Affinity capture using vancomycin-bound magnetic nanoparticles for the MALDI-MS analysis of bacteria

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides a straightforward means to differentiate microorganism species based on mass spectral fingerprinting. The pathogen cell concentration in an infected sample, however, is generally lower than that capable of being detected directly by MALDI-MS. Furthermore, the presence of proteins or metabolites in biological fluids always causes unavoidable interference for the identification of microorganism species. Vancomycin, which binds to D-Ala-D-Ala moieties on the cell walls of Gram-positive bacteria and, therefore, inhibits peptidoglycan synthesis, is one of the most potent antibiotics. Thus, we have employed vancomycin-modified magnetic nanoparticles as affinity probes to selectively trap Gram-positive pathogens from sample solutions; i.e., these bacteria can be isolated from sample solutions by applying a magnetic field. The isolated cells could then be characterized by MALDI-MS. This approach effectively reduces the interference of protein and metabolite signals in the mass spectra of Gram-positive bacteria because vancomycin has such high specificity for the D-Ala-D-Ala units of the cell walls. The lowest cell concentration we detected for both Staphylococcus saprophyticus and Staphylococcus aureus in a urine sample (3 mL) was approximately 7 x 10(4) cfu/mL.     

Analysis for TNF-alpha using solid-phase affinity capture with radiolabel and MALDI-MS detection.

Screening of mutant mice for subtle phenotypes requires sensitive, high-throughput analyses of sentinel proteins in functional pathways. The cytokine TNF-alpha is upregulated during inflammatory reactions associated with autoimmune diseases. We have developed a method to monitor the concentration of TNF-alpha under physiological conditions. TNF-alpha is captured, purified, and concentrated using monoclonal antibody-coated microbeads. The capture is efficient (> 80%) and can be used in the concentration range < 100 pg/mL to > 50 ng/mL, as determined by detection of 125I-labeled TNF-alpha. The bead capture of TNF-alpha can be combined with direct detection by MALDI-MS for sample concentrations of > 10 ng/mL. TNF-alpha can be captured and detected from diluted mouse serum, with minimal interferences observed in the MALDI spectrum. This method is adaptable to high-throughput sample handling with microfluidic devices and automated mass spectrometric analysis.     

Increasing surface capacity for on-probe affinity capture MALDI-MS via gold particle attachment to allyl amine plasma polymers

In this study, we demonstrate that the protein binding capacity of a surface modified matrix-assisted laser desorption/ionization (MALDI) target can be increased significantly by architecturing the surface of the MALDI probe using gold microparticles. In the present approach, a MALDI target, initially modified via pulsed rf plasma deposition of an allyl amine polymer thin film, is subsequently architectured via reaction with 2-iminothiolane and surface attachment of gold microparticles. The modified probe is then exposed to thiolated biotin to introduce an avidin binding element on the surface of the gold beads. The protein binding capacity of this architectured target is compared with a similarly plasma polymer modified MALDI target that is directly biotinylated. Application of various surface concentrations of avidin to the two probes and MALDI-MS analysis of avidin contained in the solution removed from the probe reveals that saturation of the gold-particle architectured target occurs at a factor of 15-30 higher applied surface concentration, as compared with the unarchitectured target. Furthermore, MALDI-MS analysis of the avidin retained on the two probes reveals that the limit of detection is lowered by a factor of 15-20 on the gold-particle architectured target as compared with the unarchitectured target.     

Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation. A protocol for MALDI-MS peptide analysis in proteomics

We present a new MALD1 sample preparation technique for peptide analysis using the matrix alpha-cyano-4-hydroxy-cinnamic acid (CHCA) and prestructured sample supports. The preparation integrates sample purification, based on the affinity of microcrystalline CHCA for peptides, thereby simplifying the analysis of crude peptide mixtures. Enzymatic digests can thus be prepared directly, without preceding purification. Prepared samples are homogeneous, facilitating automatic spectra acquisition. This method allows preparation of large numbers of samples with little effort and without the need for automation. These features make the described preparation suitable for cost-efficient high-throughput protein identification. Performance of the sample preparation is demonstrated with in situ proteolytic digests of human brain proteins separated by two-dimensional gel electrophoresis.     

Lectin and carbohydrate affinity capture surfaces for mass spectrometric analysis of microorganisms

In a preliminary report (Bundy, J. L.; Fenselau, C. Anal. Chem 1999, 71, 1460-1463), we demonstrated the use of lectin-derivatized surfaces to capture and concentrate complex carbohydrates as well as microorganisms from sample matrixes unamenable to direct MALDI mass spectrometry. Here, we extend the work to include samples representative of a wider variety of microorganisms of importance to human health and of enveloped viruses. In this study, lectins were immobilized directly to a membrane surface via primary amines. A complementary approach was also explored, using immobilized carbohydrates to capture bacteria via microbial lectins expressed on their surfaces. The carbohydrate-based surfaces were constructed by first immobilizing streptavidin to the membrane, followed by attachment of a commercially produced biotin/carbohydrate polymer. Acid treatment of the sample prior to mass spectrometric analysis permits the observation of protein biomarkers from the captured microbial samples in the 5-20 kDa mass range. Bacteria samples were detected from physiological buffers, urine, milk, and processed chicken samples using the biocapture probes. Viral samples were detected from culture based on glycoprotein moieties desorbed directly from the surface. The carbohydrate-based system provided greater sensitivity than the lectin system, possibly due to the larger number of accessible saccharide ligands on the polymer.     

Photoimmobilization of proteins for affinity capture combined with MALDI TOF MS analysis

Affinity capture surfaces can be prepared in a number of ways. A method of obtaining such surfaces through UV-activated immobilization of binding proteins using a benzophenone derivative is reported. Photoimmobilized protein G was used to selectively capture and preconcentrate bovine IgG from a mixture with BSA, and the affinity of photoattached concanavalin A toward ovalbumin was compared with that of commercially available concanavalin A on agarose beads. The results of the capture after tryptic digestion were analyzed by MALDI TOF MS. Immobilized trypsin was also prepared through photoimmobilization and later used to digest hemoglobin. Immobilized enzyme digestion resulted in more partial cleavages than solution-phase digestion. More methionine and tryptophan oxidation was also observed. Photoimmobilization was shown to be a quick and easy way of immobilizing ligands on surfaces.     

Silica nanostructured platform for affinity capture of tumor-derived exosomes

Early diagnosis of prostate cancer and evaluation of appropriate treatment options requires development of effective and high-throughput selective capture technology for exosomes that are positive for the expression of enzyme-biomarker, prostate-specific membrane antigen (PSMA). Exosomes are small secreted vesicles that play a key role in intercellular communication and cancer progression. PSMA is highly enriched in exosomes excreted by PSMA+ prostate cancer cells. Using PSMA+ cells from the well-established prostate cancer cell line (LNCaP), the secreted exosomes were collected and isolated from the culture medium. The tumor-derived exosomes were selectively captured using a novel silica nanostructure support that had been functionalized with the small-molecule ligand TG97, a known inhibitor of PSMA enzymatic activity that binds irreversibly in the active site of PSMA. The concept was demonstrated using a single cancer type (i.e., prostate cancer), but based on the data obtained the approach may be applicable to a broad panel of biomarker ligands for selective capture of biomarker-positive exosomes from an array of cell types. The approach demonstrated herein overcomes many of the limitations of alternative methods that are often ineffective in isolating tumor-derived exosomes from those derived from normal tissue because of the low yield recovery and the time required for the process. A further advantage is the ability to isolate a specific subpopulation of exosomes relying on the expression of a specific surface marker as well as improved exosome recovery rate.     

Integrated affinity capture, purification, and capillary electrophoresis microdevice for quantitative double-stranded DNA analysis

A novel injection method is developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification, and injection of dsDNA for quantitative microchip capillary electrophoresis analysis. The affinity capture matrix consists of a 20 base acrydite modified oligonucleotide copolymerized into a 6% linear polyacrylamide gel that captures ssDNA or dsDNA analyte including PCR amplicons and synthetic oligonucleotides. Double stranded PCR amplicons with complementarity to the capture probe up to 81 bases from their 5' terminus are reproducibly captured via helix invasion. By integrating the oligo capture matrix directly with the CE separation channel, the electrophoretically mobilized target fragments are quantitatively captured and injected after thermal release for unbiased, efficient, and quantitative analysis. The capture process exhibits optimal efficiency at 44 degrees C and 100 V/cm with a 20 microM affinity capture probe (TM = 57.7 degrees C). A dsDNA titration assay with 20 bp fragments validated that dsDNA is captured at the same efficiency as ssDNA. Dilution studies with a duplex 20mer show that targets can be successfully captured and analyzed with a limit of detection of 1 pM from 250 nL of solution (approximately 150,000 fluorescent molecules). Simultaneous capture and injection of amplicons from E. coli K12 and M13mp18 using a mixture of two different capture probes demonstrates the feasibility of multiplex target capture. Unlike the traditional cross-injector, this method enables efficient capture and injection of dsDNA amplicons which will facilitate the quantitative analysis of products from integrated nanoliter-scale PCR reactors.     

Application of linear and nonlinear discrete wavelet transforms to MALDI-MS measurements of bacteria for classification

The linear and nonlinear discrete wavelet transforms (DWTs) were used to compress matrix-assisted laser desorption/ionization mass spectra to address two key challenges: the relatively high noise level and the underdetermined format of the data set. By applying the DWT to MALDI-MS spectra, the spectra were simultaneously smoothed and compressed. Multivariate projected difference resolution was used to evaluate the effects of the linear and nonlinear DWT on classification. The cross-validation study using bootstrapped Latin partition and partial least-squares (PLS-2) has proved that the classification accuracy increased after data compression. The best result was obtained when using Fisher's criterion to choose wavelet coefficients for compression. With the aid of principal component analysis (PCA), different wavelet filters may provide different mathematical perspectives to visualize the clustering of bacteria. The effect of growth time was directly observed with wavelet transform, which could not be observed using the original spectra.     

Monitoring the growth of a bacteria culture by MALDI-MS of whole cells.

We have probed the time evolution of a growing bacteria culture by extracting samples periodically and performing matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) on whole cells. The mass spectra generated by this method contain tens of peaks in the 3-11-kDa mass range. Cultures of E. coli strain K-12 were grown in two types of containers and at two nutrient concentrations and sampled periodically from 6 to 84 h after inoculation. The relative intensities of several of the stronger peaks vary quite dramatically as a function of time. These temporal characteristics must be taken into account when MALDI-MS is applied to identify bacteria. The results also suggest that MALDI-MS can be used to follow the aging of a bacteria culture.     

Polymeric inverse micelles as selective peptide extraction agents for MALDI-MS analysis

Analyses of peptides in complex mixtures are significant challenges in proteomics applications. Here, we report an amphiphilic polymer-based nanoassembly that is capable of selectively extracting peptides, on the basis of their isoelectric points, into an immiscible organic phase from an aqueous solution. The isoelectric point (pI) cutoff in these extractions depends on the pH of the aqueous solution, and thus, sequential fractionation of peptide mixtures based on pI can be accomplished by varying the pH of the aqueous solution. Additionally, we observe an unexpected enhancement in the MALDI-MS signal for extracted peptides ionized in the presence of the polymer, which allows us to obtain reproducible ion signals for some peptides at concentrations as low as 10 pM.     

Direct MALDI-MS/MS of phosphopeptides affinity-bound to immobilized metal ion affinity chromatography beads

Immobilized metal ion affinity chromatography (IMAC) is a useful method to selectively isolate and enrich phosphopeptides from a peptide mixture. Mass spectrometry is a very suitable method for exact molecular weight determination of IMAC-isolated phosphopeptides, due to its inherent high sensitivity. Even exact molecular weight determination, however, is not sufficient for identification of the phosphorylation site if more than one potential phosphorylation site is present on a peptide. The previous method of choice for sequencing the affinity-bound peptides was electrospray tandem mass spectrometry (ESI-MS/MS). This method required elution and salt removal prior to MS analysis of the peptides, which can lead to sample loss. Using a matrix-assisted laser desorption/ionization (MALDI) source coupled to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer with true MS/MS capabilities, direct sequencing of IMAC-enriched peptides has been performed on IMAC beads applied directly to the MALDI target. The utility of this new method has been demonstrated on a protein with unknown phosphorylation sites, where direct MALDI-MS/MS of the tryptic peptides bound to the IMAC beads resulted in the identification of two novel phosphopeptides. Using this technique, the phosphorylation site determination is unambiguous, even with a peptide containing four potentially phosphorylated residues. Direct analysis of phosphorylated peptides on IMAC beads does not adversely affect the high-mass accuracy of an orthogonal injection QqTOF mass spectrometer, making it a suitable technique for phosphoproteomics.     

Radio frequency plasma polymer coatings for affinity capture MALDI mass spectrometry

Surface modification of MALDI probes is an attractive approach for combining bioaffinity isolation of targeted biomolecules with mass spectrometric analysis of the captured species. In this work, we demonstrate that a polymer thin film, produced by pulsed rf plasma polymerization of allylamine and deposited directly on a MALDI probe, can be subsequently biotinylated to develop a bioaffinity capture MALDI probe. The synthesis and characterization of the probe by XPS, FT-IR, and AFM is described, and the selective isolation of avidin from a three-component mixture of avidin, lysozyme, and cytochrome c is presented. These initial results offer encouragement for the further exploration of rf plasma polymer deposition as a novel approach for the development of on-probe affinity capture MALDI probes.     

Metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides

We report a new method called metal affinity capture that when coupled with tandem mass spectrometry (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with Ga(III) or Fe(III) and N(alpha),N(alpha)-bis(carboxymethyl)lysine (LysNTA) in solution and electrosprayed as doubly or triply charged positive ions. The gas-phase complexes uniformly dissociate to produce a common (LysNTA + H)+ ion that is used as a specific marker in precursor ion scans. The advantages of MAC-MSMS over the current methods of phosphopeptide detection are as follows. (1) MAC-MSMS uses metal complexes that self-assemble in solution at pH <5, which is favorable for the production of positive ions by electrospray. (2) Phosphorylation at tyrosine, serine, and threonine is detected by MAC-MSMS. (3) The phosphopeptide peaks in the mass spectra are encoded with the 69Ga-71Ga isotope pattern for selective recognition in mixtures. Detection by MAC-MSMS of singly and multiply phosphorylated peptides in tryptic digests is demonstrated at low-nanomolar protein concentrations.     

Mixed monolayer-protected gold nanoclusters as selective peptide extraction agents for MALDI-MS analysis

Cationic and anionic nanoparticles selectively target peptides with low and high isoelectric points, respectively. Additionally, their high surface area-to-volume ratios make these nanoparticles (approximately 2-nm core diameter) very efficient extraction and concentration agents. Upon extraction, the peptide-bound nanoparticles can be analyzed by MALDI-MS to provide highly sensitive detection of the targeted peptides. We demonstrate that MALDI-MS can detect peptide concentrations as low as 500 pM from 250-microL solutions using these nanoparticle scaffolds as extraction and concentration agents.     

On-probe digestion of bacterial proteins for MALDI-MS

On-probe digestion combined with MALDI mass spectrometry is studied as a rapid method for the analysis and identification of bacterial proteins. The use of trypsin adsorbed to the probe surface reduces the digestion time from hours to minutes. A high amount of trypsin must be applied to the probe for the successful digestion of bacterial proteins. Mass spectra of the digest contain a number of low-mass digest fragments. Several components of a B. subtilis bacterial digest can be identified through postsource decay and database searching.     

Two-layer sample preparation: a method for MALDI-MS analysis of complex peptide and protein mixtures

The analytical performance of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for direct analysis of peptide and protein mixtures is strongly dependent on the sample and matrix preparation. A two-layer sample preparation method is demonstrated to be very effective for analyzing complex mixtures. In this method, the first layer on the MALDI probe is the densely packed matrix microcrystals formed by fast solvent evaporation of a matrix solution. A mixture solution containing both matrix and sample is then deposited onto the first layer to form uniform analyte/matrix micrococrystals. It is found that the addition of matrix to the second-layer sample solution proves to be critical in analyzing mixtures of peptides and proteins covering a broad mass range. The effect of solvent conditions for preparing the second-layer solution is discussed. The application of this method is demonstrated for the analysis of cow's milk where milk proteins as well as peptide fragments produced from proteins by indigenous proteinases are detected. Direct analyses of peptides and proteins from a bacteria extract and crude egg white are also illustrated.     

Patterned monolayer/polymer films for analysis of dilute or salt-contaminated protein samples by MALDI-MS

This paper describes a surface science/mass spectrometry effort to develop and characterize a patterned gold surface that serves as a MALDI sample platform capable of concentrating and purifying proteins. Using microcontact printing, small (200-microm diameter) hydrophilic spots of bare gold or chemically anchored poly(acrylic acid) (PAA) are patterned at 5-mm intervals in a hydrophobic field consisting of a self-assembled monolayer of hexadecanethiol. Building on recent innovations by others, the small hydrophilic spots concentrate the sample to achieve good reproducibility and high sensitivity in the MALDI signal. One of the key features in this work is the combination of the high density of carboxylate groups in PAA with a small spot size to afford both concentration and purification of proteins via ionic interactions. This translates into detection limits for salt-contaminated proteins that are 20-100 times lower (low femtomole) than those reported for previous polymer- or monolayer-modified MALDI probes (using proteins in the 3-15-kDa range). Reflectance FT-IR spectroscopy and ellipsometry were used to determine the amount of protein adsorbed to a PAA-modified sample plate as a function of pH and salt concentration. Amide absorbances in IR spectra correlate well with MALDI-MS signals measured after addition of 2,5-dihydroxybenzoic acid as a matrix.     

Derivatization for stabilizing sialic acids in MALDI-MS

While matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is useful in oligosaccharide analysis, the sialic acid, or N-acetylneuraminic acid (NANA), moiety of an oligosaccharide is liable to dissociation in- or postsource during mass measurement. In this study, we tried to stabilize the moiety by amidation, as in the case of peptides (Sekiya, S.; Wada, Y. Tanaka, K. Anal. Chem. 2004, 76, 5894-5902), and found 4-(4,6-dimethoxy-1,3,5-triazin-2yl)-4-methylmorpholinium chloride to be a desirable condensing agent. Amidation stabilized the glycosidic bond with NANA and suppressed its preferential cleavage by in-source decay, postsource decay, or collision-induced dissociation. In addition, the suppressed dissociation considerably improved the yield of the B/Y type ions for structural analysis by MS/MS. These results demonstrate that amidation is an effective derivatization to reinforce the structural analysis of sialylated oligosaccharides by MALDI-MS. In addition, amidation with (15)N-labeled ammonium chloride decreases the mass shift from the acid to amide form to just 0.013, reducing the complexity of mass spectral interpretation and database searching.     

2,5-Dihydroxybenzoic acid butylamine and other ionic liquid matrixes for enhanced MALDI-MS analysis of biomolecules

The performance of the new ionic liquid MALDI-MS matrix 2,5-dihydroxybenzoic acid butylamine (DHBB) was assessed and compared to results obtained with the ionic liquid MALDI-MS matrixes alpha-cyano-4-hydroxycinnamic acid butylamine (CHCAB), 3,5-dimethoxycinnamic acid triethylamine (SinTri), and the frequently used solid MALDI matrixes 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA). The vacuum-stable, liquid consistency of ionic liquid matrix sample preparations considerably enhanced MALDI-MS analysis in terms of shot-to-shot reproducibility. Consequently, relative standard deviations serving as a measure for reproducibility of intensity-values acquired from 90 different spots on one MALDI-MS preparation were approximately one-half as high when solid DHB was replaced by the ionic liquid DHBB and eight times lower after exchange of solid CHCA by ionic liquid CHCAB. Interestingly, the ionic liquid MALDI matrix DHBB conserved the broad applicability of its solid analogue DHB, reduced MALDI induced fragmentation of monosialylated glycans and gangliosides, and was the superior ionic liquid matrix for MALDI-MS analysis of oligosaccharides and polymers, such as poly(ethylene glycol). It also worked well with glycoconjugates, peptides, and proteins; however, the tendency of DHBB to form multiple alkali adduct ions with peptides and proteins made CHCAB the ionic liquid matrix of choice for peptides. SinTri was the best ionic liquid matrix for proteins of high molecular weight, such as IgG. Furthermore, it was demonstrated for the first time that solvent properties and MALDI matrix properties of ionic liquids, such as DHBB, can be combined to enable fast, direct screening of an enzymatic reaction. This was proven by the desialylation of sialylactose with sialidase from Clostridium perfringens in the presence of diluted aqueous DHBB and subsequent direct MALDI-MS analysis of the reaction mixture.