Preparative separation of mixtures by mass spectrometry

A specially designed mass spectrometer which allows for preparative separation of mixtures is described. This mass spectrometer allows for large ion currents, on the order of nanoamperes, to be produced by electrospray and transmitted into a high vacuum. Accumulation of nanomole quantities of collected and recovered material in several hours is demonstrated. The use of high-velocity ions reduces space charge effects at high ion currents. Separation of mass occurs simultaneously for all ions, providing a 100% duty cycle. The use of a linear dispersion magnet avoids compression at higher m/z ratios. A deceleration lens slows the ions to allow for soft landing at low kinetic energy. The ions are neutralized by ion pairing on an oxidized metal surface. Retractable landing plates allow for easy removal of the separated components.     

Chiral morphing and enantiomeric quantification in mixtures by mass spectrometry

A novel mass spectrometric method is introduced for rapid and accurate chiral quantification by examining a tetracoordinated transition metal complex into which a reference and a fixed ligand are incorporated simultaneously with the analyte. Chiral analysis is performed by measuring the dissociation kinetics of these trimeric cluster ions [(M(II) + L(fixed) - H)(ref)(An)]+ (M(II) = a transition metal ion, L(fixed) = chiral peptide fixed ligand, ref = chiral reference ligand, and An = chiral analyte) in an ion trap mass spectrometer. The ratio of the product ion branching ratios measured when a pair of pure chiral fixed ligands and chiral reference ligands (/ref(D) and /ref(L); or /ref(L) and /ref(D)) are employed in separate experiments is related, via the kinetic method formalism, to the enantiomeric composition of the chiral mixture. This fixed-ligand quotient ratio (QR(fixed)) is logarithmically proportional to enantiomeric purity allowing construction of a calibration curve for chiral analysis when the analyte is only available in one form of known optical purity. There are reciprocal relationships when switching the chirality of the fixed/reference ligands. Improved quantification accuracy (due to simplified dissociation kinetics) and ready construction of two or more single-point calibration curves allow data to be cross-checked and represent an advantage of this approach. These features and the matrix tolerance of the kinetic method are demonstrated using the QR(fixed) method for determinations of enantiomeric excess of the drug DOPA in the presence of the co-drug compound L-carbidopa. The chiral selectivity of DOPA was found to vary from 0.0581 to 0.337 using this method, depending on the choices of fixed-ligand and reference chirality. The average relative errors are less than 1.2%. The potential of chiral morphing (changing chiral centers in the ligands) to further refine the chiral interactions and hence to maximize chiral recognition is shown.     

Processing complex mixtures of intact proteins for direct analysis by mass spectrometry

For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl sulfate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5-20 components (all within approximately 5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of approximately 400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of approximately 19,000 protein forms. This ALS-PAGE/RPLC approach to proteome processing ameliorates the "front end" problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.     

Quantitation of oligonucleotides by phosphodiesterase digestion followed by isotope dilution mass spectrometry: proof of concept

The importance of DNA as a regulatory analyte is well-known. Recent years have seen an increased interest in the quantitation of this analyte. Accurate quantitative measurements have been hampered by the lack of well-characterized standards and pure materials for this large-molecular-weight analyte. Outlined here is an approach for the accurate and reproducible quantitation of an oligonucleotide that is solely reliant on the availability of pure, well-characterized deoxynucleotides and not a sequence-specific pure DNA standard. The proposed procedure is intended to provide an accurate and definitive method for the quantitation of DNA for reference measurements as an improved alternative to the more conventional UV absorbance-based methods. For proof of concept, a gravimetrically prepared oligonucleotide solution was enzymatically digested to its constituent monomer-deoxynucleotide monophosphates (dNMPs), of which there are four different types. Qualitative mass spectrometry was used to confirm the 100% successful completion of the enzymatic digestion step. The dNMPs were then separated by liquid chromatography (LC) before being detected by electrospray ionization (ESI) mass spectrometry (MS). The method of quantitation was based on isotope dilution mass spectrometry (IDMS) analysis of the four different monomer units. The concentrations of the four dNMP residues were then summed to obtain the original concentration of the oligonucleotide. The concentrations determined by liquid chromatography/mass spectrometry (LC/MS) and also by liquid chromatography-tandem mass spectrometry (LC/MS/MS) differed by <2.5 and 1%, respectively, from the gravimetrically assigned value. These differences were well within the uncertainty of the gravimetrically assigned value. This highly accurate method, suitable for the definitive quantitation of oligonucleotides, should be ideal for characterizing primary calibration standards and certified reference materials that can then be used to underpin the more conventional quantitative techniques of UV and fluorescence spectroscopy.     

Identification of microbial mixtures by capillary electrophoresis/selective tandem mass spectrometry

In this paper, we propose a new strategy for identifying specific bacteria in bacterial mixtures by using CE-selective MS/MS of peptide marker ions associated with the bacteria of interest. We searched the CE-MS/MS spectra acquired from the proteolytic digests of pure bacterial cell extracts against protein databases. The identified peptides that match the protein associated with the corresponding species were selected as marker ions for bacterial identification. Specific peptide marker ions were obtained for each of the following three pathogens: Pseudomonas aeruginasa, Staphylococcus aureus, and Staphylococcus epidermidis. To identify a bacterial species in a sample, we performed CE-MS/MS analysis of the selected marker ions in the proteolytic digest of the cell extract and then performed protein database searches. The selected peptides that we identified correctly from Xcorr values ranking at the top of the search results allowed us to identify the corresponding bacterial species present in the sample. We have applied this method successfully to the identification of various mixtures of the three pathogens. Even minor bacterial species present at a concentration of 1% can be identified with great confidence. This method for CE-MS/MS analysis of bacteria-specific marker peptides provides excellent selectivity and high accuracy when identifying bacterial species in complex systems. In addition, we have used this approach to identify P. aeruginasa in a saliva sample spiked with E.coli and P. aeruginasa.     

Detection of malaria parasites in blood by laser desorption mass spectrometry

A novel method for the in vitro detection of the protozoan Plasmodium, the causative agent of malaria, has been developed. It comprises a protocol for cleanup of whole blood samples, followed by direct ultraviolet laser desorption (LD) time-of-flight mass spectrometry. Intense ion signals are observed from intact ferriprotoporphyrin IX (heme), sequestered by malaria parasites during their growth in human red blood cells. The LD mass spectrum of the heme is structure-specific, and the signal intensities are correlated with the sample parasitemia (number of parasites per unit volume of blood). Parasitemia levels on the order of 10 parasites/microL blood can be unambiguously detected by this method. Consideration of laser beam parameters (spot size, rastering across the sample surface) and actual sample consumption suggests that the detection limits can be further improved by at least an order of magnitude. The influence of experimental factors, such as desorbed ion polarity, laser exposure and fluence, sample size, and parasite growth stage, on the threshold for parasite detection is also addressed.     

Detection of plasmid insertion in Escherichia coli by MALDI-TOF mass spectrometry

A proteomics approach is reported for the rapid recognition of genetically modified Escherichia coli bacteria. The approach targets a class of proteins required for genetic manipulation of bacteria with plasmids and alleviates the need to construct extensive libraries of toxins and other predicted payload proteins. Detection was performed using MALDI-TOF MS to monitor peptide products after an on-probe enzymatic digestion. Digestion products were identified by searching their postsource decay spectra using MASCOT. A 5 min digestion time was required to observe peptide products from the genetic insert as well as the host bacterium. This proteomics approach enables rapid detection of genetic manipulation along with information about the host organism, both of which have forensic applications.     

Qualitative and semiquantitative analysis of composite mixtures of antibodies by native mass spectrometry

Native mass spectrometry was evaluated for the qualitative and semiquantitative analysis of composite mixtures of antibodies representing biopharmaceutical products coexpressed from single cells. We show that by using automated peak fitting of the ion signals in the native mass spectra, we can quantify the relative abundance of each of the antibodies present in mixtures, with an average accuracy of 3%, comparable to a cation exchange chromatography based approach performed in parallel. Moreover, using native mass spectrometry we were able to identify, separate, and quantify 9 antibodies present in a complex mixture of 10 antibodies, whereas this complexity could not be unraveled by cation exchange chromatography. Native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and semiquantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and the relative abundance through comparison of relative ion signal intensities. Native mass spectrometry is a particularly effective tool for characterization of heterogeneous biopharmaceutical products such as bispecific antibodies and antibody mixtures.     

Analysis of microbial mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Many different laboratories are currently developing mass-spectrometric techniques to analyze and identify microorganisms. However, minimal work has been done with mixtures of bacteria. To demonstrate that microbial mixtures could be analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), mixed bacterial cultures were analyzed in a double-blind fashion. Nine different bacterial species currently in our MALDI-MS fingerprint library were used to generate 50 different simulated mixed bacterial cultures similar to that done for an initial blind study previously reported (Jarman, K. H.; Cebula, S. T.; Saenz, A. J.; Petersen, C. E.; Valentine, N. B.; Kingsley, M. T.; Wahl, K. L. Anal. Chem. 2000, 72, 1217-1223). The samples were analyzed by MALDI-MS with automated data extraction and analysis algorithms developed in our laboratory. The components present in the sample were identified correctly to the species level in all but one of the samples. However, correctly eliminating closely related organisms was challenging for the current algorithms, especially in differentiating Serratia marcescens, Escherichia coli, and Yersinia enterocolitica, which have some similarities in their MALDI-MS fingerprints. Efforts to improve the specificity of the algorithms are in progress.     

Quantitative sequencing of complex mixtures of heterochitooligosaccharides by vMALDI-linear ion trap mass spectrometry

Heterochitooligosaccharides possess interesting biological properties. Isobaric mixtures of such linear heterochitooligosaccharides can be obtained by chemical or enzymatic degradation of chitosan. However, the separation of such mixtures is a challenging analytical problem which is so far unresolved. It is shown that these isobaric mixtures can be sequenced and quantified simultaneously using standard derivatization and multistage tandem mass spectrometric techniques. A linear ion trap mass spectrometer equipped with a vacuum matrix-assisted laser desorption ionization (vMALDI) source is used to perform MS2 as well as MS3 experiments.     

Accurate mass multiplexed tandem mass spectrometry for high-throughput polypeptide identification from mixtures

We report a new tandem mass spectrometric approach for the improved identification of polypeptides from mixtures (e.g., using genomic databases). The approach involves the dissociation of several species simultaneously in a single experiment and provides both increased speed and sensitivity. The data analysis makes use of the known fragmentation pathways for polypeptides and highly accurate mass measurements for both the set of parent polypeptides and their fragments. The accurate mass information makes it possible to attribute most fragments to a specific parent species. We provide an initial demonstration of this multiplexed tandem MS approach using an FTICR mass spectrometer with a mixture of seven polypeptides dissociated using infrared irradiation from a CO2 laser. The peptides were added to, and then successfully identified from, the largest genomic database yet available (C. elegans), which is equivalent in complexity to that for a specific differentiated mammalian cell type. Additionally, since only a few enzymatic fragments are necessary to unambiguously identify a protein from an appropriate database, it is anticipated that the multiplexed MS/MS method will allow the more rapid identification of complex protein mixtures with on-line separation of their enzymatically produced polypeptides.     

Chiral analysis by electrospray ionization mass spectrometry/mass spectrometry. 1. Chiral recognition of 19 common amino acids

Chiral recognition of 19 common amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed from the electrospray ionization of amino acids in the presence of one of the following chiral selectors: L- or D-N-tert-butoxycarbonylphenylalanine, L- or D-N-tert-butoxycarbonylproline, and L- or D-N-tert-butoxycarbonyl-O-benzylserine. The protonated trimers were dissociated to protonated dimers, and the intensity ratios of the protonated dimer (product ion) to the protonated trimer (precursor ion), i.e., the observed dissociation efficiency, was found to be strongly dependent on the chirality of the amino acids with respect to that of the chiral selectors. The results showed that the chirality of all 19 common amino acids can be definitely differentiated. The method was demonstrated as rapid, sensitive, precise, robust, and requiring no reference standards and only minimal sample preparation. The chirality of all three amino acids in a mixture was determined without prior separation of the amino acids, consuming only 70 pmol of sample and requiring only approximately 14 min of mass spectrometric measurements. A cyclodipeptide with unknown chirality was determined to be cyclo-(L-Pro-L-Leu) by acid hydrolysis followed by the present method, and the results were consistent with the physiochemical properties and NMR data of the compound. This study suggested that ESI-MS/MS can be a promising approach for the chiral recognition of other compounds.     

Detection and quantification of anthrax lethal factor in serum by mass spectrometry

The lethal toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to an optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. anthracis. Serum samples obtained at day 2 postinfection contained 30-250 ng/mL LF and illustrated the clear potential to detect LF earlier in the infection cycle. This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax.     

Improving spot homogeneity by using polymer substrates in matrix-assisted laser desorption/ionization mass spectrometry of oligonucleotides

We describe a method for improving the homogeneity of MALDI samples prepared for analysis of small, single-stranded oligonucleotides using the widely used DNA matrix system, 3-hydroxypicolinic acid/picolinic acid/ ammonium citrate. This matrix system typically produces large crystals around the rim of the dried sample and requires tedious searching of this rim with the laser. However, when a substrate is prepared using both Nafion and a hydrophilic, high-molecular-weight polymer, such as linear polyacrylamide, linear poly(ethylene oxide), or methyl cellulose, oligonucleotide-doped matrix crystals tend to be smaller and more uniformly distributed across the entire spot, thus decreasing the time that is required for locating a usable signal. In addition to MALDI characterization of the spatial distribution of "sweet spots," fluorescence microscopy allows for imaging dye-labeled DNA in dried MALDI spots. The mechanism of enhanced uniformity may involve increased viscosity in the MALDI sample droplet due to partial solubilization of the substrate by the MALDI sample solvent as well as partitioning of the matrix or DNA between the solvent and the undissolved portion of the polymer substrate.     

Detection limit of isotope dilution mass spectrometry

The detection limit is an important figure of merit for evaluating instrumentation and analytical methods. While the detection limit for techniques using linear calibration functions has been studied extensively, this fundamental metric has rarely been discussed for mass spectrometry that bases the calibration on the principle of isotope dilution. We have developed a formulation for the detection limit for isotope dilution mass spectrometry (IDMS) after a thorough analysis of the uncertainty of IDMS measurements. The new formulation describes the IDMS detection limit as a function of the enrichment of the isotopic spike and the linear calibration detection limits measured at the masses for the isotope ratio measurement.     

Detection of functional ricin by immunoaffinity and liquid chromatography-tandem mass spectrometry

The toxin ricin is a biological weapon that may be used for bioterrorist purposes. As a member of the group of ribosome-inactivating proteins (RIPs), ricin has an A-chain possessing N-glycosidase activity which irreversibly inhibits protein synthesis. In this paper, we demonstrate that provided appropriate sample preparation is used, this enzymatic activity can be exploited for functional ricin detection with sensitivity similar to the best ELISA and specificity allowing application to environmental samples. Ricin is first captured by a monoclonal antibody directed against the B chain and immobilized on magnetic beads. Detection is then realized by determination of the adenine released by the A chain from an RNA template using liquid chromatography coupled to tandem mass spectrometry. The immunoaffinity step combined with the enzymatic activity detection leads to a specific assay for the entire functional ricin with a lower limit of detection of 0.1 ng/mL (1.56 pM) after concentration of the toxin from a 500 microL sample size. The variability of the assay was 10%. Finally, the method was applied successfully to milk and tap or bottled water samples.     

Detection of double-stranded DNA by IR- and UV-MALDI mass spectrometry.

Double-stranded DNA ranging from 9 kDa to over 500 kDa were desorbed and analyzed by MALDI TOF mass spectrometry. IR-MALDI with glycerol as matrix yielded excellent results for larger double-stranded DNA by adjustment of the ionic strength through the addition of salts. Very little fragmentation and a routine sensitivity in the subpicomole range were observed in IR-MALDI when double-stranded analytes harboring 70 base pairs or more were probed. In the lower mass range (up to approximately 70 base pairs), UV-MALDI with 6-aza-2-thiothymine as matrix was the ionization method of choice because it allowed specific double-stranded complexes containing relatively few base pairs to be desorbed intactly. In this mode, an essentially quantitative detection of the double-stranded form was observed for a 70-mer. The UV-MALDI was accompanied by a significant fragmentation and a resulting reduced sensitivity and mass resolution. The methods described open MALDI-MS for the analysis of large DNA-DNA and DNA-protein complexes.     

Detection of trace impurities by time-of-flight mass spectrometry with laser-induced evaporation

A method for the determination of trace impurities of tens of ppm in solid materials is proposed, based on laser-induced evaporation of the substance in a vacuum in the forced congruence mode with simultaneous analysis of the evaporation products using a time-of-flight mass spectrometer. The results of measurements of the relative composition of impurities in α-corundum obtained from aluminum hydroxide (AlOOH) powder by high-temperature annealing (1500 and 1800°C) are presented. The necessity of comparative analysis is caused by a substantial difference in the measurement results for impurities in the initial material obtained by conventional methods. The reported values of the relative composition of impurities are in a good agreement with the results of inductively coupled plasma mass spectrometry.     

Ion-molecule reactions for the characterization of polyols and polyol mixtures by ESI/FT-ICR mass spectrometry

A mass spectrometric method is described for the identification and counting of hydroxyl groups in an analyte. Analytes introduced into a FT-ICR mass spectrometer and ionized by positive mode ESI were allowed to react with the neutral reagent diethylmethoxyborane. This results in derivatization of the hydroxyl groups of the analytes by replacement of a proton with a diethylborenium ion. Protonated polyols react by consecutive derivatization reactions, wherein all, or nearly all, of the hydroxyls are derivatized. The polyol derivatization products are separated by 68 mass units in the mass spectrum. This 68 Da mass shift, along with 30 Da mass shifts arising from intramolecular derivatization of the primary derivatization products, makes it easy to count the number of functional groups present in the analyte. The utility of this method for the analysis of polyols as single-component solutions, as mixtures, or in HPLC effluent (LC-MS analysis) is demonstrated.