电喷雾质谱法研究谷胱甘肽与L型芳香性氨基酸非共价复合物

为探索谷胱甘肽和L型芳香性氨基酸的非共价相互作用, 将一定化学剂量比的还原型γ-谷胱甘肽分别与L型芳香性氨基酸(包括苯丙氨酸、酪氨酸和色氨酸)在室温和生理pH条件下混合后, 温育1 h, 生成非共价复合物, 并使反应完全. 电喷雾质谱测量结果揭示谷胱甘肽和L型芳香性氨基酸反应可以生成非共价复合物. 在二级串级质谱MS2测得的复合物碎片离子峰中, 除芳香性氨基酸离子峰外, 还包括谷胱甘肽及其它再次碎裂产生的b2和y2碎片离子, 进一步确认了非共价复合物的形成. 紫外光谱也证实了电喷雾质谱的实验结果. 为避免严重的离子化效率差异和质谱信号的相互抑制作用, 定量评估了谷胱甘肽和酪氨酸的相互作用, 结果显示反应物的初始浓度应该选择在5×10-5~3.00×10-4 mol/L范围内. 用质谱滴定法测定了谷胱甘肽与3个芳香性氨基酸非共价复合物的解离常数, 结果表明, 谷胱甘肽复合物的稳定性按Tyr, Trp和Phe次序依次增大.     

Use of electrospray ionization mass spectrometry for the investigation of radical cation chain reactions in solution: detection of transient radical cations

Electrospray ionization mass spectrometry (ESI-MS) coupled to a microreactor is an excellent tool for the investigation of reactions in solution. Here, we report the first results of our investigations into preparatively interesting electron-transfer-initiated chain reactions in solution which proceed via radical cations as reactive intermediates. The tris(p-bromophenyl)aminium hexachloroantimonate (1)-mediated [2+2] cycloaddition of trans-anethole (2) to give 1,2-bis(4-methoxyphenyl)-3,4-dimethylcyclobutane (3) was investigated. The reaction proceeds as a radical cation chain reaction via transient intermediates 2 ⁺ and 3 ⁺ that could be detected and characterized unambiguously directly in the reacting solution by ESI-MS/MS. The identity of the intermediates was confirmed by comparison with authentic MS/MS spectra of 2 ⁺ and 3 ⁺ obtained by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). In addition, substrate and product can be monitored easily in the reacting solution by APCI-MS.Part of this work was presented at the 36th Conference of the German Society for Mass Spectrometry (DGMS).     

Electrospray ionization mass spectrometry of a novel family of complexes in which various nitroso compounds are stabilized via coordination to [IrCl5]2-

Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) data of a unique family of complexes of nitroso compounds coordinated to pentachloroiridate(III), [Cl5IrN(O)XR]2- (X=NH, S, CH and R=alkyl, aryl) are presented. These novel complexes are obtained by nucleophilic attack of primary amines, thiols. and alkenes to the coordinated nitrosyl. Despite their lability and low volatility, MS analysis of complexes of the type MN(O)X was done for the first time, complementing other spectroscopic techniques. The intrinsic dissociation chemistry of the gaseous diagnostic ions was studied via ESI-MS/MS and found to be very useful to confirm the proposed connectivities of the parent complexes. In particular, ESI-MS of their solutions allows the detection of series of diagnostic ions, mainly, [M-Cl]-, [M+K]-, [M-NO]-*, and [M-Cl+AcN]- (AcN=acetonitrile), which confirmed the identity of the analyzed complexes to be M=[Cl5IrN(O)XR]2-. Major fragments were formed by losses of NO or N(O)XR. ESI-MS and ESI-MS/MS measurements are therefore shown to be the proper techniques to complement the spectroscopic characterization of this important class of nitroso complexes. An interesting rearrangement that does not take place in solution was observed in the gaseous phase, and a plausible mechanism is discussed.     

Characterization,stoichiometry, and stability of salivary protein–tannin complexes by ESI-MS and ESI-MS/MS

Numerous protein–polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein–ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP–tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP–tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5·EgCG complexes are maintained intact in the gas phase.      

Electrospray ionization mass spectrometry: a major tool to investigate reaction mechanisms in both solution and the gas phase

Electrospray ionization mass spectrometry (ESI-MS), in conjunction with its tandem version ESI-MS/MS, is now established as a major tool to study reaction mechanisms in solution. This suitability results mainly from the ability of ESI to "fish" ions directly from solution to the gas phase environment of mass spectrometers. In this review, we summarize recent studies from our laboratory on the use of on-line monitoring by ESI-MS ion fishing of several types of reactions that permitted us to follow how these reactions progress as a function of both time and conditions using the ultra-high sensitivity and speed of ESI-MS to detect and even characterize transient reaction intermediates. We also show that the intrinsic reactivity of each key gaseous species fished by ESI can be further investigated via ESI-tandem mass spectrometry experiments, searching for the most active species via gas-phase ion/molecule reactions. In the gas-phase, solvent and counter-ion effects are absent. These studies often permit a detailed overview of major steps via the interception, characterization and reactivity investigation of key reaction players.     

Liquid chromatography/tandem mass spectrometry analysis of long-chain oxidation products of cardiolipin induced by the hydroxyl radical

The anionic phospholipid cardiolipin (CL) is found almost exclusively in the inner membrane of mitochondria, playing an important role in energy metabolism. Oxidation of CL has been associated with apoptotic events and various pathologies. In this study, electrospray ionization mass spectrometry coupled with liquid chromatography (LC/ESI-MS) was used to identify tetralinoleoyl-cardiolipin (TLCL) modifications induced by the OH(·) radical generated under Fenton reaction conditions (H(2)O(2) and Fe(2+)). The identified oxidation products of TLCL contained 2, 4, 6 and 8 additional oxygen atoms. These long-chain oxidation products were characterized by LC/ESI-MS/MS as doubly [M-2H](2-) and singly charged [M-H](-) ions. A detailed analysis of the fragmentation pathways of these precursor ions allowed the identification of hydroperoxy derivatives of CL. MS/MS analysis indicated that CL oxidation products with 4, 6 and 8 oxygen atoms have one fatty acyl chain bearing 4 oxygen atoms ([RCOO+4O](-)). Even when the TLCL molecule was oxidized by the addition of eight oxygen atoms, one of the acyl chains remained non-modified and one fatty acyl chain contained three or four oxygen atoms. This led us to conclude that under oxidative conditions by the OH(·) radical, the distribution of oxygens/peroxy groups in the CL molecule is not random, even when CL has the same fatty acyl chains in all the positions. Using mass spectrometry, the oxidation products have been unequivocally assigned, which may be useful for their detection in biological samples.     

Formation of metal-nicotianamine complexes as affected by pH, ligand exchange with citrate and metal exchange. A study by electrospray ionization time-of-flight mass spectrometry

Nicotianamine (NA) is considered as a key element in plant metal homeostasis. This non-proteinogenic amino acid has an optimal structure for chelation of metal ions, with six functional groups that allow octahedral coordination. The ability to chelate metals by NA is largely dependent on the pK of the resulting complex and the pH of the solution, with most metals being chelated at neutral or basic pH values. In silico calculations using pKa and pK values have predicted the occurrence of metal-NA complexes in plant fluids, but the use of soft ionization techniques (e.g. electrospray), together with high-resolution mass spectrometers (e.g. time-of-flight mass detector), can offer direct and metal-specific information on the speciation of NA in solution. We have used direct infusion electrospray ionization mass spectrometry (time-of-flight) ESI-MS(TOF) to study the complexation of Mn, Fe(II), Fe(III), Ni, Cu by NA. The pH dependence of the metal-NA complexes in ESI-MS was compared to that predicted in silico. Possible exchange reactions that may occur between Fe-NA and other metal micronutrients as Zn and Cu, as well as between Fe-NA and citrate, another possible Fe ligand candidate in plants, were studied at pH 5.5 and 7.5, values typical of the plant xylem and phloem saps. Metal-NA complexes were generally observed in the ESI-MS experiments at a pH value approximately 1-2 units lower than that predicted in silico, and this difference could be only partially explained by the estimated error, approximately 0.3 pH units, associated with measuring pH in organic solvent-containing solutions. Iron-NA complexes are less likely to participate in ligand- and metal-exchange reactions at pH 7.5 than at pH 5.5. Results support that NA may be the ligand chelating Fe at pH values usually found in phloem sap, whereas in the xylem sap NA is not likely to be involved in Fe transport, conversely to what occurs with other metals such as Cu and Ni. Some considerations that need to be addressed when studying metal complexes in plant compartments by ESI-MS are also discussed.     

Relative binding affinities of molecular capsules investigated by ESI-mass spectrometry

ESI-MS(/MS) has been used as a method which allows the fast, unambiguous and sensitive simultaneous detection and relative stability approximation of supramolecular assemblies in mixtures. In spite of the obvious fundamental differences between solution and gas phase, ESI-MS in the case of self-assembled molecular capsules has been shown to produce very similar results to single binding experiments monitored by NMR titrations as well as conformational searches performed by Monte-Carlo simulations. MS/MS experiments reveal the same relative order of gas phase stabilities as previously found in solution. Moreover, proton transfer reactions which lead to new molecular capsules, are not detectable in the time-averaged NMR spectrum. However, the newly produced species are found in the complex mixtures by ESI-MS and can be conveniently characterized by subsequent MS/MS experiments: in a collision-induced dissociation the single half-spheres are easily discovered and structurally assigned. Thus, ESI-MS has worked as a valuable tool for the rapid screening of complex supramolecular mixtures and in combination with MS/MS experiments elucidated both the path of unexpected side reactions as well as the thermodynamic gas-phase stabilities of all components in the mixture.     

Interactions of platinum(II) complexes with sulfur-containing peptides studied by electrospray ionization mass spectrometry and tandem mass spectrometry

Reactions of two platinum(II) complexes, cis-[Pt(NH3)2(H2O)2]2+ (Pt1) and cis-[Pt(en)(H2O)2]2+ (Pt2), with several sulfur-containing peptides, have been investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). The species produced in the reactions were detected with ESI-MS, and MS/MS analysis was performed to probe structural information. Collision-induced dissociation revealed different dissociation pathways for the main reaction products of the two platinum(II) complexes with the same peptides. The major difference is the prominent loss of ammonia ligand for complexes of Pt1 due to the strong trans effect of sulfur, whereas the loss of ethylenediamine (en) ligand from Pt2 complexes is less favored, reflecting the chelating effect of the bidentate ligand. Despite the differences in dissociation patterns, Pt1 and Pt2, in general, form structurally similar complexes with the same peptides. In the reactions with Met-Arg-Phe-Ala they both produce a N,S-chelate ring through the N-terminal NH2 and sulfur of the Met residue, and in the reactions with Ac-Met-Ala-Ser they bind to the sulfur of Met and deprotonate an amide nitrogen upstream from the anchor site. Both of them are able to promote hydrolysis of the peptides. In reactions with glutathione they both form four-membered Pt2S2 rings and Pt-S-Pt bonding through the bridging thiolate ligand, although the reaction rate is much slower for Pt2 due to steric hindrance of the en ligand.     

Comparison of flow injection analysis electrospray mass spectrometry and tandem mass spectrometry and electrospray high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry for the determination of underivatized amino acids

Twenty proteinogenic amino acids (AAs) were determined without derivatization using flow injection analysis followed by electrospray ionization mass spectrometry and tandem mass spectrometry (ESI-MS and ESI-MS/MS) and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry (ESI-FAIMS-MS and ESI-FAIMS-MS/MS), in positive and negative ionization modes. Three separate sets of ESI-FAIMS conditions were used for the separation and detection of the 20 AAs. Typically ESI-FAIMS-MS showed somewhat improved sensitivity and significantly better signal-to-noise ratios than ESI-MS mainly due to the elimination of background noise. However, the difference between ESI-FAIMS-MS and ESI-MS/MS was significantly less. ESI-FAIMS was able to partially or completely resolve all the isobaric amino acid overlaps such as leucine, isoleucine and hydroxyproline or lysine and glutamine. Detection limits for the amino acids in ESI-FAIMS-MS mode ranged from 2 ng/mL for proline to 200 ng/mL for aspartic acid. Overall, ESI-FAIMS-MS is the preferred method for the quantitative analysis of AAs in a hydrolyzed yeast matrix.     

Electrospray ionization mass spectrometry coupled to liquid chromatography for detection of cisplatin and its hydrated complexes

Electrospray ionization mass spectrometry (ESI-MS) coupled to liquid chromatography (LC) has been applied to investigate cisplatin and its hydrated complexes. Three hydrolysis products were identified following incubation of cisplatin in aqueous solution: monohydrated species, dihydrated species and the hydroxo-bridged dimer, each of which exhibited characteristic mass spectrometric behavior. The technique was employed to investigate the time- and pH-dependent hydrolysis of cisplatin in aqueous solution. The results have demonstrated that LC/ESI-MS is a powerful technique for the analysis of Pt(II) complexes and for monitoring their hydrolysis reactions.     

Characterization of linear and cyclic polylactic acids and their solvolysis products by electrospray ionization mass spectrometry

Linear and cyclic polylactic acids (PLAs) were characterized using electrospray ionization mass spectrometry (ESI-MS) as part of our ongoing investigation of the hydrolysis mechanism of biodegradable polymers. The condensation oligomers of linear polylactic acid (LPLA) were synthesized by thermal dehydration of L-lactic acid. The trimer and tetramer base polymers of cyclic polylactic acid (CPLA) were obtained by cyclization reactions of lactic acid trimers and tetramers, respectively. In the ESI-MS/MS measurement, LPLA yielded three types of product ion series, while CPLA yielded only one type, from which the repeated units of CPLA were removed. The MS/MS spectrum of the NH4+ adduct ion for both cyclic and linear PLA showed loss of one ammonia molecule. The postsource decay (PSD) spectrum of CPLA by matrix-assisted laser desorption ionization (MALDI) mass spectrometry was similar to the ESI-MS/MS spectrum, while that of LPLA was different. In addition, the degradation of cyclic and linear PLAs by solvolysis was investigated. Solvolysis with anhydrous MeOH was quite feasible, but did not readily occur in the presence of even a small amount of water in the MeOH solvent.     

Negative-ion electrospray ionization tandem mass spectrometry of N-phosphoryl amino acids and dipeptides

The negative-ions of N-phosphoryl amino acids were studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The negative-ion ESI-MS/MS of N-phosphoryl amino acids showed characteristic fragmentation patterns different from those observed in the corresponding positive-ion ESI-MS/MS and negative-ion fast-atom bombardment mass spectra. For negative-ion ESI-MS/MS, a unique fragmentation from the N-terminal of N-phosphoryl amino acids or peptides containing a free beta-OH or CO(2)H group was observed to yield the characteristic fragment ion (RO)(2)P(O)O(-). The ease of the rearrangement depended on the position of the hydroxyl group in amino acids or peptides, and the N --> O rearrangement mechanism was proposed to involve the participation of the hydroxyl group. From previous solution-phase experiments and theoretical calculations, it was found that the beta-OH group was more active than gamma-OH, and the corresponding difference in negative-ion ESI-MS/MS was consistent with those previous findings.     

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.     

Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry

The potential of electrospray ionization (ESI) mass spectrometry (MS) to detect non-covalent protein complexes has been demonstrated repeatedly. However, questions about correlation of the solution and gas-phase structures of these complexes still produce vigorous scientific discussion. Here, we demonstrate the evaluation of the gas-phase binding of non-covalent protein complexes formed between bovine pancreatic trypsin inhibitor (BPTI) and its target enzymes over a wide range of dissociation constants. Non-covalent protein complexes were detected by ESI-MS. The abundance of the complex ions in the mass spectra is less than expected from the values of the dissociation constants of the complexes in solution. Collisionally activated dissociation (CAD) tandem mass spectrometry (MS/MS) and a collision model for ion activation were used to evaluate the binding of non-covalent complexes in the gas phase. The internal energy required to induce dissociation was calculated for three collision gases (Ne, Ar, Kr) over a wide range of collision gas pressures and energies using an electrospray ionization source. The order of binding energies of the gas-phase ions for non-covalent protein complexes formed by the ESI source and assessed using CAD-MS/MS appears to differ from that of the solution complexes. The implication is that solution structure of these complexes was not preserved in the gas phase.     

Analysis of metal complex azo dyes by high-performance liquid chromatography/electrospray ionization mass spectrometry and multistage mass spectrometry

Five metal complex azo compounds were analyzed using negative-ion electrospray ionization mass spectrometry (ESI-MS). Mass spectra of all compounds yield intense peaks corresponding to [M - H](-) ions without any fragmentation, where M denotes the neutral compound with a proton as the counterion. Under collision induced dissociation (CID) conditions, structurally important fragment ions were studied using the ion trap analyzer with a multistage mass spectrometry (MS(n) facility. Synthesized compounds with (15)N atoms in the azo group facilitated the fragmentation pattern recognition. A reversed-phase high-performance liquid chromatography (HPLC) method using 5 mM ammonium acetate in 70% aqueous acetonitrile as mobile phase was developed making possible the separation of all complex compounds tested. The lower detection limits of the ESI-MS method are in the range 10-20 ng of each compound. The HPLC/ESI-MS method makes possible the monitoring of ligand exchange in aqueous solutions of metal complex azo dyes, and also investigation of the stabilities of the complexes in solution. Copyright 2000 John Wiley & Sons, Ltd.     

Identification of clusters from reactions of ruthenium arene anticancer complex with glutathione using nanoscale liquid chromatography fourier transform ion cyclotron mass spectrometry combined with <Superscript>18</Superscript>O-labeling

Reactions of the anticancer complex [(eta(6)-bip)Ru(en)Cl](+) (where bip is biphenyl and en is ethylenediamine) with the tripeptide glutathione (gamma-L-Glu-L-Cys-Gly; GSH), the abundant intracellular thiol, in aqueous solution give rise to two ruthenium cluster complexes, which could not be identified by electrospray mass spectrometry (ESI-MS) using a quadrupole mass analyzer. Here we use Fourier transform ion cyclotron mass spectrometry (nanoLC-FT-ICR MS) to identify the clusters separated by nanoscale liquid chromatography as the tetranuclear complex [{(eta(6)-bip)Ru(GSO(2))}(4)](2-) (2) and dinuclear complex [{(eta(6)-bip)Ru(GSO(2))(2)}(2)](8-) (3) containing glutathione sulfinate (GSO(2)) ligands. Use of (18)OH(2) showed that oxygen from water can readily be incorporated into the oxidized glutathione ligands. These data illustrate the power of high-resolution MS for identifying highly charged multinuclear complexes and elucidating novel reaction pathways for metallodrugs, including ligand-based redox reactions.     

Electrospray tandem mass spectrometry of new porphyrin amino acid conjugates

Porphyrin amino acid conjugates with one or two porphyrin units were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The ESI-MS spectra of all the porphyrins studied, obtained in positive ion mode, show the presence of the corresponding protonated molecule [M+H]+; ESI-MS spectra of diporphyrinyl compounds also show the doubly charged ions [M+2H]2+. The fragmentations of these ions induced by collision with argon were studied (ESI-MS/MS). ESI-MS/MS gives detailed structural information about the amino acids associated with the porphyrin. Cleavage of the bonds in the vicinity of the porphyrin moiety and those involving the side chain of amino acid residues gives structural information about this type of association. A fragmentation common to all derivatives corresponds to the cleavage of the phenyl-CO bond. The expected cleavage of the amide bond, that links the porphyrin to the amino acid moiety, is a minor fragmentation, which in some cases is even absent. The MS/MS spectra of the monoporphyrinyl derivatives show product ions characteristic of the amino acid linked to the porphyrin; the fragmentation also indicates when the amino acids has a terminal carboxylic group or a terminal ester group. The fragmentations of the diporphyrinyl compounds occur mainly by the cleavage of the spacer, leading, in the case of the doubly charged ions, to predominantly mono-charged ions, indicating a preferential location of the two protons in separated porphyrinic units.     

Fluoride anion binding by cyclic boronic esters: influence of backbone chelate on receptor integrity

A systematic investigation of fluoride anion binding properties as a function of chelate backbone has been carried out for ferrocene functionalised boronic esters of the types FcB(OR)2 and fc[B(OR)2]2 [Fc = ferrocenyl = (eta5-C5H5)Fe(eta5-C5H4); fc = ferrocendiyl = Fe(eta5-C5H4)2]. Cyclic boronic esters containing a saturated five- or six-membered chelate ring are readily synthesized from ferrocene, and selectively bind fluoride via Lewis acid/base chemistry in chloroform solution. The resulting complexes are characterized by relatively weak fluoride binding (e.g.K = 35.8 +/- 9.8 M(-1) for FcBO2C2H2Ph2-S,S), and by cathodic shifts in the ferrocene oxidation potential that form the basis for electrochemical or colorimetric fluoride detection. The fluoride selectivity of these systems is attributed to relatively weak Lewis acidity, resulting in weak F- binding, and essentially no binding of potentially competitive anions. By contrast, more elaborate Lewis acid frameworks based on calix[4]arene (calixH4), such as (FcB)2calix or fcB2calix, do not survive intact exposure to standard fluoride sources (e.g. [nBu4N]F.xH2O solutions in chloroform or acetonitrile). Instead B-O bond cleavage occurs yielding the parent calixarene; the differences between alkoxo- and aryloxo-functionalised derivatives can be rationalised, at least in part, by consideration of the differences in electron donating capabilities of RO- (R = alkyl, aryl).     

Detection of noncovalent complex between alpha-amylase and its microbial inhibitor tendamistat by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is now routinely used for detection of noncovalent complexes. However, detection of noncovalent protein-protein complexes is not a widespread practice and still produces some challenges for mass spectrometrists. Here we demonstrate the detection of a noncovalent protein-protein complex between alpha-amylase and its microbial inhibitor tendamistat using ESI-MS. Crude porcine pancreatic alpha-amylase was purified using a glycogen precipitation method. Noncovalent complexes between porcine pancreatic alpha-amylase and its microbial inhibitor tendamistat are probed and detected using ESI-MS. The atmosphere-vacuum ESI conditions along with solution conditions and the ratio of inhibitor over enzyme strongly affect the detection of noncovalent complexes in the gas phase. ESI mass spectra of alpha-amylase at pH 7 exhibited charge states significantly lower than that reported previously, which is indicative of a native protein conformation necessary to produce a noncovalent complex. Detection of noncovalent complexes in the gas phase suggests that further use of conventional biochemical approaches to provide a qualitative, and in some cases even quantitative, characterization of equilibria of noncovalent complexes in solution is possible.