Dissociation Fourier Transform Ion Cyclotron Research Articles

Molecular Structures of Refractory Sulfur Compounds in Heavy Oil Hydrodesulfurization Characterized by Collision-Induced Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Molecular Structures of Refractory Sulfur Compounds in Heavy Oil Hydrodesulfurization Characterized by Collision-Induced Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

The mechanism of reduced IgG/IgE-binding of β-lactoglobulin by pulsed electric field pretreatment combined with glycation revealed by ECD/FTICR-MS.

Bovine β-lactoglobulin (β-Lg) is a major allergen existing in milk and causes about 90% of IgE-mediated cow's milk allergies. Previous studies showed that pulsed electric field (PEF) treatment could partially unfold the protein, which may contribute to the improvement of protein glycation. In this study, the effect of PEF pretreatment combined with glycation on the IgG/IgE-binding ability and the structure of β-Lg was investigated. The result showed that PEF pretreatment combined with glycation significantly reduced the IgG and IgE binding abilities, which was attributed to the changes of secondary and tertiary structure and the increase in glycation sites and degree of substitution per peptide (DSP) value determined by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD/FTICR-MS). Unexpectedly, glycation sites (K47, K91 and K135) added by two mannose molecules were identified in glycated β-Lg with PEF pretreatment. Moreover, the results indicated that PEF pretreatment at 25 kV cm-1 for 60 μs promoted the reduction of IgG/IgE-binding capacity by increasing the glycation degree of β-Lg, whereas single PEF treatment under the same conditions markedly enhanced the IgG/IgE-binding ability by partially unfolding the structure of β-Lg. The results suggested that ECD/FTICR-MS could help us to understand the mechanism of reduction in the IgG/IgE-binding of β-Lg by structural characterization at the molecular level. Therefore, PEF pretreatment combined with glycation may provide an alternative method for β-Lg desensitization.

Fragmentation study of major spirosolane‐type glycoalkaloids by collision‐induced dissociation linear ion trap and infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry

Glycoalkaloids play a key role in the plant protection system against phytopathogens including fungi, viruses, bacteria, insects and worms. They can be toxic to humans if consumed in high concentrations causing gastrointestinal disturbances. The structural characterization of the major spirosolane glycoalkaloids, solasonine, solamargine, α-tomatine and dehydrotomatine, were investigated by positive electrospray ionization (ESI) coupled with a hybrid linear ion trap (LIT) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Tandem mass spectrometric analysis of spirosolane glycoalkaloids was performed by both collision-induced dissociation (CID) within the LIT and infrared multiphoton dissociation (IRMPD) in conjunction with the FTICR cell. Several common product ions were observed, generated by losses of the sugar moiety or aglycone fragmentation in the B- or E-ring, that can provide information on the accurate mass of aglycone and the primary sequence and branching of the oligosaccharide chains. Thanks to the multistage CID it was possible to understand the fragmentation pathways and thanks to the high resolution of IRMPD-FTICR the elemental compositions of product ions were obtained. Because the investigated tandem mass spectra data were acquired with high mass accuracy, unambiguous interpretation and determination of the chemical compositions for the majority of detected fragment ions were feasible. From these data, generalized fragmentation pathways were proposed, providing guidance for the characterization of unknown glycoalkaloids in plants. Copyright © John Wiley & Sons, Ltd.

Electron detachment dissociation of synthetic heparan sulfate glycosaminoglycan tetrasaccharides varying in degree of sulfation and hexuronic acid stereochemistry

Glycosaminoglycan (GAG) carbohydrates provide a challenging analytical target for structural determination due to their polydisperse nature, non-template biosynthesis, and labile sulfate modifications. The resultant structures, although heterogeneous, contain domains which indicate a sulfation pattern or code that correlates to specific function. Mass spectrometry, in particular electron detachment dissociation Fourier transform ion cyclotron resonance (EDD FT-ICR MS), provides a highly sensitive platform for GAG structural analysis by providing cross-ring cleavages for sulfation location and product ions specific to hexuronic acid stereochemistry. To investigate the effect of sulfation pattern and variations in stereochemistry on EDD spectra, a series of synthetic heparan sulfate (HS) tetrasaccharides are examined. Whereas previous studies have focused on lowly sulfated compounds (0.5–1 sulfate groups per disaccharide), the current work extends the application of EDD to more highly sulfated tetrasaccharides (1–2 sulfate groups per disaccharide) and presents the first EDD of a tetrasaccharide containing a sulfated hexuronic acid. For these more highly sulfated HS oligomers, alternative strategies are shown to be effective for extracting full structural details. These strategies include sodium cation replacement of protons for determining the sites of sulfation, and desulfation of the oligosaccharides for the generation of product ions for assigning uronic acid stereochemistry.

Determination of Structural Building Blocks in Heavy Petroleum Systems by Collision-Induced Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Collision-induced dissociation Fourier Transform ion cyclotron resonance mass spectrometry (CID-FTICR MS) was developed to determine structural building blocks in heavy petroleum systems. Model compounds with both single core and multicore configurations were synthesized to study the fragmentation pattern and response factors in the CID reactions. Dealkylation is found to be the most prevalent reaction pathway in the CID. Single core molecules exhibit primarily molecular weight reduction with no change in the total unsaturation of the molecule (or Z-number as in chemical formula C(c)H(2c+Z)N(n)S(s)O(o)VNi). On the other hand, molecules containing more than one aromatic core will decompose into the constituting single cores and consequently exhibit both molecular weight reduction and change in Z-numbers. Biaryl linkage, C(1) linkage, and aromatic sulfide linkage cannot be broken down by CID with lab collision energy up to 50 eV while C(2)+ alkyl linkages can be easily broken. Naphthenic ring-openings were observed in CID, leading to formation of olefinic structures. Heavy petroleum systems, such as vacuum resid (VR) fractions, were characterized by the CID technology. Both single-core and multicore structures were found in VR. The latter is more prevalent in higher aromatic ring classes.

Porcine P2 myelin protein primary structure and bound fatty acids determined by mass spectrometry

Complementary collision-induced/electron capture dissociation Fourier-transform ion cyclotron resonance mass spectrometry was used to fully sequence the protein P2 myelin basic protein. It is an antigenic fatty-acid-binding protein that can induce experimental autoimmune neuritis: an animal model of Guillain-Barré syndrome, a disorder similar in etiology to multiple sclerosis. Neither the primary structure of the porcine variant, nor the fatty acids bound by the protein have been well established to date. A 1.8-A crystal structure shows but a bound ligand could not be unequivocally identified. A protocol for ligand extraction from protein crystals has been developed with subsequent gas chromatography MS analysis allowing determination that oleic, stearic, and palmitic fatty acids are associated with the protein. The results provide unique and general evidence of the utility of mass spectrometry for characterizing proteins from natural sources and generating biochemical information that may facilitate attempts to elucidate the causes for disorders such as demyelination.

Identification of aspartic and isoaspartic acid residues in amyloid beta peptides, including Abeta1-42, using electron-ion reactions.

Amyloid beta peptides are the major components of the vascular and plaque amyloid filaments in individuals with Alzheimer's disease (AD). Although it is still unclear what initiates the disease, isomerization of aspartic acid residues in Abeta peptides is directly related to the pathology of AD. The detection of isomerization products is analytically challenging, due to their similar chemical properties and identical molecular mass. Different methods have been applied to differentiate and quantify the isomers, including immunology, chromatography, and mass spectrometry. Typically, those methods require comparative analysis with the standard peptides and involve many sample preparation steps. To understand the role of Abeta isomerization in AD progression, a fast, simple, accurate, and reproducible method is necessary. In this work, electron capture dissociation (ECD) Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS) was applied to detect isomerization in Abeta peptides. ECD generated diagnostic fragment ions for the two isomers of Abeta17-28, [M + 2H - 60]+* and z6*-44 when aspartic acid was present and z6*-57 when isoaspartic acid was present. Additionally, the z(n)-57 diagnostic ion was also observed in the electron ionization dissociation (EID) spectra of the modified Abeta17-28 fragment. ECD was further applied toward Abeta1-40 and Abeta1-42. The diagnostic ion c6 + 57 was observed in the ECD spectra of the Abeta1-42 peptide, demonstrating isomerization at residue 7. In conclusion, both ECD and EID can clearly determine the presence and the position of isoaspartic acid residues in amyloid beta peptides. The next step, therefore, is to apply this method to analyze samples of Alzheimer's patients and healthy individuals in order to generate a better understanding of the disease.

Characterization and optimization of electron detachment dissociation Fourier transform ion cyclotron resonance mass spectrometry

We have demonstrated that electron detachment dissociation (EDD) can provide extensive oligonucleotide backbone fragmentation, complementary to that of other MS/MS techniques. In addition, we have shown that, for oligosaccharides, EDD provides additional cross-ring fragments compared to collision-activated dissociation and infrared multiphoton dissociation. In our EDD implementation, the potential difference between a hollow cathode electron source and an extraction lens located in between the cathode and the ion cyclotron resonance (ICR) cell was crucial for successful fragmentation with changes as small as 0.2 V drastically altering fragmentation efficiency, a behavior that was not fully understood. Here, we present a detailed characterization of the electron current passing through the ICR cell as a function of this potential difference, the cathode bias voltage, extraction lens voltage, and the cathode heating current under EDD conditions. Our results show that the extraction lens voltage serves to regulate the number of electrons passing through the ICR cell. Thus, similar electron numbers passing through the cell can be obtained at low (1.2 A) and high (1.8 A) heating current as well as at different cathode bias voltages by adjusting the extraction lens voltage. This characteristic allowed us to investigate the influence of electron energy at fixed electron number and we found that optimum EDD efficiency was obtained with 16–22 eV electrons. We also investigated the influence of charge state on oligonucleotide EDD efficiency and sequence coverage and found that higher charge states provided improved data for a DNA 10-mer, presumably due to a more extended gas-phase structure.

Identification of Single and Double Sites of Phosphorylation by ECD FT-ICR/MS in Peptides Related to the Phosphorylation Site Domain of the Myristoylated Alanine-Rich C Kinase Protein

A series of phosphorylated test peptides was studied by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS). The extensive ECD-induced fragmentation made identification of phosphorylation sites for these peptides straightforward. The site(s) of initial phosphorylation of a synthetic peptide with a sequence identical to that of the phosphorylation site domain (PSD) of the myristoylated alanine-rich C kinase (MARCKS) protein was then determined. Despite success in analyzing fragmentation of the smaller test peptides, a unique site on the PSD for the first step of phosphorylation could not be identified because the phosphorylation reaction produced a heterogeneous mixture of products. Some molecules were phosphorylated on the serine closest to the N-terminus, and others on one of the two serines closest to the C-terminus of the peptide. Although no definitive evidence for phosphorylation on either of the remaining two serines in the PSD was found, modification there could not be ruled out by the ECD fragmentation data.

Site-specific amide hydrogen exchange in melittin probed by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

Electron capture dissociation (ECD) has been proposed to be a non-ergodic process, i.e. to provide backbone dissociation of gas-phase peptides faster than randomization of the imparted energy. One potential consequence could be that ECD can fragment deuterated peptides without causing hydrogen scrambling and thereby provide amino acid residue-specific amide hydrogen exchange rates. Such a feature would improve the resolution of approaches involving solution-phase amide hydrogen exchange combined with mass spectrometry for protein structural characterization. Here, we explore this hypothesis using melittin, a haemolytic polypeptide from bee venom, as our model system. Exchange rates in methanol calculated from consecutive c-type ion pairs show some correlation with previous NMR data: the amide hydrogens of leucine 13 and alanine 15, located at the unstructured kink surrounding proline 14 in the melittin structure adopted in methanol, appear as fast exchangers and the amide hydrogens of leucine 16 and lysine 23, buried within the helical regions of melittin, appear as slow exchangers. However, calculations based on c-type ions for other amide hydrogens do not correlate well with NMR data, and evidence for deuterium scrambling in ECD was obtained from z*-type ions.

Peptide and Protein Characterization by High-Rate Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Peptide and Protein Characterization by High-Rate Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Determination of Aberrant O-Glycosylation in the IgA1 Hinge Region by Electron Capture Dissociation Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry

In a number of human diseases of chronic inflammatory or autoimmune character, immunoglobulin molecules display aberrant glycosylation patterns of N- or O-linked glycans. In IgA nephropathy, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the Gal deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. To develop experimental approaches to address this question, the synthetic IgA1 hinge region and hinge region from a naturally Gal-deficient IgA1 myeloma protein have been analyzed by 9.4 tesla Fourier transform-ion cyclotron resonance mass spectrometry. Fourier transform-ion cyclotron resonance mass spectrometry offers two complementary fragmentation techniques for analysis of protein glycosylation by tandem mass spectrometry. Infrared multiphoton dissociation of isolated myeloma IgA1 hinge region peptides confirms the amino acid sequence of the de-glycosylated peptide and positively identifies a series of fragments differing in O-glycosylation. To localize sites of O-glycan attachment, synthetic IgA1 HR glycopeptides and HR from a naturally Gal-deficient polymeric IgA1 myeloma protein were analyzed by electron capture dissociation and activated ion-electron capture dissociation. Multiple sites of O-glycan attachment (including sites of Gal deficiency) in myeloma IgA1 HR glycoforms were identified (in all but one case uniquely). These results represent the first direct identification of multiple sites of O-glycan attachment in IgA1 hinge region by mass spectrometry, thereby enabling future characterization at the molecular level of aberrant glycosylation of IgA1 in diseases such as IgA nephropathy.

Characterization of Oligodeoxynucleotides by Electron Detachment Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Electron detachment dissociation (EDD), recently introduced by Zubarev and co-workers for the dissociation of multiply charged biomolecular anions via a radical ion intermediate, has been shown to be analogous to electron capture dissociation (ECD) in several respects, including more random peptide fragmentation and retention of labile posttranslational modifications. We have previously demonstrated unique fragmentation behavior in ECD compared to vibrational excitation for oligodeoxynucleotide cations. However, that approach is limited by the poor sensitivity for oligonucleotide ionization in positive ion mode. Here, we show implementation of EDD on a commercial Fourier transform ion cyclotron resonance mass spectrometer utilizing two different configurations: a heated filament electron source and an indirectly heated hollow dispenser cathode electron source. The dispenser cathode configuration provides higher EDD efficiency and additional fragmentation channels for hexamer oligodeoxynucleotides. As in ECD, even-electron d/w ion series dominate the spectra, but we also detect numerous a/z (both even-electron and radical species), (a/z - B), c/x, (c/x - B), and (d/w - B) ions with minimal nucleobase loss from the precursor ions. In contrast to previous high-energy collision-activated dissociation (CAD) and ion trap CAD of radical oligonucleotide anions, we only observe minimum sugar cross-ring cleavage, possibly due to the short time scale of EDD, which limits secondary fragmentation. Thus, EDD provides fragmentation similar to ECD for oligodeoxynucleotides but at enhanced sensitivity. Finally, we show that noncovalent bonding in a DNA duplex can be preserved following EDD, illustrating another analogy with ECD. We believe the latter finding implies EDD has promise for characterization of nucleic acid structure and folding.

Peptide and protein characterization by high-rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry. J. Mass Spectrom. 2004;39(7): 719–729

The original article to which this Erratum refers was published in Journal of Mass Spectrometry 39 (7) 2004; 719–729. Copyright © 2004 John Wiley & Sons, Ltd.

Characterization of Tetrahymena Histone H2B Variants and Posttranslational Populations by Electron Capture Dissociation (ECD) Fourier Transform Ion Cyclotron Mass Spectrometry (FT-ICR MS)

This work describes the nature and sequence information content of the electron capture dissociation mass spectra for the intact Tetrahymena histone H2B. Two major variants of this protein were present bearing nominal modifications of both +42 and +84 Da. This work describes identification of the nature of these two modifications. For example, using gas-phase selection and isolation of the +42-Da modified species, from a background of two H2B variants each present in six or more posttranslationally modified isoforms, we were able to determine that this +42-Da modification isoform bears trimethylation rather than acetylation. LC-CIDMS analysis was also employed on digested preparations to obtain complementary detail of the nature of site-specific posttranslational modifications. This study establishes that integration of the information from these two datasets provides a comprehensive map of posttranslational occupancy for each particular covalent assemblage selected for structural investigation.

Structurally related non-covalent complexes examined by quadrupole ion trap (QIT) MS 2 and infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry IRMPD -FT-ICR MS: evidence for salt-bridge structures in the gas phase

The gas-phase structures of a series of monomeric, homo- and heterodimeric sodium adduct ions of structurally related synthetic compounds M n [Gua +–NH–(CH 2) n –COO −] with n = 1, 2, 3, 5 and Gua = guanidiniocarbonyl pyrrole were investigated by various MS techniques. The compounds M n are zwitterions in solution and have a strong tendency to aggregate in polar solvents. First, quadrupole ion trap (QIT) collision induced dissociation (CID) product ion experiments with [M n + Na] + ions ( n = 1, 2, 3, 5) and [arginine + Na] + were conducted. The fragmentation behavior of the sodium adduct ions provides indirect evidence for a change in structure varying from predominantly charge-solvation of non-ionic molecules (M 1, M 2 and arginine), to salt-bridge interactions of zwitterionic structures of M n for n = 3, 5. Second, the sodium affinities (Δ H N a + ) of the compounds M n were related to the known literature value of arginine by examination of the CID fragmentation behavior of heterodimer ions [M n + arginine + Na] + and [M n + M m + Na] + ( n ≠ m and n, m = 2, 3, 5) in a QIT. The relative ordering of sodium affinities (Δ H N a + ): M 5 ≥ arginine > M 3 > M 2 can be deduced from the relative abundances of [M n , m + Na] + and [arg + Na] + product ions. The maximum sodium affinity of M 5 relative to the reference value of arginine strongly supports the assumption of a gas-phase zwitterionic structure. Third, the dimeric sodium adduct ions [2M n + Na] + of M 2, M 3 and M 5 dissociate upon IR activation in FT-ICR MS exclusively into the respective monomeric sodium adduct ion [M n + Na] +. Hence, the establishment of a relative ordering of the gas-phase dissociation energy barriers E a laser for the disruption of the non-covalent bond of the complexes by IRMPD-FT-ICR MS was conducted. We find the dimeric complex ion [2M 3 + Na] + more stable than the respective complexes of M 2 and M 5. Hence, the stability of the examined complex ions [2M n + Na] + is obviously strongly determined by the various possible non-covalent interactions between the two respective molecules M n . The MS study supports the assumption that M n molecules with n ≥ 3 are able to conserve zwitterionic structures in the gas phase.

Peptide and protein characterization by high-rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry.

The analytical utility of the electron capture dissociation (ECD) technique, developed by McLafferty and co-workers, has substantially improved peptide and protein characterization using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The limitations of the first ECD implementations on commercial instruments were eliminated by the employment of low-energy electron-injection systems based on indirectly heated dispenser cathodes. In particular, the ECD rate and reliability were greatly increased, enabling the combination of ECD/FTICR-MS with on-line liquid separation techniques. Further technique development allowed the combination of two rapid fragmentation techniques, high-rate ECD and infrared multiphoton dissociation (IRMPD), in a single experimental configuration. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation and led to improved peptide and protein characterization. The application of high-rate ECD/FTICR-MS has demonstrated its power and unique capabilities in top-down sequencing of peptides and proteins, including characterization of post-translational modifications, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation). Analysis of peptide mixtures has been accomplished using high-rate ECD in bottom-up mass spectrometry based on mixture separation by liquid chromatography and capillary electrophoresis. This paper summarizes the current impact of high-rate ECD/FTICR-MS for top-down and bottom-up mass spectrometry of peptides and proteins.

Determination of the activation energy for unimolecular dissociation of a non-covalent gas-phase peptide:substrate complex by infrared multiphoton dissociation fourier transform ion cyclotron resonance mass spectrometry

The activation energy for the unimolecular dissociation of a non-covalent supramolecular complex between an Artificial Cationic Receptor A ([Gua-Val-Val-Val-Amide] +, in which Gua is guanidiniocarbonyl pyrrole) and an Anionic Tetrapeptide B ([N-Acetyl-Val-Val-Ile-Ala] −) has been determined by measurement of the dissociation rate constant as a function of infrared CO 2 laser power density. Singly-charged quasimolecular [A + B + H] + ions are isolated, stored in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, and irradiated by IR photons. The rate constant for dissociation of the non-covalent complex is determined at five different laser power densities. A plot of the natural logarithm of the first-order rate constant versus the natural logarithm of the laser power density yields a straight line, the slope of which provides an approximate measure of the activation energy (E a laser) for dissociation. E a laser is calculated by a relationship derived earlier by Dunbar and with a newly proposed equation by Paech et al. The results of the two approaches deliver significantly different activation energy values for the unimolecular dissociation of the non-covalent complex. We obtain E aI laser = 0.67 eV (Dunbar approximation) and E aII laser = 1.12 eV (Paech et al. approximation). Differences between the two approaches are discussed with respect to non-covalent complexes.

An antibiotic linked to peptides and proteins is released by electron capture dissociation fourier transform ion cyclotron resonance mass spectrometry

Desfuroylceftiofur (DFC) is a bioactive β-lactam antibiotic metabolite that has a free thiol group. Previous experiments have shown release of DFC from plasma extracts after addition of a disulfide reducing agent, suggesting that DFC may be bound to plasma and tissue proteins through disulfide bonds. We have reacted DFC with [Arg 8]-vasopressin (which has one disulfide bond) and bovine insulin (which has three disulfide bonds) and analyzed the reaction products by use of electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS), which has previously shown preferential cleavage of disulfide bonds. We observe cleavage of DFC from vasopressin and insulin during ECD, suggesting that DFC is indeed bound to peptides and proteins through disulfide bonds. Specifically, we observed dissociative loss of one, as well as two, DFC species during ECD of [vasopressin + 2(DFC-H) + 2H] 2+ from a single electron capture event. Loss of two DFCs could arise from either consecutive or simultaneous loss, but in any case implies a gas phase disulfide exchange step. ECD of [insulin + DFC + 4H] 4+ shows preferential dissociative loss of DFC. Combined with HPLC, ECD FT-ICR-MS may be an efficient screening method for detection of drug-biomolecule binding.

Protein identification by peptide mass fingerprinting and peptide sequence tagging with alternating scans of nano-liquid chromatography/infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.

We have developed a method for protein identification with peptide mass fingerprinting and sequence tagging using nano liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). To achieve greater sensitivity, a nanoelectrospray (nano-ES) needle packed with reversed-phase medium was used and connected to the nano-ES ion source of the FTICR mass spectrometer. To obtain peptide sequence tag information, infrared multiphoton dissociation (IRMPD) was carried out in nano-LC/FTICR-MS analysis. The analysis involves alternating nano-ES/FTICR-MS and nano-ES/IRMPD-FTICR-MS scans during a single LC run, which provides sets of parent and fragment ion masses of the proteolytic digest. The utility of this alternating-scan nano-LC/IRMPD-FTICR-MS approach was evaluated by using bovine serum albumin as a standard protein. We applied this approach to the protein identification of rat liver diacetyl-reducing enzyme. It was demonstrated that this enzyme was correctly identified as 3-alpha-hydroxysteroid dehydrogenase by the alternating-scan nano-LC/IRMPD-FTICR-MS approach with accurate peptide mass fingerprinting and peptide sequence tagging.