Delayed extraction time-of-flight MALDI mass spectrometry of proteins above 25,000 Da

Matrix-assisted laser desorption/ionization (MALDI) with delayed extraction (DE) has been optimized for mass analysis of high-mass proteins and glycoproteins with masses above 25,000 Da. Under optimized experimental conditions, i.e. using a weak extraction field strength and a long delay time, a steep drop in mass resolution above 30,000 Da is no longer observed and an improvement of more than a factor of 10 is obtained compared with the non-DE case, at least up to 66 kDa in a 1.2 m time-of-flight mass analyzer. On this level of resolution the factors limiting further improvements become apparent, i.e. adduct ion formation between matrix and analyte, but also cationization and further non-matrix-related adducts, as well as prompt fragmentation. Moreover, heterogeneity of the sample is often the reason for the detection of broad signals for larger proteins. Within these limitations, DHBs (a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid) gave by far the best results.     

Peptide and protein identification by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay time-of-flight mass spectrometry

The potential of matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay (PSD) time-of-flight mass spectrometry for the characterization of peptides and proteins is discussed. Recent instrumental developments provide for levels of sensitivity and accuracy that make these techniques major analytical tools for proteome analysis. New software developments employing protein database searches have greatly enhanced the fields of application of MALDI-PSD. Peptides and proteins can be easily identified even if only a partial sequence information is determined. Derivatization procedures have been optimized for MALDI-PSD to increase the structural information and to obtain a complete peptide sequence even in critical cases. They are fast, simple and can be performed on target. MALDI-PSD is also a very powerful tool to characterize or elucidate post-translational or chemically induced modifications. In association with database searches, proteins issued from electrophoretic gels can be identified after specific enzymatic cleavages and peptide mapping.     

Effects of protein-surface interactions on protein ion signals in MALDI mass spectrometry

The influence of polymer surface-protein binding affinity on protein ion signals in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is examined. The surfaces of poly(vinylidene fluoride) and poly(ethylene terephthalate) polymer substrates are modified by pulsed rf plasma deposition of allylamine. By varying the on/off duty cycle of the pulsed rf plasma, the polymer substrate surfaces are coated with thin films having varying densities of surface amine groups. The varying surface amine density is shown to lead to systematic changes in the surface binding affinity for the 125I-radiolabeled peptides angiotensin I and porcine insulin. Unlabeled angiotensin I and porcine insulin are then deposited on the pulsed rf plasma-modified substrates and analyzed by MALDI mass spectrometry. The experimental approach involves applying the peptide to the modified polymer surface in an aqueous phosphate-buffered saline solution and allowing the peptide solution to dry completely under ambient conditions. Subsequently, the MALDI matrix alpha-cyano-4-hydroxycinnamic acid in methanol and 10% trifluoroacetic acid in water are added to the peptide-coated modified polymer surfaces. The results of these studies demonstrate that, for the sample preparation method employed, increases in the surface peptide binding affinity lead to decreases in the peptide MALDI ion signal.     

DNA sequence analysis by MALDI mass spectrometry

Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products. Gel casting and electrophoresis are the time limiting steps, and the gel separation is occasionally imperfect due to aberrant mobility of certain fragments, leading to erroneous sequence determination. Furthermore, illegitimately terminated products frequently cannot be distinguished from correctly terminated ones, a phenomenon that also obscures data interpretation. In the present work the use of MALDI mass spectrometry for sequencing of DNA amplified from clinical samples is implemented. The unambiguous and fast identification of deletions and substitutions in DNA amplified from heterozygous carriers realistically suggest MALDI mass spectrometry as a future alternative to conventional sequencing procedures for high throughput screening for mutations. Unique features of the method are demonstrated by sequencing a DNA fragment that could not be sequenced conventionally because of gel electrophoretic band compression and the presence of multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the sequence was deduced, and the nature of the non-specific termination could be determined. The method described here increases the fidelity in DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation.     

Impact energy measurement in time-of-flight mass spectrometry with cryogenic microcalorimeters

Time-of-flight mass spectrometry-most notably matrix-assisted laser-desorption-ionization time-of-flight (MALDI-TOF) spectrometry-is an important class of techniques for the study of proteins and other biomolecules. Although these techniques provide excellent performance for masses up to about 20,000 daltons, there has been limited success in achieving good mass resolution at higher masses. This is because the sensitivity of the microchannel plate (MCP) detectors used in most systems decreases rapidly with increasing particle mass, limiting the utility of MCP detectors for very large masses. It has recently been proposed that cryogenic particle detectors may provide a solution to these difficulties. Cryogenic detectors measure the thermal energy deposited by the particle impact, and thus have a sensitivity that is largely independent of particle mass. Recent experiments have demonstrated the sensitivity of cryogenic particle detectors to single biomolecules, a quantum efficiency several orders of magnitude larger than the MCP detectors, and sensitivity to masses as large as 750,000 daltons. Here we present results demonstrating an order of magnitude better energy resolution than previous measurements, allowing direct determination of particle charge state during acceleration. Although application of these detectors to practical mass spectrometry will require further development of the detectors and cryogenics, these detectors can be used to elucidate the performance-limiting processes that occur in such systems.     

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.     

Multi-anode detection in electrospray ionization time-of-flight mass spectrometry

An electrospray ionization ion source coupled to a time-of-flight mass analyzer incorporating a multi-anode time-to-digital converter is described. High-speed data acquisition (kHz mass spectral acquisition) rates are achieved. The four-anode detector produces a significant increase in detection/counting efficiency over that for a single-anode detector. In this work a 2.5 times increase in detection efficiency is demonstrated. The multi-anode detector is also used as a diagnostic tool to optimize transmission of the ion optics.     

Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry

Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.     

Capillary electrophoresis/electrospray ionization high mass accuracy time-of-flight mass spectrometry for protein identification using peptide mapping

Capillary electrophoresis/electrospray ionization (CE/ESI) high mass accuracy time-of-flight mass spectrometry was used for the first time to characterize small proteins using peptide mapping. To identify small proteins, the intact proteins were first analyzed to obtain their average molecular weights with errors less than 1 Da. On-line capillary electrophoresis mass spectrometry of the tryptic digests of these small proteins was then performed to obtain the accurate molecular weights of the peptides with accuracies of approximately 10 ppm. Next, this information was used for the identification of the proteins using a protein database. It was found that high mass accuracy is an effective tool in reducing the list of most-likely proteins generated by the database. In addition, on-line collision-induced dissociation of the completely or partially resolved capillary electrophoresis peaks of the protein digests was used to unambiguously identify the sequences of these peptides. Each CE/ESI-MS analysis used only 5 nL of sample containing approximately 120 fmol of each peptide in protein digests. The results indicate that the combination of capillary electrophoresis and high resolution, high mass accuracy time-of-flight mass spectrometry is a viable option for the identification of small proteins using peptide mapping.     

Capillary electrochromatography of biomolecules with on-line electrospray ionization and time-of-flight mass spectrometry

Capillary electrochromatography (CEC) is considered a hybrid of liquid chromatography and capillary electrophoresis. It is expected to combine the high peak efficiency of capillary zone electrophoresis with the versatility and loading capacity of HPLC to bring about another high-performance MS-compatible chromatographic system. This paper explores the potential of CEC coupled with the electrospray ionization and time-of-flight mass spectrometry in biochemical analysis. The packed columns used in this study were tapered at the outlet to retain the packing material, thereby obviating the need for an outlet frit. Electrosmotically driven solvent gradients were employed for the separation of phenylthiohydantoin (PTH)-amino acids by reversed-phase chromatography, and a time-of-flight (TOF) mass spectrometer was employed as the detector for the CEC column effluent. The effect of CEC operating parameters, such as gradient shape, column length, and electric field, on the analytical results from the separation and MS detection of a standard mixture of PTH-amino acids was investigated. Particular attention was paid to the effect of sheath flow-rate, sheath composition and mass spectra acquisition rate on the performance of the electrospray TOF-MS.     

Electrospray ionization and collision-induced dissociation time-of-flight mass spectrometry of neutral glycosphingolipids

A series of native naturally occurring neutral glycosphingolipids has been analysed by electrospray ionization tandem mass spectrometry using a hybrid magnetic sector-TOF instrument. The collision-induced dissociation products of precursor ions were detected by an orthogonal acceleration time-of-flight mass spectrometer as the second analyser. Glycosphingolipids, with mono- to hexa-saccharide chain lengths with different ceramide constituents, were studied. The result of electrospray ionization in the positive ion mode generally showed singly charged molecular ions with Na+ as adduct, [M + Na]+. The sensitivity of the electrospray ionization was greatly enhanced by addition of NaCl, LiCl (forming [M + Li]+) or KCl (yielding [M + K]+) to the sample. A comparison between the collision-induced dissociation of precursor molecular ions of monoglycosylceramides, using Na+, Li+ and K+ as adducting species, showed that the intensity of the fragment ions and the extent of the daughter ion fragmentation of the molecular ions, are dependent on the type of adduct used. The daughter ion spectra of Li+ adduct ions showed intense sequence fragment ions, both of the saccharide chain and the ceramide moiety, and were superior to those obtained using Na+ or K+. The collision-induced dissociation spectra of the [M + Li]+ ions, of glycosphingolipids containing di- to hexasaccharides, are also presented. Proposed possible fragments, resulting from the CID of the molecular ions [M + Li]+ of monoglycosylceramides, are shown.     

Exploring infrared wavelength matrix-assisted laser desorption/ionization of proteins with delayed-extraction time-of-flight mass spectrometry

We report a study of the application of delayed extraction (DE) to infrared-wavelength matrix-assisted time-of-flight mass spectrometry (IR-MALDI-TOF-MS) of proteins. The shapes of the spectral peaks obtained with DE-IR-MALDI-MS are compared with those obtained from the same samples and matrix using continuous extraction (CE) IR-MALDI-MS. Application of DE results in significant improvements in the peak resolution, revealing spectral features (in proteins with molecular masses < 12 kDa) that were not resolved in the corresponding CE-IR-Maldi mass spectra. Particularly significant is a series of peaks on the high mass side of the protonated protein peaks that arise through replacement of protons by adventitious sodium ions in the sample. We deduced that these sodium replacement species are a significant contributor to the broad tails (and resulting peak asymmetries) that are a feature of the DE-IR-MALDI mass spectra of proteins with molecular masses > or = 17 kDa. The peak width reduction observed in IR-MALDI by DE suggests that, as in UV-MALDI, the initial velocity distribution for ions produced in the MALDI process contributes to the peak broadness in the CE mass spectra. In a systematic comparison between DE UV-MALDI and DE IR-MALDI, we determined that photochemical matrix adduction is present in UV-MALDI but absent in IR-MALDI. In addition, we find that protein ions produced by IR irradiation are less internally excited (i.e., cooler), exhibiting less fragmentation, more Na+ replacement and/or unspecified noncovalent adduction, and more heme adduction with apomyoglobin. Thus, IR-MALDI appears to be a softer means for producing gas-phase protein ions than is UV-MALDI. It will be of considerable practical interest to determine whether large protein ions produced by IR-MALDI are sufficiently cool to survive transport through reflecting TOF mass spectrometers (without loss of small neutral species such as H2O, NH3, and CO2) and the extended time periods required for detection by quadrupole ion trap and Fourier transform ion cyclotron resonance mass analyzers.     

Procedures for tandem mass spectrometry on an ion trap storage/reflectron time-of-flight mass spectrometer

In this work, we demonstrate tandem mass spectrometry on an ion trap storage-reflectron time-of-flight mass spectrometer (IT/reTOFMS). Ion isolation and activation were achieved by resonant excitation using multi- and single-frequency waveforms generated from an analog circuit. Product-ion spectra of small polypeptides are obtained, which are comparable in fragmentation to those acquired on sector or hybrid mass spectrometers. Several important parameters governing the tandem mass spectrometry process, including the activation tickle voltage, type of collision gas, activation period and cooling period after the fragmentation were optimized using leucine-enkephalin as a model. Although the limited energy deposition from collisional activation in our experiments does not allow efficient fragmentation of large singly charged polypeptides with m/z higher than 1000, the problem may be partially solved by taking advantage of fragmenting the multiply charged ions produced from the electrospray ionization source as demonstrated for a synthetic polypeptide of molecular weight 2782. Compared to the singly charged form, the reduced m/z of multiply charged forms experience a greater trapping force as described by the pseudopotential well-depth model. Increased pseudopotential well-depths for multiply charged species permit the use of greater fragmentation energy at lower RF potentials. These conditions facilitate the fragmentation of large polypeptides, yet are suitable for trapping singly charged fragments. These experiments indicate that the high efficiency associated with ion dissociation and fragment-ion collection in the trap and the storage capability for detection of ions using the non-scanning mode of the IT/ reTOF analyzer may provide an alternative means for acquiring sequence-specific information of polypeptides at low picomol levels of sensitivity.     

Speciation of arsenic oxides using laser desorption/ionization time-of-flight mass spectrometry

Positive and negative ion mass spectra of arsenic trioxide (As2O3) and arsenic pentaoxide (As2O5) have been obtained by single-step laser desorption/ionization time-of-flight mass spectrometry. Pulsed UV radiation at 266 nm was used for the simultaneous desorption and ionization of the solid sample. High-mass cluster ions that are unique to the oxidation state of each oxide sample appear in the negative ion mass spectra. The As2O3 produces As3O5-, while the As2O5 yields As3O8-. The formation of unique negative cluster ions presents the capability for arsenic oxidation state speciation by laser desorption/ionization mass spectrometry. The ability of time-of-flight mass spectrometry to examine the relative amounts of each arsenic oxide present in a series of mixtures is discussed. Application of our speciation technique to a model incinerator sample is demonstrated.     

Identification of urinary oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A new method of urinary oligosaccharides identification by matrix-assisted laser desorption time-of-flight mass spectrometry is presented. The method involves three steps: coupling of the urinary oligosaccharides with 8-aminonaphthalene-1,3,6-trisulfonic acid; fast purification over a porous graphite carbon extraction column; and mass spectrometric analysis. Identification of urinary oligosaccharides is based on the patterns and values of the pseudomolecular ions observed. We report here the patterns in urines from patients with Pompe disease, alpha and beta mannosidoses, galacto-sialidosis, and GM1 gangliosidosis. The protocols described here allowed facile and sensitive identification of the pathognomonic oligosacchariduria present in lysosomal diseases and can be extended to any pathological oligosacchariduria.     

Monitoring protein refolding induced by disulfide formation using capillary isoelectric focusing-electrospray ionization mass spectrometry

Rapid growth in the biotechnology industry has led to a dramatic increase in attention to the protein folding problem. Understanding protein-folding pathways is essential to the production of biopharmaceuticals since commercial production of recombinant proteins often requires a protein-refolding process for recovery of high yields. Protein folding coupled to the formation of disulfide bonds presents one of the simplest approaches to studying folding intermediates. On-line capillary isoelectric focusing-electrospray ionization mass spectrometry (CIEF-ESIMS) is demonstrated for kinetic studies of disulfide bond-induced protein refolding. Refolding intermediates of bovine pancreatic ribonuclease A, a model system for this study, are blocked at different stages by alkylating free thiols with iodoacetate. The alkylation reaction results in the introduction of charge (-1) and mass (59) differences for each alkylation site, providing the means for predictable separation and direct identification of refolding intermediates using CIEF-ESIMS. Besides the observation of refolding intermediates containing different numbers of disulfide bonds and even mixed disulfides, the two-dimensional resolving power of CIEF-ESIMS allows the determination of conformational heterogeneity among groups of refolding intermediates.     

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).     

Determination of insulin content in pancreatic beta cell line MIN6 cells by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The insulin content in mouse insulinoma MIN6 cells was determined using matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOF/MS). A mass spectrum of cellular insulin was obtained with a cell burst of MIN6 by hypotonic water. Signal intensities of intracellular insulin were proportional to the number of MIN6 cells. The present method was applied to the determination of intracellular insulin content of MIN6 before and after glucose stimulation.     

Rapid peptide mapping by reversed-phase liquid chromatography on nonporous silica with on-line electrospray time-of-flight mass spectrometry

Reversed-phase liquid chromatography (LC) using a nonporous silica support has been combined with electrospray (ES) time-of-flight (TOF) mass spectrometry (MS) for the fast separation and mass detection of peptides. Using this LC method, the resolution of a peptide mixture can be completed is less than 35 s. The resulting chromatographic peak widths are less than 1 s wide. Because of the unique nature of a TOF mass analyzer, complete mass spectra can be acquired at a rate which is sufficient to sample these narrow peaks. When compared with conventional LC, the same separation takes nearly 20 min to complete, and the signal-to-noise ratio observed in the total ion chromatogram is dramatically lower due to the influence of increased background noise in the mass spectra. The limit of detection for a low molecular weight peptide, Val-Pro-Leu, was found to be 6 pmol with the total ion chromatogram and 500 fmol with the reconstructed ion chromatogram. A peptide map of horse heart myoglobin, completed in 3.5 min, is shown as an example of the results which can be obtained from combining this fast LC method with fast ES/TOF/MS detection capability.