Size exclusion chromatography/mass spectrometry applied to the analysis of polysaccharides.

A novel method for analysing polysaccharide materials is described which employs size-exclusion chromatography (SEC) followed by detection by on-line electrospray ionisation mass spectrometry (ESI-MS) and off-line matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). It is demonstrated through SEC/ESI ion trap mass spectrometry that the formation of multiply charged oligomer ions, which bind up to five sodium cations, allows the rapid analysis of polysaccharide ions with molecular weights in excess of 9 kDa. MALDI spectra generated from fractionation of the effluent collected from the same SEC separation are shown to be in good agreement with the ESI spectra with respect to molecular weight distributions and types of ions generated. ESI and MALDI mass spectra of samples obtained from sequential graded ethanol precipitation and SEC fractionation of acid and enzymatically digested arabinoxylan polysaccharides show important structural differences between polysaccharide fragments. In addition, a comparison is made between the mass spectra of native and permethylated SEC-separated fragments of acid and enzymatically treated arabinogalactan. Linkage information of the permethylated arabinogalactan oligomers can be rapidly established through the use of on-line SEC/ESI-MS( n) experiments.     

Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization

We applied electrospray ionization (ESI) tandem quadrupole mass spectrometry to establish the fragmentation pathways of ceramides under low energy collisional-activated dissociation (CAD) by studying more than thirty compounds 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 molecules, and thus, the structure of ceramides can be easily determined. Fragment ions specific to each ceramide subclasses are also observed. 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 and isomer identification. We also investigated the tandem mass spectra of the fragment ions generated by in-source CAD (pseudo-MS3) and of the deuterium-labeling molecular species obtained by H/D exchange to support the ion structure assignments and the proposed fragmentation pathways that lead to the ion formation.     

Synthesis and Electrospray Ionization Mass Spectra of N-（1,3, 2-Dioxaphosphorinan-2-ylmethyl）thiophosphoramidates

N-（1,3,2-Dioxaphosphorinan-2-ylmethyl）thiophosphoramidates were synthesized and determined by NMR spectra and positive ion electrospray ionization mass spectrometry （ESI-MS） in conjunction with tandem mass spectrometry （MS/MS）. The fragmentation pathways were investigated. The results show that these characteristic ions in ESI mass spectra are useful in the structural determination of N-（1,3,2-dioxaphosphorinan-2-ylmethyl）- thiophosphoramidates.     

A comparative study of the fragmentation of neutral lactooligosaccharides in negative-ion mode by UV-MALDI-TOF and UV-MALDI ion-trap/TOF mass spectrometry

Structure analyses of underivatized neutral lacto oligosaccharides are systematically performed by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI TOF MS) and UV-MALDI ion-trap time-of-flight mass spectrometry (ion-trap/TOF MS) acquired in negative-ion mode. Interestingly, their fragmentation significantly differ each other. In postsource decay (PSD) in UV-MALDI TOF MS, cross-ring cleavage at the reducing terminal predominates. On the other hand, glycosyl bond cleavage (C-type fragmentation) takes place preferentially in collision induced dissociation (CID) in UV-MALDI ion-trap/TOF MS. The cross-ring cleavage in PSD similar to that in in-source decay occurs via a prompt reaction path characteristic of the UV-MALDI process itself. The product ion spectra of UV-MALDI ion-trap/TOF MS are similar to the electrospray ionization (ESI) ion-trap or quadrupole/TOF CID product ion spectra. During ion-trap/TOF MS experiments, the deprotonated molecular ions survive for several tens of milliseconds after CID event because the high internal energy chlorinated precursor ions are cooled by collisional cooling in the ion trap. The results obtained suggest that the PSD from the chlorinated precursor ion in UV-MALDI TOF MS might proceed as a two-step reaction; in the first, a high internal energy deprotonated molecular ion is generated as a reaction intermediate during the flight in the drift tube, and in the second, the rapid decomposition from the deprotonated molecular ion takes place.     

Ionic liquid-assisted electrospray ionization of polysaccharides

In this work, we give the report of significant detection sensitivity improvement of electrospray ionization (ESI) mass spectra of polysaccharides by adding various ionic liquid compounds into samples. Mass spectra obtained were greatly simplified and appeared to be similar to spectra from matrix-assisted laser desorption/ionization due to the narrow charge number distribution. Mass spectra of polysaccharides with the attachment of either anion or cation of ionic liquid compounds were observed. No protonated or deprotonated polysaccharide ions were detected when ionic liquid compounds were added into samples. Little alkali-attached polysaccharide ions were observed. Ionic liquid-assisted ESI (ILA-ESI) mass spectrometry has significantly improved the detection sensitivity of large neutral polysaccharide compounds.     

Electrospray ionization mass spectrometry for identification and structural characterization of pregnane glycosides

Pregnane glycosides are a class of naturally occurring substances characterized by some interesting biological activities and widely distributed in the plant kingdom and in some marine organisms. Their toxicity and use in herbal drugs and folk medicines has generated great interest in the chemical characterization of these molecules. In the study reported here the potential of electrospray ionization mass spectrometry (ESI-MS) in the identification and structural characterization of pregnane glycosides was examined. ESI-MS/MS and ESI-MS(n) analyses were performed on 27 different compounds employing two mass spectrometers equipped with a triple-quadrupole or an ion-trap analyzer. The data illustrate the ability of the ESI techniques in the identification of pregnane glycosides, including the nature of the pregnane core, the kind of ester substituents, the types of sugar residues (hexose, deoxyhexose, dideoxyhexose, O-methyldeoxyhexose and O-methyldideoxyhexose), and the primary structure of the saccharide chain. From these data, a generalized fragmentation pathway was proposed by comparing the spectra acquired for all the compounds. Interestingly, similar results were obtained from the two instruments, thus demonstrating that detailed analyses of product ion spectra obtained using a triple-quadrupole mass spectrometer led to structural information comparable to those obtainable in MSn experiments using an ion trap. Different and complementary information was deduced by fragmenting the [M+H]+ or the [M+Na]+ ions, or the protonated aglycone [Agl+H]+ generated by in-source fragmentation. The present evidence clearly suggests that, in order to obtain a complete characterization of pregnane glycosides by MS, all three of these species should be accurately analyzed.     

Mass spectrometric studies of the gas phase retro-<Emphasis Type="Italic">Michael</Emphasis> type fragmentation reactions of 2-hydroxybenzyl-<Emphasis Type="Italic">N</Emphasis>-pyrimidinylamine derivatives

The gas-phase fragmentation reactions of 2-hydroxybenzyl-N-pyrimidinylamine derivatives (Compounds 1 to 6), the O-N-type acid-catalyzed Smiles rearrangement products of 2-pyrimidinyloxy-N-arylbenzylamine derivatives, have been examined via positive ion matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation (IRMPD) mass spectrometry in FT-ICR MS and via negative ion electrospray ionization (ESI) in-source collision-induced dissociation (CID) mass spectrometry, respectively. The major fragmentation pathway of protonated 1 to 6 gives the F ions under IRMPD; theoretical results show that the retro-Michael reaction channel is more favorable in both thermodynamics and kinetics. This explanation is supported by H/D exchange experiments and the MS/MS experiment of acetylated 1. Deprotonated 1 to 6 give rise to the solitary E ions (aromatic nitrogen anions) in the negative ion in-source CID; theoretical calculations show that a retro-Michael mechanism is more reasonable than a gas-phase intramolecular nucleophilic displacement (SN2) mechanism to explain this reaction process.     

The even-electron rule in electrospray mass spectra of pesticides

A study of the fragmentation and ion formation of three major families of pesticides (including herbicides, insecticides, and fungicides) by liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) and liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF-MS) was carried out using positive electrospray ionization (ESI) and the results compared with those by gas chromatography (GC)/TOF-MS with electron ionization (EI) in order to test the validity of the even-electron rule in electrospray ionization. First, the majority of the fragmentations by positive ion ESI were even electron (EE) ions (93% of the fragment ions). Secondly, the formation of odd-electron (OE) fragment ions was greater with EI, where the fragment ions were present in a ratio of approximately 1:2 (35% OE ions and 65% EE ions). Thirdly, in-source collision-induced dissociation (CID) fragmentation by LC/TOF-MS and CID fragmentation in the collision cell by LC/Q-TOF-MS/MS resulted in 95% of the fragment ions being identical between the two types of fragmentation. As ESI in the positive ion mode yields an EE precursor ion (normally a protonated molecule), this commonly leads to EE fragment ions by elimination of molecules - a process called the even-electron rule. Neutral radical losses were less common in ESI but were common in the EI spectra of the same compounds. The structures that did lead to OE ions in ESI (exceptions to the even-electron rule approximately 7% of all ESI ions) favored electronegative radical losses in approximately the following order: .SO(2)CH(3), .NO(2), .CH(3), .Cl, .SCH(3), .CH(2)CH, and .OH.     

Mass-spectrometric studies of new 6-nitroquipazines—serotonin transporter inhibitors

Six synthesized 6-nitroquipazine derivatives were examined by electron ionization (EI) and electrospray ionization (ESI) mass spectrometry in positive and negative ion mode. The compounds exhibit high affinity for the serotonin transporter (SERT) and belong to a new class of SERT inhibitors. The EI mass spectra registered in negative ion mode showed prominent molecular ions for all the compounds studied. All EI mass spectra and all ESI mass spectra showed similar fragmentation pathways of molecular ions, but the pathways differed between EI and ESI. The differences were explained with the aid of theoretical evaluation of the stability of the respective radical ions (EI MS) and protonated ions (ESI MS).     

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.     

Identification and structural characterization of steroidal glycosides in Hoodia gordonii by ion-trap tandem mass spectrometry and liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry

Electrospray ion-trap tandem mass spectrometry (ESI-MS/MS) and high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOFMS) were used to identify and characterize eight C-21 steroidal glycosides in Hoodia gordonii. A generalized fragmentation pathway was proposed by comparing the spectra acquired for eight C-21 steroidal glycosides. The steroidal glycosides in Hoodia gordonii have been classified into two major core groups: hoodigenin A and calogenin. Using the ESI-TOF method, the major core peak ions generated by hoodigenin A glycosides are m/z 313 and 295 and by calogenin glycosides are m/z 479, 461, 299 and 281, respectively. In the MS/MS spectra, fragmentation reactions of the [M+Na](+) ion were recorded to provide structural information about the glycosyl and aglycone moieties. The data illustrates the ability of positive mode ESI for the identification of hoodigenin A and calogenin glycosides, including the nature of the hoodigenin A and calogenin core, the number of sugar residues and the type of saccharide moiety.     

Structural characterization of iridoid glucosides by ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

The mass spectral fragmentation behavior of ten iridoid glucosides (IGs) has been studied using electrospray ionization (ESI), collision-induced dissociation (CID), and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). In the negative ESI mass spectra, the deprotonated [M-H](-) ion was observed for all of the ten IGs except gardoside methyl ester, while the formate adduct [M+HCOO](-) ion appeared to be favored by the presence of a methyl ester or a lactone group in the C-4 position when formic acid was added to the mobile phase. The CID MS/MS spectra of the [M-H](-) ions have been used for structural elucidation. Ring cleavages of the aglycone moiety have been observed in the MS/MS spectra, corresponding to (1,4)F(-), (2,6)F(-), (2,7)F(-), and (2,7)F(0) (-) ions, based on accurate mass measurements and the elemental compositions of the product ions. These characteristic ions gave valuable information on the basic structural skeletons. Furthermore, on the basis of the relative abundances of the fragment ions (1,4)F(-) and (2,7)F(-), different sub-classes, such as cyclopentane-type and 7,8-cyclopentene-type IGs, can be differentiated. Ring cleavage of the sugar moieties was also observed, yielding useful information for their characterization. In addition, the neutral losses, such as H(2)O, CO(2), CH(3)OH, CH(3)COOH, and glucosidic units, have proved useful for confirming the presence of functional substituents in the structures of the IGs. Based on the fragmentation patterns of these standard IGs, twelve IGs have been characterized in an extract of Hedyotis diffusa Willd. by means of ultra-performance liquid chromatography/Q-TOF MS/MS, of which six have been unambiguously identified and the other six have been tentatively identified.     

Investigation of the tris(trimethoxyphenyl)phosphonium acetyl charged derivatives of peptides by electrospray ionization mass spectrometry and tandem mass spectrometry

Charged derivatives of peptides are useful in obtaining simpler collision-activated dissociation (CAD) mass spectra. An N-terminal charge-derivatizing reagent capable of reacting with picomole levels of peptide has been recently reported (Huang et al. Anal. Chem. 1997, 69, 137-144) in the contexts of analyses by fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Electrospray ionization (ESI) mass spectrometric investigation of these tris(trimethoxyphenylphosphonium) acetyl derivatives are described in this article, including studies by in-source fragmentation (ISF) and tandem mass spectrometry (MS/MS). Results from ISF are compared with those from MS/MS. Similarities and differences between ESI-ISF, MALDI-post-source decay (PSD), and FAB-CAD data are presented. Differences in fragmentation of these charged derivatives in the triple quadrupole and ion trap mass spectrometers also are discussed. Application of this derivatizing procedure to tryptic digests and subsequent analysis by liquid chromatography-mass spectrometry is also shown.     

Positive-ion ESI mass spectrometry of regioisomeric nonreducing oligosaccharide fatty acid monoesters: in-source fragmentation of sodium adducts

Structural characterization and differentiation of a novel group of regioisomeric monolaurate esters of the nonreducing trisaccharides raffinose and melezitose, and the nonreducing tetrasaccharide stachyose has been obtained using positive electrospray ionization (ESI) mass spectrometry with in-source fragmentation. The surfactant nature and high polarity of these compounds make them appropriate analytes for being studied by conventional ESI-MS. The position of the acyl chain in each particular regioisomer has been used as a reporter group that allows unambiguous rationalization of the fragmentation routes of the corresponding natriated molecular ions [M + Na](+). In all cases, the main fragment ions were produced through cleavage of the glycosidic bond involving two anomeric carbons, the C-1' and C-2' of the alpha-D-Glcp-(1-2)-beta-D-Fruf bond, and it was observed that sodium cation retention occurred on the heavier mass fragment of the two formed fragments, (e.g. di- or trisaccharide type vs monosaccharide type). Our results may help to better understand the fragmentation behavior of nonreducing oligosaccharides (as sodium adducts) in positive ESI mass spectrometry.     

In-source fragmentation and accurate mass analysis of multiclass flavonoid conjugates by electrospray ionization time-of-flight mass spectrometry

In-source collision-induced dissociation (CID) fragmentation features of multiclass flavonoid glycoconjugates were examined using liquid chromatography electrospray time-of-flight mass spectrometry. Systematic experiments were performed to search for optimal conditions for in-source fragmentation in both positive and negative ion modes. The objective of the study was to attain uniformly appropriate conditions for a wide range of analytes independently of the aglycone, the attached sugar part and the type of bond between the aglycone and the glycan moieties (O- or C-glycosides). Studied substances included representatives of flavonols, flavones, flavanones and anthocyanins and, regarding their glycan parts, mono-, di- and triglycosides with varying distribution of carbohydrate moieties (di-O-glycosides, O-diglycosides, O,C-diglycosides). The breakdown properties of the analytes along with the abundances of the characteristic diagnostic ions required for structural elucidation of complex flavonoid derivatives were evaluated. An optimized value was found for the instrument parameter (fragmentor voltage) affecting the in-source CID fragmentation of the analytes [230 V (ESI+) and 330 V (ESI-)]. Thus, appropriate performance in terms of both highly sensitive full-scan acquisition and fragmentation information was obtained for all the investigated flavonoids. In addition, singularities in the abundance of selected diagnostic ions (e.g. Y(0), Y(1) and Y*) due to variations in the interglycosidic linkage (rutinoside-neohesperidoside) in the glycan part were found and are also evaluated and discussed in detail. The combination of in-source CID fragmentation with high mass accuracy MS detection establishes a working basis for the development of versatile and useful LC-MS methods for wide-scope screening, non-targeted detection and tentative identification of flavonoid derivatives.     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Pyrolysis–desorption chemical ionization and high-energy tandem mass spectrometry of the O-specific antigen of Yersinia ruckeri

Application of pyrolysis-desorption chemical ionization mass spectrometry (Py-DCI-MS) is demonstrated for the structural characterization of the native O-specific antigen of Yersinia ruckeri. Under proper pyrolytic and chemical ionization conditions this antigenic polysaccharide yields anhydrohexosamine fragments which facilitate identification of the trimeric repeating unit sequence. Tandem mass spectrometric analyses of the trimeric repeating unit fragments provided additional evidence for the proposed structure and permitted rationalization of the observed fragment ions. Furthermore, Py-DCI-MS of smaller hexosamine subunits isolated during off-line pyrolysis experiments showed fragmentation characteristics almost identical with those observed in the mass spectra of the whole antigen, hence confirming the stability of the pyrolysis fragments.     

Elucidation of the fragmentation pathways of azaspiracids, using electrospray ionisation, hydrogen/deuterium exchange, and multiple-stage mass spectrometry

Collision-induced dissociation (CID) mass spectra were generated for azaspiracids using electrospray ionisation (ESI), and hydrogen/deuterium (H/D) exchange was used to ascertain the number and type of replaceable hydrogens in the three predominant azaspiracid toxins. H/D exchange was conveniently achieved using deuterated solvents for liquid chromatography (LC). Using ion-trap mass spectrometry, multiple-stage CID experiments (MS(n)) 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 molecules from different regions during MS(n) experiments and it was possible to distinguish between the water losses from different molecular regions. These studies confirmed that the first water-loss ion in the spectra of azaspiracids resulted from dehydration at the vicinal diol at C20-C21. Five MS dissociation pathways were identified that resulted from fragmentation of the carbon skeleton of azaspiracids producing nitrogen-containing 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 analogues. The same product ions from backbone fragmentation were also observed 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/MS(n) methods for the analysis of azaspiracids.     

Structural analysis of 2,6-[bis(alkyloxy)methyl]-phenyltin derivatives using electrospray ionization mass spectrometry

Electrospray ionization (ESI) mass spectrometry was applied to the structural analysis of 23 2,6-[bis(alkyloxy)methyl]phenyltin(IV) derivatives. The mass spectra were measured in both polarity modes and multistage tandem mass spectrometric (MS(n)) measurements were performed on the ion trap analyser for positively charged tin-containing ions. The sum of complementary ions observed in the positive-ion mode (i.e. [M-R(3)](+) ion) and in the negative-ion mode (i.e. [R(3)](-) ion) permits molecular mass determination in spite of the fact that the molecular adducts were often missing even in the first-order mass spectra. The subsequent fragmentation of [M-R(3)](+) ions studied by MS(n) and the correlation of observed fragment ions with the expected structures of synthesized organotin(IV) compounds allowed us to understand the fragmentation behaviour and the mechanism of the ion formation for studied compounds. The typical neutral losses are alkenes, alcohols and aldehydes. The fragmentation pattern of one selected compound was supported by MS(n) measurements of an isotopically labelled analogue to confirm unusual ion-molecule reactions of some fragment ions with water in the ion trap.     

Capillary electrophoresis/mass spectrometry for the separation and characterization of bovine Cu,Zn‐superoxide dismutase

The native form of Cu,Zn‐superoxide dismutase (SOD‐1) is a homodimer that coordinates one Cu²⁺ and one Zn²⁺ per monomer. Cu²⁺ and Zn²⁺ ions play crucial roles in enzyme activity and structural stability, respectively. In addition, dimer formation is essential for SOD‐1 functionality, and in humans several SOD‐1 mutant isoforms have been associated with certain types of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disorder. In this paper we used capillary electrophoresis and mass spectrometry to study the different structures of bovine SOD‐1. The metal ions of the native enzyme (Cu₂,Zn₂‐dimer SOD‐1) were released in acidic medium in order to obtain apo‐SOD‐1, which is a monomer. Both substances were analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) and capillary electrophoresis with ultraviolet and electrospray ionization mass spectrometry detection (CE/UV and CE/ESI‐MS, respectively). With MALDI‐TOF‐MS, using matrices of sinapinic acid (SA) or 2,5‐dihydroxybenzoic acid (DHB) with or without trifluoroacetic acid (TFA), similar mass spectra were obtained for the metalated and non‐metalated samples. In both cases, an average molecular mass corresponding to the apo‐monomer SOD‐1 was calculated. This finding indicated that the metals were released from the Cu₂,Zn₂‐dimer SOD‐1 during sample preparation or ionization. For CE/UV and CE/ESI‐MS, two background electrolytes (BGEs) potentially compatible with ESI‐MS detection were used, namely 1 M of acetic acid (pH 2.3) and 10 mM of ammonium acetate (pH 7.3). Using a sheath liquid of 2‐propanol/water (60:40 v/v), with or without 0.1% v/v of formic acid, CE/ESI‐MS sensitivity was enhanced when the acidic BGE and the acidic sheath liquid were used. However, the electrophoretic profiles and the mass spectra obtained suggested that the metals of Cu₂,Zn₂‐dimer SOD‐1 were released, which generated the apo‐monomer during the electrophoretic separation. The neutral BGE provided enhanced conditions for the detection of the native enzyme. The differences between the mass spectra obtained for the Cu₂,Zn₂‐dimer and the apo‐monomer forms were significant and the presence of formic acid in the sheath liquid affected only sensitivity. Our results highlight the importance of selecting appropriate non‐denaturing separation and detection conditions to obtain reliable structural information about non‐covalent protein complexes by CE/ESI‐MS. Copyright © 2010 John Wiley & Sons, Ltd.