A CE-MALDI interface based on the use of prestructured sample supports

We have developed an off-line coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) based on CE fraction collection onto prestructured MALDI sample supports. Analyte carryover and detection sensitivity were investigated using a standard peptide mixture. Low femtomole amounts were detected, and no noticeable carryover was discovered. The performance of the method was evaluated with a mixture of tryptic digests of proteins from a human fetal brain cDNA expression library. The total number of identified peptides was increased from 47 to 211 when the CE-MALDI interface was used compared to direct MALDI-MS analysis. Sequence coverage with CE-MALDI was in the 25-60% range for the different proteins, corresponding to an increase of 1.3-4.9 times relative to that obtained with MALDI-MS of the crude mixture. Fractionation of sample components also facilitated protein identification by MALDI postsource decay analysis. Our initial results suggest this CE-MALDI interface can be used for the analysis of complex peptide mixtures isolated from biological tissues.     

One-step procedure for peptide extraction from in-gel digestion sample for mass spectrometric analysis

Protein identification from samples resolved by one-dimensional and two-dimensional gel electrophoresis is highly dependent on the recovery of trypsin-digested peptides prior to mass spectrometric analysis. The commonly used two-step protocol for extracting tryptic peptides, involving high-volume organic solvent extraction and cleanup via microscale reversed-phase micropipet tip or microcolumn is not only limited by significant sample loss but is also costly and very labor-intensive. We report here a simple one-step procedure for simultaneous peptide extraction and cleanup by incubating a small piece of C18 Empore Disk (3M) with the in-gel digested solution. We show that the direct Empore Disk-based peptide extraction procedure is convenient, economical, and has higher efficiency as compared with the commonly used two-step protocol for peptide preparation prior to MS analysis.     

MALDI MS sample preparation by using paraffin wax film: systematic study and application for peptide analysis

Recently developed sample preparation techniques employing hydrophobic sample support have improved the detection sensitivity and mass spectral quality of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). These methods concentrate the samples on target by minimizing the sample area via the solvent repellent effect of the target surface. In the current study, we employed the use of paraffin wax film (Parafilm M) for improved MALDI MS analysis of low-abundance peptide mixtures, including neuronal tissue releasate and protein tryptic digests. This thin film was found to strongly repel polar solvents including water, methanol, and acetonitrile, which enabled the application of a wide range of sample preparation protocols that involved the use of various organic solvents. A "nanoliter-volume deposition" technique employing a capillary column has been used to produce tiny ( approximately 400 microm) matrix spots of 2,5-dihydroxybenzoic acid on the film. By systematically optimizing the sample volume, solvent composition, and film treatment, the Parafilm M substrate in combination with the nanoliter-volume matrix deposition method allowed dilute sample to be concentrated on the film for MALDI MS analysis. Peptide mixtures with nanomolar concentrations have been detected by MALDI time-of-flight and MALDI Fourier transform ion cyclotron resonance mass spectrometers. Overall, the use of Parafilm M enabled improved sensitivity and spectral quality for the analysis of complex peptide mixtures.     

Submicrosecond surface-induced dissociation of peptide ions in a MALDI TOF MS

Surface-induced dissociation (SID) has been implemented in a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI TOF MS), allowing production of tandem mass spectrometric information for peptide ions (MALDI TOF SID TOF). The instrument retains the standard operational modes such as the reflectron monitoring of the MALDI-generated intact ions and postsource decay. We show through ion trajectory simulations and experimental results that implementing SID in a commercial MALDI TOF spectrometer is feasible and that the SID products in this instrument fall in an observation time frame that allows the specific detection of fast-fragmentation channels. The instrument design, pulse timing sequence, and high-voltage electronics together with SID spectra of MALDI-generated peptide ions are presented. Standard peptides such as YGGFLR, angiotensin III, fibrinopeptide A, and des-Arg1-bradykinin were dissociated by means of hyperthermal collisions with a gold surface coated with a self-assembled monolayer of 2-(perfluorodecyl)ethanethiol. With the extraction fields and the short observation times used, the spectra obtained show intense low-mass ion signals such as immonium, b2, b3, and y2 ions. TOF data analysis involved matching simulated and experimental flight times and indicates that the observed fragments are produced at approximately 250 ns after the precursor ion collides with the surface. This submicrosecond gas-phase fragmentation time frame is complementary to the observation time frame of existing SID spectrometers, which are on the order of 10 micros for tandem quadrupoles and are larger than a few milliseconds for SID implemented in Fourier transform ion cyclotron resonance spectrometers.     

Bacillus spore identification via proteolytic peptide mapping with a miniaturized MALDI TOF mass spectrometer

An approach is tested here as a rapid screening method for Bacillus spore species employing bacterial peptide analysis with a miniaturized MALDI TOF mass spectrometer. A limited set of tryptic peptides was generated in situ following selective solubilization of the small, acid-soluble protein family (SASP) from spore samples on the MALDI sample holder. To facilitate species identification, a compact database was created comprising masses of the tryptic cleavage products generated in silico from all Bacillus and Clostridium SASPs whose sequences are available in public databases. Experimental measurements were matched against the custom-made database, and a published statistical model was then used to evaluate the probability of false identifications.     

Subtyping botulinum neurotoxins by sequential multiple endoproteases in-gel digestion coupled with mass spectrometry

Botulinum neurotoxin (BoNT) is one of the most toxic substances known. BoNT is classified into seven distinct serotypes labeled A-G. Among individual serotypes, researchers have identified subtypes based on amino acid variability within a serotype and toxin variants with minor amino acid sequence differences within a subtype. BoNT subtype identification is valuable for tracing and tracking bacterial pathogens. A proteomics approach is useful for BoNT subtyping since botulism is caused by botulinum neurotoxin and does not require the presence of the bacteria or its DNA. Enzymatic digestion and peptide identification using tandem mass spectrometry determines toxin protein sequences. However, with the conventional one-step digestion method, producing sufficient numbers of detectable peptides to cover the entire protein sequence is difficult, and incomplete sequence coverage results in uncertainty in distinguishing BoNT subtypes and toxin variants because of high sequence similarity. We report here a method of multiple enzymes and sequential in-gel digestion (MESID) to characterize the BoNT protein sequence. Complementary peptide detection from toxin digestions has yielded near-complete sequence coverage for all seven BoNT serotypes. Application of the method to a BoNT-contaminated carrot juice sample resulted in the identification of 98.4% protein sequence which led to a confident determination of the toxin subtype.     

Integrated sample processing system involving on-column protein adsorption, sample washing, and enzyme digestion for protein identification by LC-ESI MS/MS

An automated system has been developed for protein identification using mass spectrometry that incorporates sample cleanup, preconcentration, and protein digestion in a single stage. The procedure involves the adsorption of a protein or a protein mixture from solution onto a hydrophobic medium that is contained within a microcolumn. The protein is digested while still bound to the hydrophobic support. The peptides are then eluted from surface digestion to an electrospray ionization mass spectrometer for detection and sequencing. The entire system is fully automated wherein the mass spectrometer is collecting data continuously. We demonstrate that this system is capable of identifying standard protein samples at concentrations down to 100 nM. Further development of this technique may offer a potential solution for proteomics applications that require unattended operation, such as on-line monitoring and identification of microorganisms on the basis of the detection of their protein biomarkers.     

An on-line protein digestion device for rapid peptide mapping by electrospray mass spectrometry

A simple laboratory-constructed device has been developed for fast on-line protein digestion followed by peptide mapping by use of electrospray mass spectrometry. Taking advantage of its nonsolubility properties at near-neutral pH values, pepsin could be nonpermanently attached to the PEEK tube commonly employed as transfer capillary between the syringe and the electrospray ion source. After optimization of experimental conditions such as pH, solvent, and exposure time, efficient digestion of several model proteins of molecular weights between 14,000 and 66,000, some having disulfide bridges, was successfully carried out. This technique provided reliable and reproducible sequence information by means of C-terminal-specific cleavages of aromatic and hydrophobic residues. As an application, protein identification could be achieved using a protein database search software.     

Retrograde labeling of single neurons in conjunction with MALDI high-energy collision-induced dissociation MS/MS analysis for peptide profiling and structural characterization

To reveal the peptide contents of the visually nonidentifiable neurons from a neuronal circuit of interest, we combined retrograde labeling of neurons with mass spectrometric single cell analysis. We used the neuronal circuit involved in the copulation behavior of a freshwater snail, Lymnaea stagnalis, as a model. Central neurons that control this behavior are known to send their axons to the penis nerve and innervate the penis complex. By retrograde filling from the penis nerve with nickel-lysine, these neurons were selectively labeled darkish blue. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric analyses of single stained neurons in the parietal ganglion from different animals reveal consistently the presence of several molecular ion species in the range of 800-1200 Da. From a single neuron, six molecular ion species were further characterized with MALDI time-of-flight/time-of-flight mass spectrometry, which demonstrates that the peptides are derived from a previously reported -FLRFamide precursor.     

Integrated sample preparation and MALDI mass spectrometry on a microfluidic compact disk

High-throughput microfluidic processing of protein digests integrated with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on a compact disk (CD) is described. Centrifugal force moves liquid through multiple microstructures, each containing a 10-nL reversed-phase chromatography column. The CD enables parallel preparation of 96 samples with volumes ranging from one to several microliters. The peptides in the digests are concentrated, desalted, and subsequently eluted from the columns directly into MALDI target areas (200 x 400 microm) on the CD using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into the MALDI instrument for peptide mass fingerprinting and database identification at a routine sensitivity down to the 200-amol level. Detection of proteolytic peptides down to the 50-amol level is demonstrated. The success rate of the CD technology in protein identification is about twice that of the C(18) ZipTips and standard MALDI steel targets. The CDs are operated using robotics to transfer samples and reagents from microcontainers to the processing inlets on the disposable CD and spinning to control the movement of liquid through the microstructures.     

An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation

A comprehensive on-line two-dimensional 2D-HPLC system with integrated sample preparation was developed for the analysis of proteins and peptides with a molecular weight below 20 kDa. The system setup provided fast separations and high resolving power and is considered to be a complementary technique to 2D gel electrophoresis in proteomics. The on-line system reproducibly resolved approximately 1000 peaks within the total analysis time of 96 min and avoided sample losses by off-line sample handling. The low-molecular-weight target analytes were separated from the matrix using novel silica-based restricted access materials (RAM) with ion exchange functionalities. The size-selective sample fractionation step was followed by anion or cation exchange chromatography as the first dimension. The separation mechanism in the subsequent second dimension employed hydrophobic interactions using short reversed-phase (RP) columns. A new column-switching technique, including four parallel reversed-phase columns, was employed in the second dimension for on-line fractionation and separation. Gradient elution and UV detection of two columns were performed simultaneously while loading the third and regenerating the fourth column. The total integrated workstation was operated in an unattended mode. Selected peaks were collected and analyzed off-line by MALDI-TOF mass spectrometry. The system was applied to protein mapping of biological samples of human hemofiltrate as well as of cell lysates originating from a human fetal fibroblast cell line, demonstrating it to be a viable alternative to 2D gel electrophoresis for mapping peptides and small proteins.     

High-sensitivity peptide mapping by capillary zone electrophoresis and microcolumn liquid chromatography, using immobilized trypsin for protein digestion

Procedures for the reduced-scale analysis of proteins by peptide mapping have been developed, allowing peptide maps to be obtained from picomole to femtomole quantities of protein. The use of trypsin immobilized on agarose gel and placed in a small reactor column has made it possible to reproducibility digest as little as 50 ng of protein. This represents a decrease in sample size of approximately 3 orders of magnitude from conventional tryptic digestion schemes. Separations of tryptic digests were accomplished by using either microcolumn high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). Separations of 100 ng (4 pmol) of tryptic digest samples of beta-casein were achieved with microcolumn HPLC, while separations of approximately 2 ng (80 fmol) of beta-casein tryptic digest (from a total sample size of 50 ng) were possible with CZE. Peptide maps from phosphorylated and dephosphorylated forms of beta-casein were readily distinguishable using both separation methods, demonstrating an ability to detect a single amino acid modification in a protein. Relative standard deviations of peak retention or migration times were less than 3% for microcolumn HPLC and less than 1% for CZE.     

Protein identification by MALDI-TOF-MS peptide mapping: a new strategy

A new strategy for identifying proteins by MALDI-TOF-MS peptide mapping is reported. In contrast to current approaches, the strategy does not rely on a good relative or absolute mass accuracy as the criterion that discriminates false positive results. The protein sequence database is first searched for all proteins that match a minimum five of the submitted masses within the maximum expected relative errors when the default or externally determined calibration constants are used, for instance, +/-500 ppm. Typically, this search retrieves many thousand candidate sequences. Assuming initially that each of these is the correct protein, the relative errors of the matching peptide masses are calculated for each candidate sequence. Linear regression analysis is then performed of the calculated relative errors as a function of m/z for each candidate sequence, and the standard deviation to the regression is used to distinguish the correct sequence among the candidates. We show that this parameter is independent of whether the mass spectrometric data were internally or externally calibrated. The result is a search engine that renders internal spectrum calibration unnecessary and adapts to the quality of the raw data without user interference. This is made possible by a dynamic scoring algorithm, which takes into account the number of matching peptide masses, the percentage of the protein's sequence covered by these peptides and, as new parameter, the determined standard deviation. The lower the standard deviation, the less cleavage peptides are required for identification and vice versa. Performance of the new strategy is demonstrated and discussed. All necessary computing has been implemented in a computer program, free access to which is provided in the Internet.     

Enhancement of the quality of MALDI mass spectra of highly acidic oligosaccharides by using a nafion-coated probe

Spectra of highly acidic oligosaccharides obtained by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be greatly enhanced in quality by coating the MALDI probe surface with a film consisting of a perfluorosulfonated ionomer (Nafion) prior to the addition of the sample-matrix mixture. For comparison, three mixtures containing highly acidic oligo- and polysaccharides derived from partial acidic hydrolysis of alginate, pectin, or carboxymethyl cellulose (CMC) were analyzed by employing probes with an uncoated gold surface or a surface coated with a Nafion or nitrocellulose film. The negative ion-mode MALDI spectra of the oligouronates (oligomers containing mannuronic/guluronic and galacturonic acid residues) obtained using uncoated or nitrocellulose-coated probes consisted of a series of broad, multiple peaks. These multiple peaks were assigned to the molecular ions of the nondissociated [M - H]- and partially sodiated [MnNa - H]-, where n = 1, 2, or 3, oligomers. In contrast, the corresponding spectra obtained with Nafion-coated probes contained only a single series of sharp peaks originating from the molecular ions ([M - H]-) of nondissociated oligomers exhibiting chain lengths of as many as approximately 15 uronic acid residues. The Nafion coating was apparently capable of removing the sodium counterions remaining in the deposit of the sample-matrix mixture on the probe, thereby greatly enhancing the signal-to-noise ratios of the peaks in the spectra. In a similar manner, higher quality spectra could also be obtained by using Nafion-coated probes for analysis of the oligouronates and CMC oligomers by positive ion-mode MALDI-MS.     

Ammonium dodecyl sulfate as an alternative to sodium dodecyl sulfate for protein sample preparation with improved performance in MALDI mass spectrometry

Sodium dodecyl sulfate (SDS) is a strong surfactant that is widely used in protein sample preparation. While protein and peptide samples containing up to approximately 1% SDS can be analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) using a two-layer matrix/sample deposition method, the presence of SDS in a protein sample generally degrades mass resolution and mass measurement accuracy. This degradation in performance is found to be related to the formation of sodium-protein adducts in the MALDI process. If the instrument resolving power is insufficient to separate these adduct peaks from the protonated molecular ion peak, peak broadening is observed in the protein molecular ion region, and as a result, the peak centroid shifts to a higher mass. In this work, we present a method using ammonium dodecyl sulfate as a viable alternative to SDS for protein sample preparation with much improved MALDI MS performance. Three non-sodium-based dodecyl sulfate surfactants, ammonium dodecyl sulfate (ADS), hydrogen dodecyl sulfate, and tris(hydroxymethyl)aminomethane dodecyl sulfate were investigated. Of the three surfactants tested, it is found that ADS gives the best performance in MALDI. For proteins with moderate molecular masses (i.e., up to approximately 25 kDa), the presence of ADS in a protein sample does not result in significant degradation in mass resolution and accuracy, and the protonated molecular ion peak is the dominant peak in the MALDI spectrum. The ammonium adduct ions dominate the MALDI spectra when the protein mass exceeds approximately 25 kDa; however, ADS still gives better results than SDS. The behavior of ADS in gel electrophoresis was also investigated. It is shown that cell extracts dissolved in ADS can be separated by normal SDS-polyacrylamide gel electrophoresis by simply mixing them with the SDS sample buffer. The application of ADS as the surfactant for protein solubilization with improved performance in MALDI analysis is demonstrated in the study of a detergent insoluble fraction from a Raji/CD9 B-cell lymphocyte lysate.     

Electrostatic interaction of an atomic force microscope probe with a sample surface

Electrostatic forces acting on the charged probe of an atomic-force microscope near a grounded conducting sample surface with or without a thin dielectric coating are calculated by numerically solving the Laplace equation for the electric potential in this system. It is shown that correct interpretation of the data of contact force spectroscopy requires calculating the electrostatic forces with increased accuracy and allowance for the dielectric characteristics of the sample surface layer.     

Depth profiling of peptide films with TOF-SIMS and a C60 probe

A buckminsterfullerene ion source is employed to characterize peptide-doped trehalose thin films. The experiments are designed to utilize the unique sputtering properties of cluster ion beams for molecular depth profiling. The results show that trehalose films with high uniformity can be prepared on Si by a spin-coating technique. Bombardment of the film with C60+ results in high quality time-of-flight secondary ion mass spectrometry spectra, even during ion doses of up to 3 x 10(14) ions/cm2. This result is in contrast to atomic bombardment experiments in which the dose of incident ions must be kept below 10(12) ions/cm2 so as to retain mass spectral information. Moreover, since the films are of uniform thickness, it is possible to depth-profile through the film and into the Si substrate. This experimental protocol allows the yield of trehalose molecular equivalents and the degree of interface mixing to be evaluated in detail. When doped with a variety of small peptides up to a molecular weight of m/z 500, we find that the peptide molecular ion intensity remains stable under continuous C60+ bombardment, although some decrease in intensity is observed. The results are interpreted in terms of a model whereby the high trehalose yield and low damage depth of the C60 projectile combine to prevent damage accumulation. In general, the peptide-trehalose system provides a valuable model for evaluating the parameters that lead to effective 3-dimensional characterization of biomaterials.     

Dual-function microanalytical device by in situ photolithographic grafting of porous polymer monolith: integrating solid-phase extraction and enzymatic digestion for peptide mass mapping

Microfluidic devices with a dual function containing both a solid-phase extractor and an enzymatic microreactor have been prepared, and their operation has been demonstrated. The devices were fabricated from a 25-mm-long porous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith prepared within a 50-microm-i.d. capillary. This capillary with a pulled 9-12-microm needle tip was used as a nanoelectrospray emitter coupling the device to a mass spectrometer. Photografting with irradiation through a mask was then used to selectively functionalize a 20-mm-long portion of the monolith, introducing reactive poly(2-vinyl-4,4-dimethylazlactone) chains to enable the subsequent attachment of trypsin, thereby creating an enzymatic microreactor with high proteolytic activity. The other 5 mm of unmodified hydrophobic monolith served as micro solid-phase extractor (microSPE). The dual-function devices were used in two different flow directions; concentration of myoglobin that was absorbed from its dilute solution, followed by elution and digestion or digestion, followed by concentration. Operations in both directions afforded equal sequence coverage. Different volumes of myoglobin solution ranging from 2 to 20 microL were loaded on the device. Very high sequence coverages of almost 80% were achieved for the highest loading. Despite the very short length of the extractor unit, the device operated in the digest-solid-phase extraction direction also enabled the separation of peaks that mostly contained undigested protein and peptides.     

石墨炉原子吸收光谱法测定水样消解研究

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