Complete and rapid peptide and glycopeptide mapping of mouse monoclonal antibody by LC/MS/MS using ion trap mass spectrometry

Complete and rapid peptide and glycopeptide mapping of a mouse monoclonal immunoglobulin (IgG2b) were carried out by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry (LC/ ESI IT-MS/MS). It was possible to obtain spectra of a minor glycopeptide at a quantity as low as 1.8 pmol. Reduced and carboxymethylated mouse antidansyl monoclonal IgG2b (RCM-IgG2b) was digested with lysyl-endopeptidase. Proteolytic peptides were subjected to capillary HPLC separation followed by analysis with an ion trap mass spectrometer. The complete amino acid sequence of the IgG2b was characterized by using LC/ ESI IT-MS/MS. The structures of 12 different types of O-linked oligosaccharides attached to Thr-221AH in the hinge region and those of three major types of N-linked oligosaccharides attached to Asn-297H have been characterized.     

Microfabricated device coupled with an electrospray ionization quadrupole time-of-flight mass spectrometer: protein identifications based on enhanced-resolution mass spectrometry and tandem mass spectrometry data

We describe the coupling of a microfabricated fluidic device to an electrospray ionization (ESI) quadrupole time-of-flight mass spectrometer (QqTOFMS) for the identification of protein samples. The microfabricated devices consisted of three reservoirs connected via channels to a main capillary, which in turn was linked via a microspray interface to the QqTOFMS. Here we present preliminary results obtained using this system. Standardized solutions of myoglobin tryptic digest were analyzed indicating a limit of detection at the low to sub fmol/microL. The combination of the microfabricated device for rapid sample delivery and the rapid acquisition capability, enhanced resolution and mass accuracy of the QqTOF offers unique possibilities for the rapid identification of proteins by database searching. This platform can generate MS data suitable for protein database searching by the peptide-mass fingerprinting approach and MS/MS data suitable for protein database searching. Here the results of the two database-searching approaches are compared and the possibilities of combining the two approaches for rapid identification of protein are discussed. Also, we present a comparison of the results obtained using the three-position microfabricated device coupled to the ESI-QqTOFMS and to an ESI-ion trap MS. Finally the combination of C-terminal 18O labeling of peptides and the microfabricated system for automated combined peptide-mass fingerprinting and sequence-tag database searching is discussed.     

Accurate mass determinations for the confirmation and identification of organic microcontaminants in surface water using on-line solid-phase extraction liquid chromatography electrospray orthogonal-acceleration time-of-flight mass spectrometry

The identification of polar microcontaminants in surface water is an important issue in environmental analysis. Liquid chromatography/mass spectrometry (LC/MS) is frequently applied for this purpose. However, even in combination with tandem mass spectrometry (MS/MS), unambiguous identification of the compounds detected is often difficult. The potential of an alternative strategy, based on the ability of an orthogonal-acceleration time-of-flight mass spectrometer to routinely perform accurate mass determination at 10 ppm in on-line LC/MS, is explored. On-line solid-phase extraction LC electrospray orthogonal-acceleration time-of-flight mass spectrometry is shown to enable the determination of pesticides from various compound classes in surface water in the concentration range of 0.1 to 10 micrograms/L. In addition, the ability to discriminate and unambiguously identify pesticides in mixtures of isobaric and/or isomeric compounds is investigated.     

Rapid identification of comigrating gel-isolated proteins by ion trap-mass spectrometry

In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) followed by an initial database search with the peptide masses failed to identify the proteins. The peptides were separated by reversed-phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional sequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence of multiple matching peptide masses in the MALDI-MS spectra. In addition, a novel sequence, not found in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.     

Screening for high-affinity ligands to the Src SH2 domain using capillary isoelectric focusing-electrospray ionization ion trap mass spectrometry

A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.     

Determination of n-hexane metabolites by liquid chromatography/mass spectrometry. 2. Glucuronide-conjugated metabolites in untreated urine samples by electrospray ionization

A liquid chromatography atmospheric pressure electrospray mass spectrometry (ESI-LC/MS) system was evaluated for the identification and characterization of n-hexane conjugated metabolites (glucuronides) in untreated urine samples. Chromatography of glucuronides was obtained under ion-suppressed reversed-phase conditions, by using high-speed (3 cm, 3 microns) columns and formic acid (2 mM) as modifier in the mobile phase. The mass spectrometer was operated in negative ion (NI) mode. For the first time, four glucuronides were identified by ESI-LC/MS in untreated urine samples of rats exposed to n-hexane: 2-hexanol-glucuronide, 5-hydroxy-2-hexanone-glucuronide, 2,5-hexanediol-glucuronide and 4,5-dihydroxy-2-hexanone-glucuronide. Confirmation of the conjugated metabolites was obtained by LC/MS/MS experiments. Gas chromatography/mass spectrometry (GC/MS) and atmospheric pressure chemical ionization (APCI) LC/MS analyses were performed on the same samples. An integrated approach GC/MS-LC/MS for the semi-quantitative analysis of n-hexane glucuronides, whose standards are not commercially available, is discussed and proposed here. In order to understand the fate of the metabolites during sample pre-treatment, a study about the effects of enzymatic and acid hydrolysis on urine samples was conducted on glucuronides isolated by solid-phase extraction. Combined analyses by GC/MS and LC/MS enabled us to distinguish 'true' n-hexane metabolites from compounds resulting from sample treatment and handling (i.e. enzymatic and acid hydrolysis, extraction and GC injection).     

Effect of symplasmic isolation and antigibberellin treatment on morphogenesis in Chara

We characterized the particle size distribution and the analyte transmission efficiency of a liquid chromatography/particle beam/mass spectrometry (LC/PB/MS) system as a function of experimental variations normally used to optimize the LC/PB/MS system. The particle size distribution was evaluated using an electrical differential mobility particle sizer (DMPS) and both the DMPS and the mass spectrometer were used to evaluate transmission. The latter results were correlated to provide evidence related to mechanisms which contribute to poor sample transmission. Addition of ammonium acetate buffer did not increase the aerosol particle mean diameter. However, it did lead to significant increases in caffeine transmission efficiency observed in both the DMPS and the mass spectrometer. Our results were interpreted to suggest a possible electrostatic cause for quantitative anomalies in LC/PB/MS rather than simple mass discrimination in the particle beam momentum separator.     

On-line coupling of micellar electrokinetic chromatography to electrospray mass spectrometry

The possibility of selectivity enhancement in capillary electrophoresis-mass spectrometry (CE-MS) by hyphenating micellar electrokinetic chromatography (MEKC) and electrospray mass spectrometry (MS) is described for two quaternary ammonium compounds. Direct coupling of MEKC to MS is hazardous because of the contamination of the ion source due to presence of an excess of micelle forming agent in the MEKC buffer. Therefore, a coupled-capillary setup with the possibilities of voltage switching and buffer renewal has been designed. Such a system allows on-line heartcutting of the zones of interest in the MEKC capillary with subsequent transfer via a second capillary to the mass spectrometer.     

Identification and relative quantitation of F2-isoprostane regioisomers formed in vivo in the rat

F2-isoprostanes are a complex mixture of isomers formed in four regioisomeric family types by free radical-initiated oxidation of arachidonic acid present in membrane phospholipids. F2-isoprostanes isolated from the livers of rats treated with carbon tetrachloride were separated by initial reverse phase HPLC and detected using electrospray ionization mass spectrometry with the characteristic loss of 44 u (C2H4O) from the common 1,3-diol cyclopropane ring found in these eicosanoids. Collision induced decomposition of the carboxylate anions from the separated F2-isoprostanes formed abundant ions characteristic for regioisomers of Type I (m/z 115), Type III (m/z 127), and Type IV (m/z 193), which made possible characterization of these three family subtypes by LC/MS/MS. Capillary GC/MS was employed to further identify the F2-isoprostane regioisomers using electron ionization mass spectrometry and to obtain characteristic mass spectra of the pentafluorobenzyl ester trimethylsilyl ether derivatives. Quantitation of the F2-isoprostanes separated by both reverse-phase HPLC and capillary GC/MS was carried out using negative ion chemical ionization mass spectrometry. The most abundant isomers identified were Type I and IV regioisomers constituting 33 and 25% of the total products, respectively. As expected, the Type II and III regioisomer products were of less abundance. Over 45 F2-isoprostanes could be separated in this complex mixture, suggesting random production of each regioisomeric subtype in this in vivo model.     

Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides

Electrospray ionization-ion trap mass spectrometry, with its capacity to perform multiple stages of fragmentation (MSn), is demonstrated as an effective method for the structural characterization of permethylated N-linked complex glycoprotein oligosaccharides. Complex glycan structural features, such as N-acetyllactosamine antenane, neuraminic acids, and nonreducing terminal GlcNAc monosaccharides, commonly suppress cross-ring and core saccharide cleavages in traditional MS/MS experiments. Using ion trap mass spectrometry, removal of these substituents permits determination of branching patterns and intersaccharide linkages by MS3 and MS4. Both sequence and linkage data are obtained for N-acetyllactosamine and sialyl-N-acetyllactosamine oligosaccharide antennae from biantennary glycans using MS3, and the location of a bisecting GlcNAc residue is also established after exposing the core pentasaccharide. Higher-order experiments further illustrate the potential of electrospray ionization-quadrupole ion trap mass spectrometry for carbohydrate analysis, as MS8 is used to produce significant and otherwise unobtainable branching information for an oligosaccharide from chicken ovalbumin. These studies constitute further evidence of the unique role that ion trap mass spectrometry can assume in the area of oligosaccharide analysis.     

Trace quantitation of the novel cholinesterase inhibitor in human plasma by capillary gas chromatography/ion trap mass spectrometry

This paper demonstrates the utility of an ion trap mass spectrometer as a detector for trace quantitative determinations of pharmaceuticals in human plasma by capillary gas chromatography/mass spectrometry. A novel acetylcholinesterase inhibitor (CI-1002) was selected as an illustrative example for the technique. When coupled with a selective solid-phase extraction, this approach was capable of quantifying as little as 34 pg (0.50 ng ml-1, RSD = 12.7%) of compound on the column, and the inter-run precision was typically 3-4% RSD over a 0.5-25 ng ml-1 linear range. The advantages and requirements of the technique, in addition to the prospects for improvements in the detection limit, are discussed.     

Capillary liquid chromatography/electrospray mass spectrometry for the separation and detection of catechins in green tea and human plasma

The separation and detection of biologically active green tea catechins has been accomplished using capillary liquid chromatography/electrospray mass spectrometry (cLC/ESI-MS). Microscale determination (approximately 20 ng) of all six catechins in a green tea infusion, and the most extensively studied catechin, (-)epigallocatechin gallate (EGCG), in human plasma is demonstrated by cLC/ESI-MS with selected ion monitoring of protonated molecular ions. The overall quality of the analysis is shown to be dependent on the use of a capillary column with a deactivated, monomeric C18 stationary phase. The high chromatographic separation efficiency of this packed-capillary column, combined with the high sensitivity and selectivity afforded by the mass spectrometer as detector, provide a reliable approach to the analysis of picomolar quantities of these interesting compounds in complex matrices.     

Quantitative subfemtomole analysis of alpha-tocopherol and deuterated isotopomers in plasma using tabletop GC/MS/MS

A rapid, high-selectivity method with subfemtomole sensitivity is reported for quantification of alpha-tocopherol in plasma-based gas chromatography/tandem mass spectrometry (GC/MS/MS) using a tabletop quadrupole ion trap mass spectrometer. Sample workup is rapid, consisting of protein precipitation followed by liquid/liquid extraction and O-trimethylsilyl derivatization of alpha-tocopherol (alpha-T-TMS) and an internal standard, 2,2,5,7,8-pentamethyl-6-chromanol (PC-TMS). Rudimentary chromatography was carried out using an 8-m DB-5 capillary column resulting in an analyte retention time of 7.2 min. No interferences from the plasma matrix were observed. The assay has a detection limit of 178 amol (89.6 fg) and a lower limit of quantification of 700 amol (350 fg) of derivatized alpha-tocopherol in diluted plasma; < 30 pL of plasma is estimated to yield sufficient alpha-tocopherol for quantitative analysis at typical concentrations found in humans. A calibration curve constructed from National Institute of Standards and Technology serum standards was linear in the working range of 1.9-1073 ng/mL (0.95-0.54 ng). Within- and between-day precision averaged 5.8% and did not exceed 11.3% for three concentrations of quality control (QC) solutions. The overall accuracy for the QC samples was within 7.2%. Storage studies showed that, alpha-T-TMS and PC-TMS are stable under conditions that might be encountered during analyses. In a test study, plasma kinetic curves for alpha-tocopherol-d6 and alpha-tocopherol-d3 were obtained for a catheterized pregnant ewe and her fetus who were simultaneously given a bolus injection of alpha-tocopherol-d6, to the ewe and alpha-tocopherol-d3 to the fetus. These data show that a tabletop GC ion trap can determine alpha-T-TMS and its isotopomers quantitatively at high selectivity in a complex matrix.     

C-terminal peptide sequencing via multistage mass spectrometry

Results are presented showing the ability to obtain C-terminal sequence information from peptides by multiple stages of mass spectrometry. Under typical low-energy collision-induced dissociation conditions of quadrupole ion trap and ion cyclotron resonance mass spectrometers, lithium- and sodium-cationized peptides dissociate predominantly by reaction at the C-terminal peptide bond or an adjacent bond. For the majority of cases studied, the dominant reaction is a rearrangement process that results in the loss of the C-terminal residue and formation of a product ion that is one amino acid shorter than the original peptide ion. Using the multistage MS/MS capabilities of quadrupole ion trap and ion cyclotron resonance mass spectrometers, a subsequent stage of MS/MS can be performed to determine the identity of the new C-terminal residue. Up to eight stage of MS/MS have been performed with both quadrupole ion trap and ion cyclotron resonance mass spectrometers. In general, the same dissociation pathways are observed with both instruments, although occasionally there are significant differences in the branching ratios of competing pathways.     

Structural assignment of permethylated oligosaccharide subunits using sequential tandem mass spectrometry

The sequential tandem mass spectrometry (MSn) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of permethylated oligosaccharides. Under collision-induced dissociation, protonated molecular species generated in the electrospray ionization mode yield simple and predictable mass spectra. Information on sequence, branching, and, to some extent, interglycosidic linkages can be deduced from fragments resulting from the cleavage of glycosidic bonds. Simple rules for the structural assignment of carbohydrates have been established for the fragmentation of protonated species and subunits thereof and corroborated by 18O-labeling experiments. Moreover, sequential tandem mass spectrometry was demonstrated to allow the straightforward structural characterization of unknown carbohydrate moieties by comparing their CID spectra with those of a set of references. As the collision-induced dissociation patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by match of the spectra. These findings establish the basis of MSn performed on a quadrupole ion trap instrument for elucidating structures of large carbohydrates, which can be virtually degraded in the mass spectrometer into smaller entities in one or several steps. This powerful technique has been applied, used in conjunction with specific CD3 labeling, to the characterization of series of subunits generated from fucosylated and sialylated oligosaccharides, which are among the most important structures as far as biological activities are concerned.     

Development of a nanoscale liquid chromatography/electrospray mass spectrometry methodology for the detection and identification of DNA adducts

In this work, the coupling of liquid nanochromatography to NanoFlow electrospray mass spectrometry was evaluated for the detection of DNA adducts. The NanoFlow ES LC/MS system was compared with the capillary and conventional ES LC/MS system by analyzing an in vitro reaction mixture resulting from the interaction of 2'-deoxyguanosine 5'-monophosphate with bisphenol A diglycidyl ether and by injecting 2'-deoxyadenosine. By using NanoFlow ES LC/MS, the mass sensitivity could be improved by a factor of 3300. Three different injection methods used in liquid nanochromatography, i.e., split, large-volume, and column-switching injections were compared in terms of sensitivity. Furthermore, NanoFlow ES LC/MS was used to detect 2'-deoxynucleotide adducts isolated from an in vitro mixture of calf thymus DNA and bisphenol A diglycidyl ether. Different 2'-deoxynucleotide adducts could be identified by monitoring typical product ions, diagnostic for the adducts.     

CD45RC isoforms define two types of CD4 memory T cells, one of which depends on persisting antigen

A method to directly identify proteins contained in mixtures by microcolumn reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) is studied. In this method, the mixture of proteins is digested with a proteolytic enzyme to produce a large collection of peptides. The complex peptide mixture is then separated on-line with a tandem mass spectrometer, acquiring large numbers of tandem mass spectra. The tandem mass spectra are then used to search a protein database to identify the proteins present. Results from standard protein mixtures show that proteins present in simple mixtures can be readily identified with a 30-fold difference in molar quantity, that the identifications are reproducible, and that proteins within the mixture can be identified at low femtomole levels. Based on these studies, methodology has been developed for direct LC/MS/MS analysis of proteins enriched by immunoaffinity precipitation, specific interaction with a protein-protein fusion product, and specific interaction with a macromolecular complex. The approach described in this article provides a rapid method for the direct identification of proteins in mixtures.     

A microscale electrospray interface incorporating a monolithic, poly(styrene-divinylbenzene) support for on-line liquid chromatography/tandem mass spectrometry analysis of peptides and proteins

A methodology is described for creating a monolithic chromatography support within a pulled fused-silica electrospray needle. The monolith was formed from a mixture of styrene, divinylbenzene, 1-dodecanol, and toluene using 2,2'-azobis(isobutyronitrile) as the catalyst. The mixture was loaded into 150-micron-i.d. fused-silica capillary tubing with a pulled 5-10-micron needle tip at one end. Polymerization at 65 degrees C followed by removal of the porogen material yielded a stable, porous, monolithic support which had excellent properties for the separation and on-line, electrospray, mass spectrometry analysis of peptides and proteins. The performance of the monolith-filled electrospray needles was compared with similar needles filled with commercial C18 silica and polymeric particulate supports. Separation efficiencies for both protein and peptide mixtures were generally equal to or better than the particulate supports at comparable pressures and flow rates. The ion chromatograms derived from the on-line MS analysis were remarkably free from chemical background signals that often complicate the LC/MS analysis of femtomole amounts of sample. Good sequence coverage was obtained by LC/MS/MS analysis of the peptide mixture obtained from a protein isolated by silver-stained gel electrophoresis. The capability of the monolith to do peak parking experiments was demonstrated by the characterization of an immunoreactive HPLC fraction. The simple fabrication method, chromatographic performance, and robust nature of these microscale integrated column electrospray sources make them ideally suited for high-sensitivity tandem LC/MS analyses.     

Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometry is shown to be a powerful tool for the elucidation of protein modifications. Low-energy covalent bonds that originate from certain posttranslational modifications dissociate preferentially to produce characteristic mass spectrometric signatures that prove useful for the accurate, confident identification and characterization of such modifications. Because the MALDI ion trap is an authentic tandem mass spectrometer, it proves feasible to acquire secondary information to test hypotheses as to the nature and site of the putative modifications--further increasing the reliability of the tool. The method combines the advantageous features of MALDI (i.e., the ability to measure the same sample repeatedly, to measure unfractionated complex mixtures without the need for sample cleaning, and to determine peptide mixtures with subpicomole sensitivity) with the ease and the speed of the ion trap measurement. We demonstrate how the unique properties of MALDI ion trap MS can be used to address problems involving the determination of both native posttranslational modifications of proteins (e.g., disulfide mapping, glycosylation determination, and phosphorylation determination) and non-native chemical modifications of proteins (e.g., methionine oxidation and photo-cross-linking of proteins with DNA).     

ESI/ion trap/ion mobility/time-of-flight mass spectrometry for rapid and sensitive analysis of biomolecular mixtures

An ion trap/ion mobility/time-of-flight mass spectrometry technique is shown to be a rapid and sensitive means of analyzing peptide/protein mixtures. In this approach, an ion trap is used to accumulate ions that have been electrosprayed from a mixture into concentrated packets. The ion packets are injected into a drift tube where components of the mixture are separated based on differences in mobility through a buffer gas. Ions that exit the drift tube are dispersed in a time-of-flight mass spectrometer for mass-to-charge (m/z) determination. The gas-phase separation strategy reduces congestion in the mass spectrum, and experimental mobilities complement m/z measurements in assigning peaks. Examples of the application of the approach to identification of peptides (from tryptic digests) and to separation of charge-state distributions from electrospray of a mixture containing ubiquitin and myoglobin are presented. Most peptides that are observed from tryptic digests of proteins such as cytochrome c and myoglobin can be identified from data that are acquired in under 1 min; studies of mixtures with known compositions indicate that detection limits are approximately 0.5-3 pmol for individual components. Factors that may influence the distributions that are observed, such as storage time in the trap, injection voltages used for the mobility experiment, and variations in ion cross section with charge state, are discussed.