Rapid profiling of induced proteins in bacteria using MALDI-TOF mass spectrometric detection of nonporous RP HPLC-separated whole cell lysates.

A method for rapid profiling of water-soluble proteins from whole cell lysates has been developed using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) following separation by reversed-phase high-performance liquid chromatography (RP HPLC). Rapid separation of proteins from cell lysates was achieved using columns packed with C18 nonporous (NP) silica beads. Using this method, the whole cell lysate water-soluble proteins of E. coli were separated in under 15 min. A method using two columns in series at different temperatures was used in order to provide high loadability without loss of separation efficiency. The nonporous packing in the columns provided for high recovery. Eluting fractions were collected and analyzed by MALDI-TOFMS to determine the molecular weights and peptide maps of the proteins. These methods provided for the rapid screening and identification of proteins from E. coli where the response of E. coli to L-arabinose induction was studied. In this work, it is demonstrated that NP RP HPLC with MALDI-TOFMS detection may serve as a rapid means of detecting and identifying changes in bacterial protein expression due to external stimuli.     

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.     

Improved sensitivity for phosphopeptide mapping using capillary column HPLC and microionspray mass spectrometry: comparative phosphorylation site mapping from gel-derived proteins

Reversible protein phosphorylation regulates many cellular processes. Understanding how phosphorylation controls a given pathway usually involves specific knowledge of which amino acid residues are phosphorylated on a given protein. This is often a nontrivial task. In addition to the difficulties involved in purifying sufficient amounts of any given protein, most phosphoproteins contain multiple, substoichiometric sites of phosphorylation. In this paper, we describe substantial improvements made to our previously reported multidimensional electrospray MS-based phosphopeptide mapping technique that have resulted in a 20-fold increase in sensitivity for the overall process. Chief among these improvements are the incorporation of capillary chromatography and a microionspray source for the mass spectrometer into the first dimension of the analysis. In the first dimension of the process, phosphopeptides present in the proteolytic digest of a protein are selectively detected and collected into fractions during on-line LC/ESMS, which monitors for phosphopeptide specific marker ions. The phosphopeptide containing fractions are then analyzed in the second dimension by either MALDI-PSD or nano-ES with precursor ion scanning. The relative merits and limitations of these two techniques for phosphopeptide detection are demonstrated. The enhancement in sensitivity of the method under the new experimental conditions makes it suitable for phosphorylation mapping (from selective detection through sequencing) on gel-separated phosphoproteins where the level of phosphorylation at any given site is <200 fmol. Furthermore, this method detects serine, threonine, and tyrosine phosphorylation equally well. We have successfully employed this new configuration to map 11 in vivo sites of phosphorylation on the Saccharomyces cerevisiae protein kinase YAK1. YAK1 peptides containing all five YAK1 PKA consensus sites are phosphorylated, suggesting that YAK1 is an in vivo substrate for PKA. In addition, four peptides containing cdk sites and the autophosphorylation site at Tyr530 were found to be phosphorylated. Because the first dimension of this method generates a phosphorylation profile that can be used for a semiquantitative evaluation of site specific phosphoxylation, we evaluated its ability to detect site-specific changes in the phosphorylation profile of a protein in response to altered cellular conditions. This comparative phosphopeptide mapping strategy allowed us to detect a change in phosphorylation stoichiometry on the motor protein myosin-V in response to treatment with either mitotic or interphase Xenopus egg extracts and to identify the single functionally significant phosphorylation site that regulates myosin-V cargo binding.     

A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and cross-linking

The combination of limited proteolysis and MALDI-TOF mass spectrometry has become an important tool for the determination of epitopes but works best with highly purified antibodies. Here we report the use of capture antibodies to reduce the need for purification of the antibody in the mass spectrometric determination of the epitope. In this new method, a secondary Fc-specific antibody, covalently bound to Sepharose beads, is used to capture the primary antibody (the antibody of interest). After capture, the two antibodies are cross-linked. The antigen is then bound to the immobilized antibodies and subjected to proteolysis using several successive proteinases. In this study, this strategy is demonstrated with a crude mouse anti-ACTH IgG solution and adrenocorticotropin (ACTH). Comparing this strategy with previous methods where the antibody is bound directly to activated beads, the new method (1) results in a higher binding capacity of the bound antibody to ACTH, (2) does not require purification of the antibody of interest, and (3) dramatically reduces the chemical background in the MALDI mass spectra.     

Analysis of glycopeptides using lectin affinity chromatography with MALDI-TOF mass spectrometry

Glycopeptides prepared from 1 nmol of a mixture of glycoproteins, transferrin, and ribonuclease B by lysylendopeptidase digestion were isolated by lectin and cellulose column chromatographies, and then they were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and MALDI-quadrupole ion trap (QIT)-TOF mass spectrometry which enables the performance of MS ( n ) analysis. The lectin affinity preparation of glycopeptides with Sambucus nigra agglutinin and concanavalin A provides the glycan structure outlines for the sialyl linkage and the core structure of N-glycans. Such structural estimation was confirmed by MALDI-TOF MS and MALDI-QIT-TOF MS/MS. Amino acid sequences and location of glycosylation sites were determined by MALDI-QIT-TOF MS/MS/MS. Taken together, the combination of lectin column chromatography, MALDI-TOF MS, and MALDI-QIT-TOF MS ( n ) provides an easy way for the structural estimation of glycans and the rapid analysis of glycoproteomics.     

Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.     

A method for the analysis of low-mass molecules by MALDI-TOF mass spectrometry

We report a novel method termed matrix suppressed laser desorption/ionization to improve the analysis of low-mass molecules by MALDI-TOF mass spectrometry. In this method, the surfactant of cetrimonium bromide (CTAB) is added to the conventional matrix of alpha-cyano-4-hydroxycinnamic acid solution to prepare the MALDI samples. During the MALDI process, the presence of CTAB could substantially or even completely suppress the matrix-related ion background. As a result, very clean mass spectra can be routinely obtained in the low-mass range. In addition, the presence of CTAB can significantly improve the mass resolution of low-mass molecules. It is seen that high-quality spectra were routinely obtained at a matrix/CTAB ratio of 1000:1. This method has been successfully used to analyze a variety of low-mass molecules.     

Analysis of a model virus using residue-specific chemical cleavage and MALDI-TOF mass spectrometry

A nonenzymatic proteomics strategy is applied to the rapid identification of viruses. The approach provides solubilization and subsequent digestion of viral coat proteins in under 30 s. Acid digestions were carried out using a laboratory-quality microwave system equipped with temperature, pressure, and power controls, which allowed for precise optimization of experimental parameters. Under optimal conditions, this method provides an efficient alternative to traditional enzymatic digestion-based methods for virus identification. Following rapid microwave heating of a suspension of a model virus, RNA bacteriophage MS2, 13 chemical digestion products were detected in parallel with the coat protein precursor using MALDI-TOF MS. Because of the high sequence coverage obtained, the bacteriophage MS2 coat protein was identified with high confidence and the specificity of the identification allowed for the discrimination between bacteriophage MS2 and other closely related RNA bacteriophages.     

Nanosecond laser-induced photochemical oxidation method for protein surface mapping with mass spectrometry

We have developed an ultrafast pulse method for protein surface footprinting by laser-induced protein surface oxidations. This method makes use of a pulse UV laser that produces, in nanoseconds, a high concentration of hydroxyl (OH) free radicals by photodissociation of a hydrogen peroxide (H2O2) solution. The OH radicals oxidize amino acid residues located on the protein surface to produce stable covalent modifications. The oxidized protein is then analyzed by mass spectrometry to map the oxidized amino acid residues. Ubiquitin and apomyoglobin were used as model proteins in this study. Our results show that a single laser pulse can produce extensive protein surface oxidations. We found that monooxidized ubiquitins were more susceptible to further oxidations by subsequent laser irradiation, as compared to nonoxidized ones. This is due to the conformational changes of proteins by oxidation that increases the solvent-accessible surface area. Therefore, it is crucial to perform this experiment with a single pulse of laser so as to avoid oxidation of proteins after conformation of the protein changes. Subsequently, to obtain a high frequency and coverage of the oxidation sites while keeping the number of laser shots to one, we further optimized the laser power and concentration of hydrogen peroxide as well as the concentration of protein. This ultrafast OH radical generation method allows for rapid and accurate detection of surface residues, enabling mapping of the solvent-accessible regions of a protein in its native state.     

Surface mass spectrometry of two component drug-polymer systems: novel chromatographic separation method using gentle-secondary ion mass spectrometry (G-SIMS)

In recent years, there has been an increase in the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) for characterizing material surfaces. A great advantage of SIMS is that the analysis is direct and has excellent spatial resolution approaching a few hundred nanometers. However, the lack of the usual separation methods in mass spectrometry such as chromatography or ion mobility combined with the complexity of the heavily fragmented ions in the spectra means that the interpretation of multicomponent spectra in SIMS is very challenging indeed. The requirements for high-definition imaging, with say 256 × 256 pixels, in around 10 min analysis time places significant constraints on the instrument design so that separation using methods such as ion mobility with flight times of milliseconds are incompatible. Clearly, traditional liquid and gas chromatographies are not at all possible. Previously, we developed a method known as Gentle-SIMS (G-SIMS) that simplifies SIMS spectra so that the dominant ions are simply related to the structure of the substances analyzed. The method uses a measurement of the fragmentation behavior under two different primary ion source conditions and a control parameter known as the g-index. Here, we show that this method may be used "chromatographically" to separate the mass spectra of a drug molecule from the matrix polymer. The method may be used in real-time and is directly compatible with the majority of TOF-SIMS instruments. The applicability to other imaging mass spectrometeries is discussed.     

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry

Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyami-domethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interface. Tandem mass spectrometry was performed on an ion trap mass spectrometer using automated data-dependent MS/MS procedures. Tandem mass spectra were collected from the five most abundant ions in each scan using dynamic and isotopic exclusion to minimize redundancy. The spectra were analyzed by a version of the SEQUEST algorithm modified to identify amino acid substations resulting from SNPs.     

Proteolysis in mixed organic-aqueous solvent systems: applications for peptide mass mapping using mass spectrometry

The rate of protein digestion imposes significant limitations on high-throughput protein identification using mass spectrometry. In this report, we demonstrate that proteins are readily digested by trypsin in the presence of organic solvents such as methanol, acetone, 2-propanol, and acetonitrile. The rates of protein digestion in organic solvents, as indicated by the abundances of digest fragment ions in the mass spectrum, are increased relative to aqueous solution. In addition, amino acid coverage for the analyzed proteins increases in the presence of the organic solvents, and proteins that are resistant to proteolysis are readily digested. For example, a 68% amino acid sequence coverage was attained from a tryptic digest of myoglobin in < 5 min from an 80% acetonitrile solution, whereas no digest fragments were detected from a 5 min digestion in an aqueous solution. Moreover, the tryptic digestion of a complex protein mixture in an organic-aqueous solvent system showed significantly enhanced digestion for nearly all of the protein components. Enzymatic digestion in an organic-aqueous solvent system is a rapid, simple, and effective peptide mass-mapping technique.     

Identification of water-soluble selenium-containing proteins in selenized yeast by size-exclusion-reversed-phase HPLC/ICPMS followed by MALDI-TOF and electrospray Q-TOF mass spectrometry

An approach to speciation of selenium incorporated in yeast proteins was developed. The tryptic digest of a water-soluble protein fraction isolated by size-exclusion chromatography was analyzed by reversed-phase HPLC/ICPMS. The selenopeptides selected owing to the detector's elemental specificity were then analyzed by MALDI-TOFMS in order to select target ions for collision-induced dissociation MS. The latter, carried out with an electrospray Q-TOF spectrometer, enabled the sequencing of the selenopeptides detected by HPLC/ICPMS. The approach allowed for the first time the identification of a family of Se-containing proteins resulting from the replacement by selenomethionine of 2-9 methionine residues in a salt-stress-induced protein SIP18 (Mr 8874). The presence of these proteins was confirmed by MALDI-TOFMS of the original (nondigested) protein fraction. Another selenium protein identified was a heat-shock protein HSP12 (Mr 11693) in which the only methionine residue was replaced by selenomethionine. These two Se-containing proteins accounted for more than 95% of selenium in the water-soluble protein fraction.     

Web and database software for identification of intact proteins using "top down" mass spectrometry

For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/).     

Protein concentration and enzyme digestion on microbeads for MALDI-TOF peptides mass mapping of proteins from dilute solutions

A method for generating peptide mass maps from dilute protein samples is presented. The method involves the concentration of proteins from aqueous solution by adsorption onto reversed-phase polymeric microbeads. These beads are then washed extensively to remove contaminants, after which the bound proteins are digested with trypsin. Analysis of the digestion products is performed by MALDI-TOF mass spectrometry following direct deposition of the beads on a MALDI target, along with the matrix solution. The procedure is demonstrated using solutions of cytochrome c, lysozyme, and bovine serum albumin. The results of these digests are compared to trypsin digestions of the protein samples without sample preconcentration. Comparative results are also presented for protein solutions contaminated with 2 M NaCl, 2 M urea, or sodium dodecyl sulfate at concentrations up to 0.02%. These results reveal that, with the microbead preconcentration procedure, peptide mass maps can routinely be generated from highly contaminated samples with a protein concentration of only 100 nM.     

Bidimensional tandem mass spectrometry for selective identification of nitration sites in proteins

Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitrite formation during development, oxidative stress, and aging. However, protein nitration might also have biological functions comparable to protein phosphorylation, mainly in redox signaling and in signal transduction. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Existing methodologies essentially rely on immunochemical techniques either using 2D-PAGE fractionation in combination with western blot analyses or exploiting immunoaffinity procedures to selectively capture nitrated proteins. Here we report a totally new approach involving dansyl chloride labeling of the nitration sites that rely on the enormous potential of MSn analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and an MS3 analysis. This new procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures.     

Monolithic column HPLC separation of intact proteins analyzed by LC-MALDI using on-plate digestion: An approach to integrate protein separation and identification

A method is developed to integrate a protein separation by monolithic capillary reversed-phase high-performance liquid chromatography to on-probe tryptic digestion for subsequent analyses by MALDI-TOF MS and MALDI-TOF/TOF MS. The method provides a means of directly interfacing separations to MALDI-MS, reducing the amount of time required for traditional procedures involving in-solution enzymatic digestion and sample cleanup prior to MALDI-MS analysis. When used with pI-based fractionation as a first dimension, it provides a means of analyzing complex mixtures of proteins with minimal sample handling and cleanup. The use of monolithic capillary columns sufficiently resolved intact proteins so that peptide mass fingerprinting analysis by MALDI-TOF MS resulted in the identification of close to 40 unique proteins from 120 ng of sample obtained from a prefractionated MCF10 cell line at pH 6.34, where the identifications of several of these proteins were also confirmed by intact MW and tandem mass spectrometric analysis. The reproducibility of this method has been demonstrated to be sufficient for the purpose of protein identifications. Experimental values of protein intact MW are obtained and compared to that expected for each protein identified.     

MALDI-TOF mass spectrometry of insoluble giant polycyclic aromatic hydrocarbons by a new method of sample preparation

The insolubility of giant polycyclic aromatic hydrocarbons (PAHs) prevents their characterization by conventional analytical methods, which require a solubilization of the analyte. Laser desorption mass spectrometry may be used to analyze insoluble samples but is limited to relatively low molecular weights (approximately 2000), in the case of PAHs. To overcome this limitation, we applied MALDI-TOF mass spectrometry. Since MALDI sample preparation also requires solubility of analyte and matrix molecules, the sample preparation needed modification. The giant PAHs (>2000 Da) were investigated after using a new sample preparation, consisting of mechanically mixing analyte and matrix without any solubilization procedures. This solvent-free process allows insoluble compounds to be characterized. Furthermore, new organic molecules can be used as a matrix. Indeed, 7,7,8,8-tetracyanoquinodimethane, a new matrix with promising properties, has proven to be particularly suitable for the measurement of PAHs. Thanks to the successful characterization with MALDI-TOF mass spectrometry, the chemical design of giant PAHs, which was hindered until now for a lack of analytical methods, can now continue to develop.     

A digital microfluidic system for the investigation of pre-steady-state enzyme kinetics using rapid quenching with MALDI-TOF mass spectrometry

A digital microfluidic system based on electrowetting has been developed to facilitate the investigation of pre-steady-state reaction kinetics using rapid quenching and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The device consists of individually addressable electrodes arranged to allow the combination of liquid droplets at well-defined time intervals and an integrated, electrohydrodynamically driven mixer. The device combines two droplets to initiate a reaction, then, with precise timing, combines a third droplet to quench the reaction, and finally combines a fourth droplet to form a matrix. Improvements to throughput when compared to traditional laboratory-scale methods, and previous MALDI-TOF MS digital microfluidic systems, were made. The device was tested against a model protein tyrosine phosphatase system, and results agreed well with published data. The system therefore allows for the analysis of reaction kinetics that were previously too rapid to analyze using MALDI-TOF MS.