiasis and Pneumocystis carinii

pneumonia. The sequential product ion (MSn) fragmentations of DB289 and selected metabolites were characterized

using ion trap mass spectrometry with electrospray ionization. An unusual homolytic bond cleavage, formation of an

odd-electron ion from an even-electron ion with the loss of a radical, was commonly seen in the fragmentation

patterns of DB289 and its metabolites. Both O-Et and N-Me homologs of DB289 were utilized to confirm this

fragmentation pathway. The labile hydrogen atoms in DB289 are readily exchanged with deuterium atoms in the


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solvent contg. deuterium oxide (D2O) instead of water. The mass shift patterns displayed in the product ion spectra

of DB289 in D2O proved useful in verifying the fragmentation pathway. Octadeuterated DB289 and DB75 (d-

labeling on the di-Ph rings) showed unequivocally that the diphenylfuran moiety is not involved in the

fragmentation. The fragmentation pathways uncovered in this work will facilitate structural characterization of all

the metabolites produced in the metabolic activation of DB289.


Structural features of interferon-aggregation revealed by hydrogen exchange. Tobler, Scott A.; Fernandez,Erik J. Department of Chemical Engineering, University of Virginia, Charlottesville, VA, USA. Protein

Science (2002), 11(6), 1340-1352. CODEN: PRCIEI ISSN: 0961-8368. Journal written in English. CAN


Abstract

Using hydrogen-deuterium exchange (HX) and electrospray ionization mass spectrometry, we have investigated the

stability and structural changes of recombinant human interferon-(IFN-) during aggregation induced by guanidinehydrochloride (GdnHCl) and potassium thiocyanate. First, HX labeling was initiated after the amorphous aggregates

were formed to probe the tertiary structure of the aggregated state. Second, labeling was performed at low protein

concns. to assess stability under aggregation prone conditions. In 1 M GdnHCl, the stability of IFN-was greatlyreduced and much less protection from HX in soln. was obsd. Exchange under these conditions was slower in helix

C than in the rest of the protein. Aggregates formed in 1 M GdnHCl showed a HX pattern consistent with a partially

unfolded state with an intact helix C. Although aggregates formed in 0.3 M KSCN exhibited a HX pattern similar to

those formed in GdnHCl, the soln. phase HX pattern in 0.3 M KSCN was surprisingly comparable to that of the

native state. Varying the aggregation time before performing HX revealed that KSCN first pptd. native protein and

then facilitated partial unfolding of the pptd. protein. These results show that helix C, which forms the hydrophobic

core of the IFN-dimer, is highly protected from HX under native conditions, is more stable in GdnHCl than the restof the protein and remains intact in both GdnHCl- and KSCN-induced aggregates. This suggests that native-state

HX patterns may presage regions of the protein susceptible to unfolding during aggregation.


Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and

electrospray ionization mass spectrometry. Kim, Moo-Young; Maier, Claudia S.; Reed, Donald J.; Deinzer, Max

L. Department of Chemistry, Oregon State University, Corvallis, OR, USA. Protein Science (2002), 11(6),

1320-1329. CODEN: PRCIEI ISSN: 0961-8368. Journal written in English. CAN 137:121297 AN


Abstract

Hydrogen/deuterium (H/D) exchange in combination with electrospray ionization mass spectrometry and near-UV

CD (CD) was used to study the conformational properties and thermal unfolding of Escherichia coli thioredoxin and

its Cys32-alkylated derivs. in 1% acetic acid (pH 2.7). Thermal unfolding of oxidized (Oxi) and reduced (Red)

-thioredoxin (TRX) and Cys-32-ethylglutathionyl (GS-ethyl-TRX) and Cys-32-ethylcysteinyl (Cys-ethyl-TRX),

which are derivs. of Red-TRX, follow apparent EX1 kinetics as charge-state envelopes, H/D mass spectral exchangeprofiles, and near-UV CD appear to support a two-state folding/unfolding model. Minor mass peaks in the H/D

exchange profiles and nonsuperimposable MS- and CD-derived melting curves, however, suggest the participation of

unfolding intermediates leading to the conclusion that the two-state model is an oversimplification of the process.

The relative stabilities as measured by melting temps. by both CD and mass spectral charge states are, Oxi-TRX, GS-

ethyl-TRX, Cys-ethyl-TRX, and Red-TRX. The introduction of the Cys-32-ethylglutathionyl group provides extra

stabilization that results from addnl. hydrogen bonding interactions between the ethylglutathionyl group and the

protein. Near-UV CD data show that the local environment near the active site is perturbed to almost an identical

degree regardless of whether alkylation at Cys-32 is by the ethylglutathionyl group, or the smaller, nonhydrogen-

bonding ethylcysteinyl group. Mass spectral data, however, indicate a tighter structure for GS-ethyl-TRX.


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Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem

mass spectrometry with electrospray ionization. Hsu, Fong-F.; Turk, John; Stewart, Mary E.; Downing, Donald

T. Mass Spectrometry Resource, Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine,

Washington University School of Medicine, St. Louis, MO, USA. Journal of the American Society for MassSpectrometry (2002), 13(6), 680-695. CODEN: JAMSEF ISSN: 1044-0305. Journal written in English. CAN


Abstract

We applied electrospray ionization (ESI) tandem quadrupole mass spectrometry to establish the fragmentation

pathways of ceramides under low energy collisional-activated dissocn. (CAD) by studying more than thirty compds.

in nine subclasses. The product-ion spectra of the [M +Li]+ ions of ceramides contain abundant fragment ions that

identify the fatty acyl substituent and the long-chain base (LCB) of the mols., and thus, the structure of ceramides

can be easily detd. Fragment ions specific to each ceramide subclasses are also obsd. These feature ions permit

differentiation among different ceramide subclasses. The ion series arising from the classical C-C bond cleavages

that were reported in the fast-atom bombardment (FAB)-high energy tandem mass spectrometry is not observable;

however, the product-ion spectra contain multiple fragment ions informative for structural characterization andisomer identification. We also investigated the tandem mass spectra of the fragment ions generated by in-source

CAD (pseudo-MS3) and of the deuterium-labeling mol. species obtained by H/D exchange to support the ion

structure assignments and the proposed fragmentation pathways that lead to the ion formation.


Mass spectral characterization of tetracyclines by electrospray ionization, H/D exchange, and multiple stage

mass spectrometry. Kamel, Amin M.; Fouda, Hassan G.; Brown, Phyllis R.; Munson, Burnaby. Department of

Drug Metabolism, Pfizer Global Research and Development, Groton Laboratories, Groton, CT, USA. Journal of

the American Society for Mass Spectrometry (2002), 13(5), 543-557. CODEN: JAMSEF ISSN: 1044-0305.


(R))

Abstract

Electrospray ionization (ESI) and collisionally induced dissocn. (CID) mass spectra were obtained for five

tetracyclines and the corresponding compds. in which the labile hydrogens were replaced by deuterium by either gas

phase or liq. phase exchange. The no. of labile hydrogens, x, could easily be detd. from a comparison of ESI spectra

obtained with N2 and with ND3 as the nebulizer gas. CID mass spectra were obtained for [M +H]+ and [M - H]-

ions and the exchanged analogs, [M(Dx) +D]+ and [M(Dx) - D]-, and produced by ESI using a Sciex API-IIIplus and

a Finnigan LCQ ion trap mass spectrometer. Compns. of product ions and mechanisms of decompn. were detd. by

comparison of the MSN spectra of the un-deuterated and deuterated species. Protonated tetracyclines dissoc. initially

by loss of H2O (D2O) and NH3 (ND3) if there is a tertiary OH at C-6. The loss of H2O (D2O) is the lower energy

process. Tetracyclines without the tertiary OH at C-6 lose only NH3 (ND3) initially. MSN expts. showed easily

understandable losses of HDO, HN(CH3)2, CH3 - N:CH2, and CO from fragment ions. The major fragment ions do

not come from cleavage reactions of the species protonated at the most basic site. Deprotonated tetracyclines had

similar CID spectra, with less fragmentation than those obsd. for the protonated tetracyclines. The lowest energy

decompn. paths for the deprotonated tetracyclines are the competitive loss of NH3 (ND3) or HNCO (DNCO).

Product ions appear to be formed by charge remote decompns. of species de-protonated at the C-10 phenol.


In electrospray ionization source hydrogen/deuterium exchange LC-MS and LC-MS/MS for characterization

of metabolites. Lam, Wing; Ramanathan, Ragu. Department of Pharmacokinetics, Dynamics, and Metabolism,


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Pfizer Global Research and Development, Ann Arbor, MI, USA. Journal of the American Society for Mass

Spectrometry (2002), 13(4), 345-353. CODEN: JAMSEF ISSN: 1044-0305. Journal written in English. CAN


Abstract

A new method is described for performing hydrogen/deuterium (H/D) exchange in an electrospray ionization (ESI)source. The use of liq. chromatog. (LC)-mass spectrometer equipped with an ESI source and deuterium oxide (D2O)

as the sheath liq. allows H/D exchange expts. to be performed online. This directly provides information for detg.

the no. and position of exchangeable hydrogens, aiding in the elucidation of the structures of drug metabolites. To

demonstrate the utility of this method, LC-mass spectrometry (MS) and LC-MS/MS expts. were performed using

either H2O or D2O as sheath liq. on a matrix metalloprotease (MMP) inhibitor (PD 0200126) and its metabolites.

Examn. of the mass shift of the deuterated mol. from that of the protonated mol. allowed the no. of exchangeable

protons to be detd. Interpretation of the product-ion-spectra helped to det. the location of the exchanged protons and

assisted in the assignment of the site(s) of modification for each metabolite.


Thermal unfolding of proteins studied by coupled reversed-phase HPLC-electrospray ionization mass

spectrometry techniques based on isotope exchange effects. Hearn, Milton T. W.; Quirino, Joselito P.;Whisstock, James; Terabe, Shigeru. Centre for Bioprocess Technology, Department of Biochemistry and Molecular

Biology, Monash University, Clayton, Australia. Analytical Chemistry (2002), 74(6), 1467-1475. CODEN:

ANCHAM ISSN: 0003-2700. Journal written in English. CAN 136:243957 AN 2002:126126 CAPLUS


Abstract

In this study, a deuterium exchange procedure has been employed to evaluate the thermal stability of globular

proteins under conditions that replicate their interactive behavior in reversed-phase high performance chromatog.

(RP-HPLC) systems. In particular, this investigation has permitted the conformational stability of two proteins, hen

egg white lysozyme (HEWL) and horse heart myoglobin (HMYO) to be examd. under different temp. and low-pH

solvent regimes. The results confirm that this exptl. approach provides an efficient strategy to explore fundamental

conformational features of polypeptides or proteins in their folded and partial unfolded states under these interactiveconditions. In particular, this anal. procedure permits insight to be readily gained into the processes that occur when

polypeptides and globular proteins interact with lipophilic liq./solid interfaces in the presence of water-org. solvent

mixts. at different temps.


Mass spectrometric studies on peptides and proteins: conformations of Escherichia coli thioredoxin and its

alkylated adducts studied by hydrogen/deuterium exchange and HPLC-electrospray ionization mass

spectrometry. Kim, Moo-Young. Oregon State Univ., Corvallis, OR, USA. Avail. UMI, Order No.

DA3005517. (2001), 161 pp. From: Diss. Abstr. Int., B 2001, 62(2), 862. Dissertation written in English.



Conformational properties of -dendrotoxin using electrospray mass spectrometry. Belva, Helene; Lange,

Catherine; Segalas, Isabelle. Spectrometrie de Masse Bio-Organique, CNRS-UMR 6014, INSERM-IFR23, UFR

des Sciences, Universite de Rouen, Mont Saint Aignan, Fr. European Journal of Mass Spectrometry (2001),

7(4&5), 373-383. CODEN: EJMSCL ISSN: 1469-0667. Journal written in English. CAN 136:65341 AN


Abstract


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To examine the conformation of -dendrotoxin, charge state distributions (CSD) and hydrogen/deuterium (H/D)

isotopic exchange using electrospray-ionization mass spectrometry [ES-MS] have been used. Here, it cannot be

deduced from the CSDs that some basic residues are hidden in the core of the structure. At neutral pH, the CSDs

reflect the equil. bulk soln. exactly. Seven basic residues seem to be shielded from the solvent, according to calcns.

from the X-ray structure. But, under acidic conditions, the CSDs obsd. in mass spectra are not a true reflection of the

equil. bulk soln., partly because of an increased intramol. electrostatic effect with the gaseous ions. H/D exchangeexpts. show that three to seven hydrogens are involved in hydrogen bonds. The resistance to urea denaturation has

also been investigated. Urea and 1,4-dithiothreitol (DTT) denaturation together lead to unfolded conformers.

During this redn. of the bridges, it is obsd. that the Cys16-Cys40 and Cys32-Cys53 linkages are more accessible to

the reducing agent than the Cys7-Cys57 linkage, which is very important by maintaining a compact fold.


Conformational changes in -endorphin as studied by electrospray ionization mass spectrometry. Lin, Hui;Dass, Chhabil. Department of Chemistry, The University of Memphis, Memphis, TN, USA. Rapid

Communications in Mass Spectrometry (2001), 15(23), 2341-2346. CODEN: RCMSEF ISSN: 0951-4198.


(R))

Abstract

Because of a wide range of physiol. functions, the structure of -endorphin (BE) is of great interest. In this study,conformational changes in BE induced by methanol are explored with electrospray ionization-mass spectrometry

(ESI-MS). Differences in the charge-state distribution (CSD) and the extent of hydrogen/deuterium (H/D) exchange

were used to monitor the conformational changes. The latter expts. were conducted via time-resolved ESI-MS in a

continuous-flow app. Both these techniques demonstrate that BE exists in a random coil open structure in aq. media,

but it acquires a more compact conformation with increased concn. of methanol. The H/D exchange expts. reveal

that BE forms 61% -helix in mixed solvents.


Structure of melittin bound to phospholipid micelles studied using hydrogen-deuterium exchange and

electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Akashi, Satoko; Takio,

Koji. Division of Biomolecular Characterization, RIKEN (The Institute of Physical and Chemical Research),

Saitama, Japan. Journal of the American Society for Mass Spectrometry (2001), 12(12), 1247-1253. CODEN:

JAMSEF ISSN: 1044-0305. Journal written in English. CAN 136:195820 AN 2001:896293 CAPLUS


Abstract

The structure of melittin bound to dodecylphosphocholine (DPC) micelles was investigated using hydrogen-

deuterium (H/D) exchange in conjunction with collision induced dissocn. (CID) in an rf-only hexapole ion guide

with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The

deuterium incorporation into backbone amide hydrogens of melittin with or without DPC micelles was analyzed atdifferent time points examg. the mass of each fragment ion produced by hexapole CID. When melittin existed alone

in aq. soln., more than 80% of amide hydrogens was exchanged within 10 s, and the deuterium content in each

fragment ion showed high values throughout the expts. When melittin was bound to DPC micelles, the percentage of

deuterium incorporation into the fragment decreased remarkably at any time point. It increased little by little as the

exchange period prolonged, indicating that some stable structure was formed by the interaction with DPC. The

results obtained here were consistent with the previous studies on the helical structure of melittin carried out by

NMR and CD analyses. The strategy using H/D exchange and MS anal. might be useful for studying structural

changes of peptides and proteins caused by phospholipid micelles. It could also be applied to membrane-bound

proteins to characterize their structure.


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Hydrogen-deuterium exchange at non-labile sites: a new reaction facet with broad implications for structural

and dynamic determinations. Reed, Dana R.; Kass, Steven R. Department of Chemistry, University of

Minnesota, Minneapolis, MN, USA. Journal of the American Society for Mass Spectrometry (2001), 12(11),

1163-1168. CODEN: JAMSEF ISSN: 1044-0305. Journal written in English. CAN 136:167055 AN2001:818453 CAPLUS (Copyright 2004 ACS on SciFinder (R))

Abstract

Hydrogen-deuterium exchange at non-labile sites is reported. The conjugate bases of isophthalic acid (m-

C6H4(CO2H)2), 2-oxoglutaric acid (HO2CCOCH2CH2CO2H), and 2-methylisophthalic acid (2-CH3-1,3-

C6H4(CO2H)2) undergo scrambling with 1, 2, and 3 carbon-centered hydrogens under a variety of conditions.

Likewise, protonated 2-(m-methoxyphenyl)ethylamine ((m-CH3OC6H4)CH2CH2NH2) undergoes up to 5 H/D

exchanges upon gentle activation whereas the conjugate acid of 2-phenylethylamine (C6H5CH2CH2NH2) requires

the presence of ammonia-d3 in order to be pushed to undergo up to 8 H/D exchanges. The very act of

electrospraying ions can result in extensive movement of deuterium to carbon centers and, in some cases, could not

be prevented. These findings offer great promise for future exploitation but also suggest that the interpretation of

many H/D exchange expts. using mass spectrometry as the anal. tool could be in error.


Intramolecular Interactions in Chemically Modified Escherichia coli Thioredoxin Monitored by

Hydrogen/Deuterium Exchange and Electrospray Ionization Mass Spectrometry. Kim, Moo-Young; Maier,

Claudia S.; Reed, Donald J.; Ho, P. Shing; Deinzer, Max L. Department of Chemistry and Department of

Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA. Biochemistry (2001), 40(48),

14413-14421. CODEN: BICHAW ISSN: 0006-2960. Journal written in English. CAN 136:65850 AN


Abstract

Site specific amide hydrogen/deuterium content of oxidized and reduced Escherichia coli thioredoxin (TRX), and

alkylated derivs., Cys-32-ethylglutathionylated and Cys-32-ethylcysteinylated thioredoxins are measured, after

exposure for 20 s to D2O/phosphate buffer (pH 5.7), by electrospray mass spectrometry. The degree of deuteration

of Oxi-TRX and Red-TRX correlated with the rates of H/D exchange measured previously by NMR. The

ethylcysteinyl modification was shown to minimally perturb the active site of the reduced protein, but showed more

global effects on structures of -helixes and -strands distant from the site of modification. In contrast, the largerethylglutathionyl group had little effect on the protein's overall conformation, but significantly affected the structure

of loops close to the active site. A mol. model of GS-ethyl-TRX derived from mol. simulati

Hydrogen/Deuterium Exchange

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