ESI Interface Research Articles

Determination of Simvastatin in human plasma by liquid chromatography–mass spectrometry

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for the determination of simvastatin (I) has been developed. After extraction by ethyl acetate, using lovastatin (II) as internal standard, solutes are separated on a C 18 column with a mobile phase consisting of methanol–water (9:1). Detection is performed on an atmospheric pressure ionization single quadruple mass spectrometer equipped with an ESI interface and operates in positive ionization mode. Simvastatin quantification was realized by computing peak area ratio (I/II) of the extracts analyzed in SIM mode ( m/ z: 441 and m/ z: 427 for I and II, respectively) and comparing them with calibration curve ( r=0.9997). Accuracy and precision for the assay were determined by calculating the intra-batch and inter-batch variation at three concentrations 0.1, 5.0, 10.0 ng/ml; the intra batch relative standard deviation (RSD) was less than 10% and ranged from 1.8 to 8.5%, respectively; the inter-batch RSD was less than 20% and ranged from 4.1 to 16.5%. The limit of detection was 0.05 ng/ml.

Study of non-covalent complexation between catechin derivatives and peptides by electrospray ionization mass spectrometry.

The recent development of electrospray ionization mass spectrometry (ESI-MS) has allowed its use to study molecular interactions driven by non-covalent forces. ESI-MS has been used to detect non-covalent complexes between proteins and metals, ligands and peptides and interactions involving DNA, RNA, oligonucleotides and drugs. Surprisingly, the study of the interaction between polyphenolic molecules and peptides/proteins is still an area where ESI-MS has not benefited. With regard to the important influence of these interactions in the biological and food domains, ESI-MS was applied to the detection and the characterization of soluble polyphenol-peptide complexes formed in model solution. The ability to observe and monitor the weak interactions involved in such macromolecular complexation phenomena was demonstrated for monomeric and dimeric flavonoid molecules (catechin-derived compounds) largely encountered in plants and plant derived products. Intact non-covalent polyphenol-peptide complexes were observed by ESI-MS using different experimental conditions. Utilizing mild ESI interface conditions allowed the detection of 1 : 1 polyphenol-peptide complexes in all tested solutions and 2 : 1 complexes for the dimers and galloylated polyphenols (flavanols). These results show that there is a preferential interaction between polymerized and/or galloylated polyphenols and peptide compared with that between monomeric polyphenols and peptides. Thus, ESI-MS shows potential for the study of small polyphenolic molecule-peptide interactions and determination of stoichiometry.

Packed capillary liquid chromatography–electrospray mass spectrometry of snow contaminated with sarin

Packed capillary liquid chromatography–electrospray mass spectrometry (LC–ESI-MS) was used for the analysis of a snow sample that was accidentally contaminated with an organophosphorus chemical warfare agent during the destruction of a chemical munition. Sarin, its hydrolysis products and a number of related compounds were identified on the basis of acquired LC–ESI-MS data. Full mass spectra were acquired for 14 compounds, with all exhibiting MH +, [MH+ACN] + ions and/or protonated dimers that could be used to confirm molecular mass. Sampling cone voltages from 20 to 70 V were utilized with the higher sampling voltages enhancing formation of structurally important product ions in the ESI interface. All data were acquired with a time-of-flight mass spectrometer with a resolution of 5000 (50% valley definition), a resolution that aided in the assignment of elemental composition of the observed ions. The application of LC–ESI-MS to snow analysis appears to be an attractive alternative to the GC–MS methods, since both chemical warfare agents and their hydrolysis products may be analysed directly, eliminating the need for additional sample handling and derivatization steps.

Determination of 2-Amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and Its Metabolite 2-Hydroxyamino-PhIP by Liquid Chromatography/Electrospray Ionization–Ion Trap Mass Spectrometry

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic aromatic amine found in cooked meat. It is metabolically activated by the human cytochrome P450 enzymes to form the carcinogenic metabolite N2-OH-PhIP. PhIP has been found to induce tumors in rats and is a suspected human carcinogen. In the present work, we have developed and validated a liquid chromatography–electrospray ionization/ion trap mass spectrometry (LC-ESI/ITMS) method for the determination of PhIP and N2-OH-PhIP. PhIP was incubated with microsomes prepared from the human liver; the PhIP and N2-OH-PhIP formed were isolated from the biomatrices by solid-phase extraction using C18 cartridges, with recoveries greater than 86%. Subsequently, the products were separated on a microbore reversed-phase C18 liquid chromatograph coupled to an ESI-ITMS. The ESI interface and the ITMS were tuned for various parameters, and data acquisition was performed in selective ion monitoring mode. The detection limit of PhIP and N2-OH-PhIP was 1 and 10 pg, respectively. The method is highly sensitive and selective, has simple sample preparation protocols, and should be applicable to the study of the metabolic activation of PhIP in various human tissues.

An integrated ten-pump, eight-channel parallel LC/MS system for automated high-throughput analysis of proteins.

An integrated 10-pump eight-channel LC/MS system has been developed for automated high-throughput analysis of intact proteins in recombinant protein purification processes. The key features of the system include (1) a compact 10-pump HPLC module that uses two pumps to generate a binary gradient and 8 pumps to deliver the mixed gradient to eight independent flow channels; (2) a TOF mass spectrometer with an eight-channel multiplexed ESI interface, which records separate data for all eight channels over each HPLC run cycle; and (3) highly automated data processing software that allows unattended calculation of protein molecular weight (in Da) from original mass spectral data (in m/z). This system was used in the routine screening of fractions from preparative scale chromatography to monitor the purification process with the required mass accuracy and throughput. As an example, the production and purification of an acylated protein with a molecular weight of 9 kDa is described. Using this off-line approach, it is practical to fully characterize protein-containing fractions from column chromatography with an overall analytical throughput of 1 min/protein sample with minimum operator involvement.

On the signal response of various pesticides in electrospray and atmospheric pressure chemical ionization depending on the flow-rate of eluent applied in liquid chromatography–tandem mass spectrometry

The API–MS signal response of several pesticides (atrazine, simazine, isoproturon, diuron, chlorfenvinphos, chlorpyrifos, alachlor, trifluralin) depending on the flow-rate of eluent entering the MS interface was investigated. The investigations were based on API–MS–MS analyses of standard pesticide mixtures in the flow injection mode (FIA) at systematically varied eluent flow-rates using both an ESI interface (Turboionspray) and a heated nebulizer type APCI source. In the result, the individual compounds included in this study showed significant differences in their signal response behaviour depending on the flow-rate of eluent applied. The most hydrophobic compounds among the investigated pesticides (chlorpyrifos and trifluralin) showed drastic losses of sensitivity with increasing eluent flow-rate in both ESI and APCI, while more hydrophilic compounds like atrazine, simazine and isoproturon showed the expected signal response (concentration-sensitive in ESI, mass-flow-sensitive in APCI) at least within a certain range of flow-rates (200–600 μl/min in ESI, 200–2000 μl/min in APCI). These findings lead to the conclusion that application of a programmed HPLC eluent flow-rate may be advantageous to achieve maximum sensitivity of API–MS detection for all pesticides of interest. This is exemplified by the implementation of a flow gradient into an online SPE–HPLC–APCI–MS/MS method for improved analysis of pesticides in drinking water.

Automated high-throughput infusion ESI-MS with direct coupling to a microtiter plate.

This paper describes the design and application of instrumentation for automated high-throughput infusion ESI-mass spectrometry. The approach, based on a subatmospheric ESI interface, allows sample introduction from a commercially available microtiter plate without the need for a separate fluid delivery system. The microtiter plate was placed vertically on a three-dimensional translation stage in front of the sampling ESI interface. A single, 7-cm, 20-microm-i.d. fused-silica capillary (total volume, 70 nL), with a tapered tip, served as a combination of sample delivery and spraying capillary. The tapered tip of the capillary was enclosed in a subatmospheric chamber attached in front of the orifice of the mass spectrometer. The sample aspiration rate (flow rate) was regulated by computer-controlled pneumatic valves, which allowed fast switching of the pressure in the subatmospheric ESI chamber. A flow-through wash device was positioned between the microtiter plate and the ESI interface. This design allowed alternate filling of the capillary with (a) sample from the wells and (b) wash solution from the wash device. Sample turnaround times of 10 s/sample, with a 120-nL sample consumption/analysis, and a duty cycle (percentage of total analysis time spent acquiring data) of 40% were achieved. The infusion system was demonstrated in the analysis of preparative HPLC fractions from a small molecule combinatorial library.

Design and performance of an ESI interface for selective external ion accumulation coupled to a Fourier transform ion cyclotron mass spectrometer.

The coupling of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) with electrospray ionization has advanced the analysis of large biopolymers and provided the basis for high-throughput protein characterization (e.g., for rapid "proteome" analyses). In this work, the combination of high-performance capillary liquid chromatography with FTICR mass spectrometry and external ion accumulation has been shown to increase both sensitivity and analysis duty cycle. Instrument versatility is further improved by ion preselection followed by ion accumulation in an external linear quadrupole ion trap. The interface was tested with a 3.5-T FTICR mass spectrometer and evaluated with a number of peptides and proteins whose molecular weights ranged from 500 to 66000. A significant increase in the sensitivity, duty cycle, and dynamic range over that of the previously used accumulated trapping was achieved, exhibiting a detection limit of approximately 10 zmol (approximately 6000 molecules) for smaller proteins such as cytochrome c. Capillary LC external accumulation interface with FTICR was successfully applied for the study of whole-proteome mouse tryptic digests.

Simulation-based optimization of the electrodynamic ion funnel for high sensitivity electrospray ionization mass spectrometry

High sensitivity is one of the most important requirements for applications of electrospray ionization mass spectrometry (ESI MS). Recent work with the electrodynamic ion funnel has demonstrated that it can provide significantly improved ion transmission through the ESI interface. Here we summarize the results of the simulations for several ion funnel configurations and their comparison with experimental measurements. We also report an alternative treatment of the ion funnel operation based on the effective potential approximation. The analytical relationships derived are used to generalize the results of computer simulations and develop an optimized ion funnel design. The new configuration reduces the spacing between ring electrodes to 1 mm and provides an optimized profile of the ring electrodes radii. It can also generate a deeper effective potential well, resulting in transmission of higher input ion currents over an extended mass range and increased operating pressure range (up to ∼20 Torr). Furthermore, light m/z ion transmission is improved due to suppression of the effective potential wells near the ion funnel exit. Simulations for an optimized ion funnel configuration indicate unit transmission efficiency in the m/z range of interest for most biomolecular research using ESI (approximately m/z 100–5000), for 1–5 Torr pressure and ion currents >∼1 nA, typical for low flow rate charge-constrained electrosprays (i.e. nanospray). Experimental results obtained with an improved electrodynamic funnel validate the model and demonstrate its general utility for designing rf-damped focusing elements.

Observation of daunomycin and nogalamycin complexes with duplex DNA using electrospray ionisation mass spectrometry.

The noncovalent binding of the antitumour drugs daunomycin and nogalamycin to duplex DNA has been studied using electrospray ionisation mass spectrometry (ESI-MS). The conditions for the preparation of drug/duplex DNA complexes and for their detection by ESI-MS have been optimised. Ions corresponding to these complexes were most abundant relative to free DNA when prepared in the pH range 8-9, and using gentle ESI interface conditions. Self-complementary oligonucleotides, 5'-d(GGCTAGCC)-3' or 5'-d(CGGCGCCG)-3', annealed in the presence of a 5-fold molar excess of either nogalamycin or daunomycin gave ESI mass spectra in which the most intense ions corresponded to three molecules of drug bound to duplex DNA, with some evidence for four drug molecules bound. For binding to 5'-d(TGAGCTAGCTCA)(2)-3', complexes containing up to four nogalamycin and six daunomycin molecules were observed. These data are consistent with the neighbour exclusion principle whereby intercalation occurs between every other base pair such that up to four bound drugs would be expected for the 8 mers and up to six for the 12 mer. Competition experiments involving a single drug in an equimolar mixture of two oligonucleotides (5'-d(TGAGCTAGCTCA)(2)-3' with either 5'-d(CGGCGCCG)(2)-3' or 5'-d(GGCTAGCC)(2)-3') showed ions arising from complexes of drug/5'-d(CGGCGCCG)(2)-3' were more intense than complexes of drug/5'-d(GGCTAGCC)(2)-3', relative to those from the 12 mer in each mixture. While this suggests ESI-MS has the potential to detect differences in sequence selectivity, more detailed experiments involving a comparison of the relative ionisation efficiency of different oligonucleotides and a wider range of intercalators are required to establish this definitively. ESI mass spectra from experiments in which both drugs were reacted with the same oligonucleotide were more complex, such that a clear preference for one drug could not be established.

Phenyl ring structures as stationary phases for the high performance liquid chromatography electrospray ionization mass spectrometric analysis of basic pharmaceuticals

To produce a large mass spectral signal in the analysis of basic drugs by high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS), a high percentage of organic solvent is needed in the mobile phase. With the commonly used reversed phase columns (C18, C8 and C4) high concentrations of organic solvents cannot be used; therefore other stationary phases were investigated. The stationary phases investigated had phenyl ring structures; phenyl (Ph) and pentafluorophenyl (PFP) which were bonded directly to the silica backbone and other phases, pentafluorophenylpropyl (PFPP), phenylpropyl (PhP) and tolylpropyl (TP), which were attached to the silica via a propyl chain. The effects of these stationary phases on retention, peak shape and size of the signal in the mass spectrometric analysis of a selected group of basic drugs, tricyclic antidepressants (TCAs) and beta-blockers, were evaluated and compared with those obtained on a C18 column. The PFPP column was found to give the best results. The basic drugs were adequately retained with retention times greater than 2 minutes when the PFPP column was used with mobile phases of high concentrations of organic solvents. Thus the endogenous interferences could be eliminated and the analysis time was still rapid enough to have a high throughput. Most importantly, a mobile phase of 90% acetonitrile could be used which allowed easy desolvation in the ESI interface and provided good peak shapes. In addition, ion-pairing reagents and ion-suppressing agents which suppress the signal in ESI-MS were not required. The best results were obtained using a 1 cm column and a flow rate of 0.9 mL/min. With the PFPP stationary phase and a mobile phase of 90% acetonitrile, the HPLC/ESI mass spectral signal of the beta-blocker, oxprenolol, was increased by a factor of 16 compared with the C18 phase with only 12% acetonitrile in the mobile phase. Copyright 1999 John Wiley & Sons, Ltd.

Multi-Residue Analysis by Liquid Chromatography-Mass Spectrometry Coupling. Application to Drinking and River Waters

The coupling between liquid chromatography and mass spectrometry with an APCI or ESI interface (in positive or negative mode) is used here for multi-residue analyses in natural waters, covering basic and neutral pesticides as well as acid pesticides. The methods developed are applied to drinking and, river waters after the samples are concentrated by liquid-liquid extraction or solid phase extraction on C18 cartridges. Comparisons are made between UV detection and mass spectrometry and between two chromatographic methods for acid substances. The quantitation limits range from 0.01 to 0.1 μg/l according to the substance.

Quantitative analysis of human serum corticosterone by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry

An original method based upon high-performance liquid chromatography coupled to electrospray ionization mass spectrometry has been developed for corticosterone (B) quantification in human serum. After extraction by diethyl ether using triamcinolone (T) as an internal standard, solutes are separated on a C 18 microbore column (250×1.0 mm, I.D.), using acetonitrile–water–formic acid (40:59.9:0.1, v/v/v) as the mobile phase (flow-rate 40 μl/min). Detection is performed on an API 1 single quadrupole mass spectrometer equipped with a ESI interface and operated in positive ionization mode. Corticosterone quantifications were realized by computing peak area ratios (B/T) of the serum extracts analyzed in SIM mode ( m/ z 347 and m/ z 395 for B and T, respectively), and comparing them with the calibration curve ( r=0.998).

Site localization of sialyl Lewis(x) antigen on alpha1-acid glycoprotein by high performance liquid chromatography-electrospray mass spectrometry.

A simple, fast and sensitive method was developed to verify the presence of the sialyl Lewis(x) antigen on an N-linked glycoprotein. High performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI/MS) was used to identify which of the five N-linked glycosylation sites of human plasma alpha1-acid-glycoprotein (orosomucoid, OMD) contain the sialyl Lewis(x) antigen. OMD was digested with proteolytic enzymes and analyzed by reversed phase chromatography coupled with on-line ESI/MS. A tandem mass spectrometry experiment was designed to detect the presence of the sialyl Lewis(x) antigen based on the observation of an 803 mass to charge ratio ( m/z ) ion produced in the intermediate pressure region of the ESI interface. The ESI/MS signal at m/z 803 is consistent with an oxonium ion for a glycan structure containing NeuAc, Gal, GlcNAc, and Fuc. The identity of the m/z 803 ion was confirmed by ESI/MS/MS analysis of the m/z 803 fragment ion and comparison with a sialyl Lewis(x) standard. The stereochemistry and linkage positions were assigned using previous NMR analysis but could be determined with permethylation analysis if necessary. The analysis of OMD gave a pattern showing signal for the sialyl Lewis(x) antigen coeluting with each of the five N-linked glycopeptides. The ability to monitor sialyl Lewis(x) expression at each of the five sites is of interest in the study of OMD's role in inflammatory diseases.

Liquid chromatographic–high-resolution mass spectrometric and tandem mass spectrometric identification of synthetic peptides using electrospray ionization

Liquid chromatography–high-resolution electrospray mass spectrometry (LC–ESI-MS) was investigated for the identification of known and unknown synthetic peptides in a research effort designed to evaluate the applicability of this and complementary MS techniques for peptide characterization and identification. The monoisotopic molecular masses of five related peptides with molecular masses between 2000 and 2500 u were acquired with a resolution of 3000 (10% valley). Under narrow and wide mass range magnetic sector scanning conditions monoisotopic molecular mass errors were typically in the 10–20 and 30–40 ppm range, respectively. Tryptic maps were generated for each peptide following LC–ESI-MS analysis and collisionally activated dissociation (CAD) in the ESI interface resulted in the production of characteristic product ions that enabled amino acid sequencing of the tryptic fragments. Unknown identification was demonstrated during analysis of an incomplete synthetic peptide reaction mixture. The synthesis of an 18 amino acid peptide, LTTAVKKVLTTGLPALIS, was not successful. In its place were six unknown peptides that were identified on the basis of monoisotopic molecular mass and amino acid sequence data. The monoisotopic molecular masses of these unknowns were determined to within 10–20 ppm with a resolution of 3500 (10% valley). Amino acid sequences for the six peptides were generated during ESI–MS–MS analysis. Finally two synthetic peptides differing only by the incorporation of a 13C at leucine were analysed with a resolution of 6000 (10% valley) to confirm that the isotopic distributions were consistent with theoretical expectations.

Sensitive high-resolution analysis of biological molecules by capillary zone electrophoresis coupled with reflecting time-of-flight mass spectrometry

Off-line and on-line capillary zone electrophoresis–electrospray ionization time-of-flight mass spectrometry (CZE–ESI-TOF-MS) experiments were conducted using uncoated fused-silica capillaries coupled to a reflecting TOF mass spectrometer via a gold-coated sheathless interface. Off-line and on-line experiments were performed on standard mixtures of proteins and peptides. Samples collected off-line electrokinetically in plastic vials were analyzed by standard ESI–TOF-MS at the pmol level. Sheathless CZE–ESI-TOF-MS was first simulated in an off-line experiment, using a test bench, in order to select a suitable running electrolyte, to find the optimal electrospray potential, and also to test the gold-coated capillary tips. This enabled an ease of transition to on-line measurements. On-line CZE–ESI-TOF-MS measurements of the total ion electropherogram (TIE) and of selected ion electropherograms (SIE) on peptide mixtures demonstrated fmol-level sensitivity, with S/ N values of 250–400 on raw data (TIE mode) and of 30–760 (SIE mode). The use of reflecting TOF-MS afforded mass resolution values R>6000 ( m/Δ m FWHM) and enabled isotopic resolution of peptide components as well as mass accuracy in the 10 ppm range. These results were comparable with values observed with the usual ESI source on the same mass spectrometer, and thus demonstrated no loss in spectral quality from using the sheathless CE interface. On-line CE separation efficiency was equivalent to that obtained off-line for the separation of a peptide mixture, with N=35 000–87 000 theoretical plates. Separations of standard proteins yielded equivalent mass spectral resolution and accuracy with separation efficiencies of N=2800–5500 and S/ N values of 110–225 on raw data. The gold-coated sheathless CE–ESI interface was found to be relatively easy to prepare with the use of gold vapour deposition. The interface produced a stable electrospray for extended periods of time and was found to be robust and reliable.

Electrospray Mass Spectrometric Characterization of Six Therapeutic Oximes: HI-6, HS-6, Obidoxime, 2-PAM, TMB-4 and HLö-7

Pyridinium and bis-pyridinium oxime salts are currently in use or under development for treatment of patients exposed to organophosphorus nerve agents. The low volatility and thermal lability of these compounds limits the number of mass spectrometric approaches that may be used to characterize them. Electrospray mass spectrometry (ESI-MS), a relatively new ionization approach, was investigated as a possible technique for the characterization and identification of these oximes and their degradation products. Six therapeutic oxime salts, the bis-pyridinium oximes, HI-6, HS-6, obidoxime, TMB-4 and HLo-7, and the pyridinium oxime, 2-PAM, were analysed by ESI-MS, making use of collisionally-activated dissociation (CAD) in the ESI interface. The CAD mass spectrum of each oxime was acquired with two different sampling cone voltage settings and the quadrupole mass analyser associated with a hybrid tandem mass spectrometer was utilized for tandem mass spectrometry (MS/MS) and CAD-MS/MS applications including the reliable differentiation of similar bis-pyridinium oximes and the identification of a HI-6 decomposition product.

Separation and identification of higher fullerenes in soot extract by liquid chromatography-mass spectrometry

Two-step liquid chromatographic separation (LC) has been applied to soot extract and the identification of higher fullerenes has been accomplished by LC-MS measurements using an ESI interface. The first separation step is preparative-scale LC using a 50 mm i.d. column packed with monomeric octadecylisilica (ODS) because elution is mainly controlled by relative molecular mass. 39 batches of five fractions each were collected and then as the second separation step each fraction was analysed by analytical-scale LC using a conventional column of a polymeric ODS phase which can elute fullerenes according to shape and structure. This stationary phase can also separate many isomers of higher fullerenes, consequently the existence of several higher fullerenes larger than C86 has been confirmed and their UV-Vis spectra were obtained by the photodiode array detection system coupled to the analytical LC.

Investigation of non‐covalent ligand binding to the intact streptavidin tetramer by electrospray ionization mass spectrometry

AbstractThe relative non‐covalent binding behavior of two small molecule ligands to the tetrameric protein streptavidin was investigated by electrospray ionization mass spectrometry (ESI‐MS). An extended m/z range quadrupole mass spectrometer was employed to observe the intact multimeric form of the protein and to probe the relative stabilities of the tetrameric protein‐ ligand complexes in the gas phase. Various protein:ligand molar ratio concentrations and incubation times were studied for the ligands biotin (Kd ≈ 10−15 M) and iminobiotin (Kd ≈ 10−7 M), in combination with the adjustment of variables in the ESI atmosphere‐vacuum interface region. Positive‐ion ESI‐MS of a 1:7 molar ratio streptvidin–biotin sample produced peaks corresponding to the intact tetrameric complex (16+ to 14+ charge states) with four ligands binding to the active from of the protein. Only the expected specific non‐covalent complexes of four ligand molecules binding to the protein tetramer were detected, consistent with known solution behavior and without the appearance of any random aggregation. However, under identical interface conditions, complete iminobiotin binding to the protein was not observed, even when higher ligand concentrations, longer complexation times and gentler interface conditions were employed. The thermally induced dissociation (TID) of the non‐covalent complexes was studied by applying more severe conditions in the ESI interface region. In addition, observation of an intact streptavidin–biotinylated oligonucleotide non‐covalent complex (17– to 14– tetrameric charge states) by negative‐ion ESI‐MS at m/z 4500–5000 is presented. The results show that the complexes observed in the mass spectrometer are representative of those that exist in solution, under the ESI conditions employed, and that the ability to observe such complexes is at least qualitatively related to stability in solution.

Observation of noncovalent complexes to the avidin tetramer by electrospray ionization mass spectrometry

Intact avidin-biotin and avidin-biotin maleimide noncovalent complexes have been observed by electrospray ionization mass spectrometry (ESI-MS) by using an extended mass range quadrupole mass spectrometer. By utilizing mild ESI interface conditions, the expected solution behavior of four biotin or biotin maleimide molecules noncovalently binding to each avidin tetramer can be preserved in the gas phase. The ESI-MS results show the appropriate mass additions of 973 ± 60 Da for biotin and 1802 ± 40 Da for biotin maleimide to the avidin tetramer species. These results support the hypothesis that substantial retention of higher order structure is possible in the gas phase by using gentle ESI conditions.