Quantitative profiling of endogenous retinoic acid in vivo and in vitro by tandem mass spectrometry

We report an improved tandem mass spectrometric assay for retinoic acid (RA) applicable to in vitro and in vivo biological samples. This liquid chromatography tandem mass spectrometric (LC/MS/MS) assay for direct RA quantification is the most sensitive to date, with a 62.5 attomol lower limit of detection and a linear range spanning greater than 4 orders of magnitude (from 250 attomol to 10 pmol). This assay resolves all-trans-RA (atRA) from its endogenous geometric isomers, is applicable to samples of limited size (10-20 mg of tissue), and functions with complex biological matrixes. Coefficients of variation are as follows: instrumental, < or =2.6%; intraday, 5.2% +/- 0.7%; interday, 6.7% +/- 0.9%. In vitro capabilities are demonstrated by quantification of endogenous RA and RA production (from retinol) in primary cultured astrocytes. Quantification of endogenous atRA and its geometric isomers in 129SV mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, and brain) reveals in vivo utility of the assay. The ability to discriminate spatial concentrations of RA in vivo is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum), as well as with Lewis rat proximal/distal mammary gland regions during various morphological stages: virgin, early pregnancy (e7), late pregnancy (e20), lactating (day 4), involuting day 1, and involuting day 11. This assay provides the sensitivity necessary for direct, endogenous RA quantification necessary to elucidate RA function, e.g., in neurogenesis, morphogenesis, and the contribution of altered RA homeostasis to diseases, such as Alzheimer's disease, type 2 diabetes, obesity, and cancer.     

Unambiguous assignment of intramolecular chemical cross-links in modified mammalian membrane proteins by Fourier transform-tandem mass spectrometry

Fourier transform tandem mass spectrometry (FT-MS/MS) can be used to unambiguously assign intramolecular chemical cross-links to specific amino acid residues even when two or more possible cross-linking sites are adjacent in the cross-linked protein. Bovine rhodopsin (Rho) in its dark-adapted state was intramolecularly cross-linked with lysine-cysteine (K-C) or lysine-lysine (K-K) cross-linkers to obtain interatomic distance information. Large, multiply charged, cross-linked peptide ions containing adjacent lysines, corresponding to Rho(50-86) (K(66) or K(67)) cross-linked to Rho(310-317) (C(316)) or Rho(318-348) (K(325) or K(339)), were fragmented by collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron capture dissociation (ECD). Complementary sequence-specific information was obtained by combining cross-link assignments; however, only ECD revealed full palmitoylation of adjacent cysteines (C(322) and C(323)) and cross-linking of K(67) (and not K(66)) to C(316), K(325), and K(339). ECD spectra contained crucial c- and z-ions resulting from cleavage of the bond between K(66) and K(67). To our knowledge, this work also presents the first demonstration that ECD can be used to characterize S-linked fatty acid acylation on cysteines. The comprehensive fragmentation of large peptides by CID, IRMPD, and particularly ECD, in conjunction with the high resolution and mass accuracy of FT-MS/MS, is shown to be a valuable means of characterizing mammalian membrane proteins with both chemical and posttranslational modifications.     

Identification of trichloroethanol visualized proteins from two-dimensional polyacrylamide gels by mass spectrometry

Proteins visualized by 2,2,2-trichloroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MS/MS. In a previous study, a method was developed that placed TCE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 min. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization. In this study, MALDI-TOF MS and LC-MS/MS are used to identify randomly selected Escherichia coli proteins. The identification of TCE visualized proteins is compared to the identification of Coomassie brilliant blue (CBB) stained proteins from two-dimensional gel electrophoresis of E. coli proteins. This study demonstrated that TCE visualized proteins are compatible with protein identification by MALDI-TOF peptide mass fingerprinting. For 10 randomly selected spots, TCE visualization lead to statistically significant identification of 5 proteins and CBB visualization lead to identification of 6 proteins. TCE visualized proteins are also shown to be well suited for protein identification using LC-MS/MS. In 16 spots selected for MS/MS analysis, TCE samples lead to the identification of 79 peptides; while CBB samples lead to the identification of 65 peptides. TCE samples also supported the identification of more proteins. The low stoichiometry of labeling of tryptophan residues does not require inclusion of this modification for database searches. In addition to being a rapid visualization technique compatible with MS, TCE visualization utilizes rapid washing conditions for sample preparation of proteins spots excised from polyacrylamide gels.     

Analysis of antibodies and other large glycoproteins in the mass range of 150,000-200,000 Da by electrospray ionization mass spectrometry.

The analytical applicability of electrospray ionization mass spectrometry (ESIMS) to large glycoproteins in the molecular weight (MW) range of 150,000-200,000 was demonstrated. Multiply charged ions (charge state as high as 150+) of several typical macrosized glycoproteins of immunological significance were generated by pneumatically-assisted electrospray (ionspray) and their masses measured on a quadrupole mass spectrometer having a mass-to-charge (m/z) range of 2400. The resolution of the quadrupole instrument was insufficient to resolve the glycocomposition microheterogeneities in the MW range studied. Nevertheless, the average MWs of three immunoglobulin G (IgG) class murine monoclonal antibodies, anti-(human alpha 1-antitrypsin) (148,484 +/- 4), anti-(human alpha 1-acid glycoprotein) (149,599 +/- 12) and anti-(beta-galactosidase) (component I, 150,544 +/- 10, and component II, 151,496 +/- 17), and human alpha 2-macroglobulin monomer (186,100 +/- 100), and human complement component C4 (196,863 +/- 29) were still determined from the fused peak profiles of their constituent glyco components (the errors given reflect the measurement precisions of the simultaneous multichannel MW determinations). The difference between the measured average MW and the unmodified sequence MW was used to assess the degree of post-transitional modification in human alpha 2-macroglobulin (13.6%) and human complement component C4 (5.3%). For the large glycoproteins studied here, glycosylation did not appear to seriously affect the effectiveness of the electrospray ionization; up to 70% of their full charge-retaining capacities were fulfilled under the usual experimental conditions. These results show that ESIMS is capable of providing analytically useful information for macrosized proteins.     

Accurate mass measurement by electrospray ionization quadrupole mass spectrometry: detection of variants differing by <6 Da from normal in human hemoglobin heterozygotes

Mass spectrometry has a basic limitation when human hemoglobin variants are analyzed, because it cannot resolve two globin chains that differ in mass by <6 Da. Several common beta-chain variants differ by 1 Da from normal and, hence, when present in heterozygotes, are not resolved from the normal beta-chain. Normal and variant chains appear together in the spectrum as a single entity, whose mass is the abundance weighted mean of the two chains. Here we show that such heterozygotes can be detected in 500-fold diluted blood by accurately measuring the mass of the beta-chain using an electrospray ionization quadrupole instrument and the alpha-chain for internal mass calibration. A statistical analysis of the normal beta-chain mass (n = 86) showed that the standard deviation (SD) of the mean was <+/-0.05 Da (<+/-3.2 ppm). Hence, at the 95% confidence level (+/-2 SD), an abnormal alpha- or beta-chain differing by 1 Da from normal should be detectable in a heterozygote provided its abundance is >10% of total alpha- or beta-chains, respectively. Variants whose masses lay between 1 and 4 Da from normal were detected in 19 heterozygotes. Moreover, the proportion of each variant estimated from the mass change correlated with the proportion determined by cation-exchange HPLC. Variants were assigned to the alpha- or beta-chain by combining the sign of the mass change with the polarity change inferred from electrophoretic data. This procedure could be used for screening clinically significant hemoglobin variants.     

Absolute quantification of proteins in solutions and in polyacrylamide gels by mass spectrometry

A combination of nanoelectrospray tandem mass spectrometry and (18)O-labeled peptide internal standards was applied for the absolute quantification of proteins from their in-solution and in-gel tryptic digests. Although absolute quantification from in-solution digests was accurate, we observed that in-gel digestion compromised the quantification accuracy by affecting the recovery of individual peptides and, therefore, the provided estimates might be strongly influenced by the selection of reference peptides. Under optimized experimental conditions, it was possible to provide a semiquantitative estimate of the absolute amount of gel separated proteins within better than 50% error margin.     

Profiling of hydrophilic proteins from Olea europaea olive pollen by MALDI TOF mass spectrometry

The antigenic profile of Olea europaea pollen from different Mediterranean cultivars was obtained by MALDI mass spectrometry. A simple procedure of chemical fractionation of the whole antigen extract was developed, whereby less complex, or pure, fractions of antigen candidate were obtained prior to mass spectrometric analysis. Some of the features of protein structure and distribution probably depend on cultivar adaptation to the environment. The profilings of pollen proteins thus obtained allow the distinction of the analyzed cultivars into three distinct groups: (i) those characterized by a low Ole e 1 content; (ii) those over-enriched in Ole e 1 and (iii) that containing Ole e 3 and Ole e 7 only. The latter consists of at least four isoforms differing by the degree of glycosilation. These results demonstrate that the proposed experimental procedure, can supply valuable information on the antigens' micro heterogeneity.     

Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry

The direct profiling of proteins present in tissue sections for several organs of the mouse has been accomplished using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Fresh tissue was sectioned and blotted on a conductive polyethylene membrane. The dried membrane blot was coated with matrix, typically sinapinic acid, and directly analyzed in the mass spectrometer. Generally, well over 100 peptide/protein signals in the 2000-30,000 Da range were observed, with 30-50 having relatively high signal intensities. Analysis of different areas of the same tissue gave remarkably similar mass spectra with greater than 90% homology. However, different parts of a segmented tissue, such as the proximal, intermediate, and distal colon, gave some unique protein signals. After treatment of the tissue blot with protease and subsequent MALDI MS analysis using postsource decay methods for peptide sequencing, some of the proteins were identified. The unique protein profiles measured from these tissue blots also showed differences from strain to strain of the mouse, with genetically similar strains having very similar patterns.     

In vivo nanoelectrospray for the localization of bioactive molecules in plants by mass spectrometry

The method for the localization of bioactive molecules in plants is highly needed since it provides a fundamental prerequisite for understanding their physiological and ecological functions. Here, we propose a simple method termed in vivo nanoelectrospray for the localization of bioactive molecules in plants without sample preparation. A capillary is partly inserted into the plant to sample liquid from a highly located region, and then, a high voltage is applied to the plant to generate an electrospray from the capillary tip for mass spectrometry analysis. Using this method, bioactive molecules such as saccharides, glycoalkaloids, flavonoids, organic acids, and glucosinolates (GLs) are detected in the target regions of living plants or fresh fruits. Original information for endogenous chemicals including liable molecules in plant can be obtained. A sketchy three-dimensional distribution of glycoalkaloids in a cherry tomato has been obtained. The present work provides a powerful tool for the study of bioactive molecules in a living plant by mass spectrometry.     

Comprehensive assignment of mass spectral signatures from individual Bacillus atrophaeus spores in matrix-free laser desorption/ionization bioaerosol mass spectrometry

We have fully characterized the mass spectral signatures of individual Bacillus atrophaeus spores obtained using matrix-free laser desorption/ionization bioaerosol mass spectrometry (BAMS). Mass spectra of spores grown in unlabeled, 13C-labeled, and 15N-labeled growth media were used to determine the number of carbon and nitrogen atoms associated with each mass peak observed in mass spectra from positive and negative ions. To determine the parent ion structure associated with fragment ion peaks, the fragmentation patterns of several chemical standards were independently determined. Our results confirm prior assignments of dipicolinic acid, amino acids, and calcium complex ions made in the spore mass spectra. The identities of several previously unidentified mass peaks, key to the recognition of Bacillus spores by BAMS, have also been revealed. Specifically, a set of fragment peaks in the negative polarity is shown to be consistent with the fragmentation pattern of purine nucleobase-containing compounds. The identity of m/z = +74, a marker peak that helps discriminate B. atrophaeus from Bacillus thuringiensis spores grown in rich media is [N1C4H12]+. A probable precursor molecule for the [N1C4H12]+ ion observed in spore spectra is trimethylglycine (+N(CH3)3CH2COOH), which produces a m/z = +74 peak when ionized in the presence of dipicolinic acid. A clear assignment of all the mass peaks in the spectra from bacterial spores, as presented in this work, establishes their relationship to the spore chemical composition and facilitates the evaluation of the robustness of "marker" peaks. This is especially relevant for peaks that have been used to discriminate Bacillus spore species, B. thuringiensis and B. atrophaeus, in our previous studies.     

Quantitative measurement of endogenous estrogens and estrogen metabolites in human serum by liquid chromatography-tandem mass spectrometry

Endogenous estrogen plays a key role in the development of human breast cancer, yet the contribution of specific estrogen metabolites and patterns of estrogen metabolism remains unclear. To determine their individual and joint roles in breast carcinogenesis, it is necessary to be able to measure quantitatively each estrogen metabolite in epidemiologic and clinical biospecimens. In this report, we detail a sensitive, specific, accurate, and precise high-performance liquid chromatography-tandem mass spectrometry method utilizing selected reaction monitoring for measuring the absolute quantities of free (unconjugated) and total (conjugated + unconjugated) endogenous estrogens and estrogen metabolites in human serum from premenopausal and postmenopausal women. The method requires a simple sample preparation and only 0.5 mL of serum, yet is capable of quantifying simultaneously 15 estrogens and estrogen metabolites (EM): estrone and its 2-, 4-, and 16alpha-hydroxy and 2- and 4-methoxy derivatives; 2-hydroxyestrone-3-methyl ether; 17beta-estradiol and its 2-hydroxy and 2- and 4-methoxy derivatives; and estriol, 17-epiestriol, 16-ketoestradiol, and 16-epiestriol. The lower limit of quantitation for each EM was 0.4 pg on-column, equivalent to 8 pg/mL (26.5-29.6 fmol/mL) in the original serum sample. Calibration curves were linear over a 10(3)-fold concentration range. For a stripped serum sample containing 8 pg/mL of each EM, accuracy (percent recovery of a known added amount) ranged from 91 to 113%. Intrabatch precision (including hydrolysis, extraction, and derivatization steps) ranged from 7 to 30% relative standard deviation (RSD), and interbatch precision ranged from 8 to 29% RSD. Since distinct roles have been proposed for many of these estrogen metabolites, an accurate, precise, sensitive, and specific method for measuring their levels in circulation should suggest new approaches to breast cancer prevention, screening, and treatment.     

Widespread existence of Cytosine methylation in yeast DNA measured by gas chromatography/mass spectrometry

DNA methylation is one of the major epigenetic modifications and has been involved in a number of biological processes in mammalian cells. Yeast is widely used as a model organism for studying cell metabolism, cell cycle regulation, and signal transduction. However, it remains controversial whether methylated cytosine (5-methylcytosine, 5mC) exists in the yeast genome. In the current study, we developed a highly sensitive method based on gas chromatography/mass spectrometry (GC/MS) and systematically examined the incidence of 5mC in 19 yeast strains, which represent 16 yeast species. Our results showed that DNA methylation is widespread in yeast and the genome-wide DNA methylation of the studied yeast strains ranged from 0.014 to 0.364%, which were 1 to 2 orders of magnitude lower than that in mammalian cells (i.e., 3-8%). Furthermore, we found that the 5mC content in yeast varied considerably at different growth stages and DNA methylation inhibitor 5-azacytidine could induce a decrease in genome-wide DNA methylation as that in mammalian cells. The demonstration of the universal presence of DNA cytosine methylation in yeast constituted the first and essential step toward understanding the functions of this methylation in yeast.     

Specific method for the determination of genomic DNA methylation by liquid chromatography-electrospray ionization tandem mass spectrometry

Herein we report a novel method for determining genomic DNA methylation that utilizes liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to measure 5-methyl-2'-deoxycytidine levels following enzymatic hydrolysis of genomic DNA. LC separation of 5-methyl-2'-deoxycytidine from the four deoxyribonucleosides, the four ribonucleosides, and 5-methyl-2'-cytidine, a RNA methylation product, has been achieved within 15 min. In combination with ESI-MS/MS detection, the reported method is highly specific and extremely sensitive with a limit of detection (LOD) of 0.2 fmol and a quantification linearity range from 1 fmol to 20 pmol. Genomic DNA methylation was expressed as the ratio of 5-methyl-2'-deoxycytidine to 2'-deoxyguanosine and was determined directly using 2'-deoxyguanosine as the internal standard. Because deoxycytidine methylation typically ranges from 2 to 6% in mammalian genomes, and pharmacological or genetic manipulations have not achieved levels lower than 0.1%, we validated the assay for methylation levels ranging from 0.05 to 10%. Importantly, both RNA contamination and incomplete DNA hydrolysis had no appreciable effect on 5-methyl-2'-deoxycytidine quantification. LOD studies indicate that only 4 ng of DNA is required for this assay. This LOD should permit the use of this method for applications having limiting amounts of DNA that were not previously candidates for global genomic DNA methylation analysis, e.g., clinical trial samples, or cells collected by laser capture microdissection.     

A programmable fragmentation analysis of proteins by in-source decay in MALDI-TOF mass spectrometry

Here we describe an algorithm for identifying peptides/ proteins of known sequence and unknown peptides from partial spectra generated by an in-source decay (ISD) technique coupled with matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. The identification of protein fragments is processed with a software program called CMATCH, which generates candidate subsequences for both known peptides/proteins and unknown peptides for the major product ions in the spectral range m/z 400-5000 and then matches these to known protein sequences contained in a reference database for the known peptides/proteins. CMATCH, which is compiled for MSDOS or WINDOWS95/NT, has two main advantages: first, the candidate subsequences are generated automatically without the need for supplementary information concerning the distribution of either N-terminal or C-terminal ions in the spectra for both known peptides/proteins and unknown peptides; second, the highest coordinated homologous sequences are picked up automatically from the reference database as the best matches with known peptides/proteins. Examples from the ISD spectra of several test proteins demonstrate the efficacy of this protein identification software.     

In-spray supercharging of peptides and proteins in electrospray ionization mass spectrometry

Enhanced charging, or supercharging, of analytes in electrospray ionization mass spectrometry (ESI MS) facilitates high resolution MS by reducing an ion mass-to-charge (m/z) ratio, increasing tandem mass spectrometry (MS/MS) efficiency. ESI MS supercharging is usually achieved by adding a supercharging reagent to the electrospray solution. Addition of these supercharging reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases the average charge of enzymatically derived peptides and improves peptide and protein identification in large-scale bottom-up proteomics applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation from a droplet in the ESI ion source.     

Modeling and analysis of the induced signal detected in electrostatic ion beam trap for mass spectrometry

The induced image charge and image current acquired by a detector tube for mass analysis are simulated using a numerical electrostatic model in the context of the electrostatic ion beam trap (EIBT). With the simulation results, the principle of mass analysis using the induced signal is demonstrated and studied systematically. The results show that the intensity of the detected signal is significantly influenced by the size and configuration of the detector, and also impacted by ion velocity, the number of ions in the ion group, and the ion beam length. The simulation results could not only be used to optimize the size and configuration of the detector and thus to improve the detected signal, but also to support the signal analysis (such as FFT) at an EIBT for mass spectrometry.     

Resonant laser ablation of metals detected by atomic emission in a microwave plasma and by inductively coupled plasma mass spectrometry

It has been shown that an increase in sensitivity and selectivity of detection of an analyte can be achieved by tuning the ablation laser wavelength to match that of a resonant gas-phase transition of that analyte. This has been termed resonant laser ablation (RLA). For a pulsed tunable nanosecond laser, the data presented here illustrate the resonant enhancement effect in pure copper and aluminum samples, chromium oxide thin films, and for trace molybdenum in stainless steel samples, and indicate two main characteristics of the RLA phenomenon. The first is that there is an increase in the number of atoms ablated from the surface. The second is that the bandwidth of the wavelength dependence of the ablation is on the order of 1 nm. The effect was found to be virtually identical whether the atoms were detected by use of a microwave-induced plasma with atomic emission detection, by an inductively coupled plasma with mass spectrometric detection, or by observation of the number of laser pulses required to penetrate through thin films. The data indicate that a distinct ablation laser wavelength dependence exists, probably initiated via resonant radiation trapping, and accompanied by collisional broadening. Desorption contributions through radiation trapping are substantiated by changes in crater morphology as a function of wavelength and by the relatively broad linewidth of the ablation laser wavelength scans, compared to gas-phase excitation spectra. Also, other experiments with thin films demonstrate the existence of a distinct laser-material interaction and suggest that a combination of desorption induced by electronic transition (DIET) with resonant radiation trapping could assist in the enhancement of desorption yields. These results were obtained by a detailed inspection of the effect of the wavelength of the ablation laser over a narrow range of energy densities that lie between the threshold of laser-induced desorption of species and the usual analytical ablation regime. Normal ablation employs high-power lasers in an attempt to create a vapor plume without selective vaporization, and with a stoichiometry that accurately represents the stoichiometry of species in the solid sample. RLA, as a method of selective vaporization, appears to provide an opportunity to exploit selective vaporization in new ways.     

Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards

A new method is presented for quantifying proteomic and metabolomic profile data by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization. This biotechnology provides differential expression measurements and enables the discovery of biological markers (biomarkers). Work presented here uses human serum but is applicable to any fluid or tissue. The approach relies on linearity of signal versus molecular concentration and reproducibility of sample processing. There is no use of isotopic labeling or chemically similar standard materials. Linear standard curves are reported for a variety of compounds introduced into human serum. As a measure of analytical reproducibility for proteome and metabolome sampling, median coefficients of variation of 25.7 and 23.8%, respectively, were determined for approximately 3400 molecular ions (not counting their numerous isotopes) from 25 independently processed human serum samples, corresponding to a total of 85000 individual molecular ion measurements.     

Plasma electron capture dissociation for the characterization of large proteins by top down mass spectrometry

For the backbone dissociation of large (29 kDa) multiply charged protein ions in the gas phase by electron capture, the main experimental challenges are juxtaposition of the electron and ion for efficient capture, dissociation of tertiary noncovalent bonds that prevent product separation, and minimization of secondary electron capture that destroys larger product ions. A simple alternative methodology is described in which electrons (0.03-100 microA, 0.1-15 eV) are first impinged on a gas pulse in the ion cell of a Fourier transform mass spectrometer, followed by ion beam introduction. For carbonic anhydrase, the resulting plasma conditions produce 87% efficiency for electron capture; a single spectrum yields 512 product ions of 237 different masses from cleavage of 183 of the 258 interresidue bonds, while two spectra cleave 197 of these bonds. The problem of secondary dissociation of product ions is reduced by plasma conditions in which product ions are formed near electrons whose velocities are unfavorable and whose capture cross sections no longer have a square dependence on charge. One plasma ECD spectrum of ubiquitin provides its sequence de novo except for two residue pairs. ECD of casein identifies 126 of 208 interresidue cleavages, providing direct and specific characterization of all its 26 Ser/Thr/Tyr phosphorylation sites.     

T3-sequencing: targeted characterization of the N- and C-termini of undigested proteins by mass spectrometry

A novel extension of the "top-down" approach is introduced for the selective characterization of protein termini that does not involve proteolytic digestion steps. N- and C-terminal peptides were generated from intact proteins in the mass spectrometer and further analyzed by MS/MS-an approach referred to as T(3)-sequencing. N-terminal and C-terminal fragment ion series were obtained by the pseudo-MS/MS technique in-source decay (ISD) on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). These ions provided near-terminal sequence tags from the undigested protein in the ISD spectrum acquired in reflector mode and allowed to screen for the proper processing state of the terminus with respect to a reference sequence. In the second step of T(3)-sequencing, the precursor ions, which have been generated by ISD and which included the N- or C-terminal sequence, were selected in the timed ion gate of a MALDI-TOF/TOF mass spectrometer for MS/MS analysis. These spectra allowed identification of the protein, the proper definition of both termini, and allowed confirmation of suspected terminal modifications. T(3)-Sequencing appears to be an alternative to classical Edman sequencing, which is fast and even permits the analysis of N-terminally blocked proteins and their C-terminus.