Direct Matrix-assisted Laser Desorption Ionization Research Articles

DHB Matrix with Additives for Direct MALDI Mass Spectrometry of Carbohydrates and Glycoconjugates

Matrix-assisted laser desorption/ionization (MALDI) is one of the soft ionization methods in mass spectrometry (MS). Its analyte compatibility with a matrix can improve analyte-selective ionization efficiency and control the fragmentation properties of the product ions. 2,5-dihydroxybenzoic acid (DHB) is a versatile matrix that can be used for various molecular species. Although DHB is commonly used as the matrix of first choice for MALDI MS of glycans, many attempts to optimize DHB for glycan analysis using additives have also been reported. This mini-review will present additives of the DHB-based matrix that promote direct analysis of carbohydrates and glycoconjugates by MALDI MS.

Direct Prosthetic Joint Infection Diagnosis from Sonication Fluid Inoculated in Blood Culture Bottles by Direct MALDI-TOF Mass Spectrometry

An accurate and fast microbiological diagnosis is key for a proper management and results when facing prosthetic joint infection (PJI). The purpose of this study is to assess the role of direct Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) for early identification of the pathogens causing PJI from sonication fluid inoculated in blood culture bottles (BCB-SF). This prospective multicentric study included 107 consecutive patients from February 2016 to February 2017. Among them, 71 prosthetic joint revision surgeries were undergone for aseptic and 36 for septic reasons. Prostheses were sonicated and the resulting fluid inoculated into blood culture bottles, regardless the suspicion for infection. We assessed the diagnostic performance of direct MALDI-TOF MS identification of the pathogens in BCB-SF and compared it with periprosthetic tissue and conventional sonication fluid cultures. The sensitivity of direct MALDI-TOF MS of BCB-SF (69%) was higher compared to conventional sonication fluid (69% vs. 64%, p > 0.05) or intraoperative tissue cultures (69% vs. 53%, p = 0.04), especially for patients receiving antimicrobial treatment. This approach also reduced the time for identification but the specificity was compromised (100% vs. 94%) and polymicrobial infections were missed. In conclusion, BCB-SF improves the sensitivity and reduces the time of PJI diagnosis when used in combination with conventional cultures under strict sterility conditions.

Direct introduction MALDI FTICR MS based on dried droplet deposition applied to non-targeted metabolomics on Pisum Sativum root exudates

Non-targeted metabolomic approaches based on direct introduction (DI) through a soft ionization source are nowadays used for large-scale analysis and wide cover-up of metabolites in complex matrices. When coupled with ultra-high-resolution Fourier-Transform ion cyclotron resonance (FTICR MS), DI is generally performed through electrospray (ESI), which, despite the great analytical throughput, can suffer of matrix effects due to residual salts or charge competitors. In alternative, matrix assisted laser desorption ionization (MALDI) coupled with FTICR MS offers relatively high salt tolerance but it is mainly used for imaging of small molecule within biological tissues. In this study, we report a systematic evaluation on the performance of direct introduction ESI and MALDI coupled with FTICR MS applied to the analysis of root exudates (RE), a complex mixture of metabolites released from plant root tips and containing a relatively high salt concentration. Classic dried droplet deposition followed by screening of best matrices and ratio allowed the selection of high ranked conditions for non-targeted metabolomics on RE. Optimization of MALDI parameters led to improved reproducibility and precision. A RE desalted sample was used for comparison on ionization efficiency of the two sources and ion enhancement at high salinity was highlighted in MALDI by spiking desalted solution with inorganic salts. Application of a true lyophilized RE sample exhibited the complementarity of the two sources and the ability of MALDI in the detection of undisclosed metabolites suffering of matrix effects in ESI mode.

Rapid diagnosis of periprosthetic joint infection from synovial fluid in blood culture bottles by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

The aim of this prospective study was to use direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to rapidly diagnose periprosthetic joint infections (PJIs). Synovial fluid was taken from 77 patients (80 joints, 41 hips and 39 knees) who met the International Consensus Meeting criteria for PJI, and inoculated into blood culture bottles (BCBs) and onto conventional swabs. Positive blood cultures were analyzed using either direct or routine MALDI-TOF MS. Pathogen identification and the time to identification was recorded. Differences between groups were analyzed using the Kruskal-Wallis test and Bonferroni's post-hoc test. Direct and routine MALDI-TOF MS both detected 64 positive results (80%), compared to 47 (59%) by conventional swabs (p = 0.002). Direct MALDI-TOF MS identified 85.3% of the gram-positive organisms and 92.3% of the gram-negative organisms. No fungi were identified by direct MALDI-TOF MS. In 17 BCBs that were flagged positive, identification by direct MALDI-TOF MS failed. Among the positive results in the direct MALDI-TOF MS group, Staphylococcus aureus accounted for 47%, followed by Staphylococcus epidermidis (17%), Escherichia coli (9%) and Klebsiella pneumoniae (9%). The median time to microorganism identification was significantly shorter with direct MALDI-TOF MS (12.7 h, IQR: 8.9-19.6 h) than with routine MALDI-TOF MS (39.5 h, IQR: 22.8-46.0 h) or swabs (44.4 h, IQR: 27.2-72.6 h) (p < 0.0001). In pairwise comparisons, there were significant differences in the time of microorganism identification between direct MALDI-TOF MS and routine MALDI-TOF MS (p < 0.0001) or swab culture (p < 0.0001). There was no significant difference between routine MALDI-TOF MS and swab culture (p = 0.0268). Compared with current laboratory practice, direct MALDI-TOF MS shortened the time to microorganism identification and had superior results compared to conventional swabs, except for fungi. Further studies should investigate whether the earlier administration of appropriate antimicrobial agents can improve the treatment outcomes of PJIs.

Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry and real-time PCR in a combined protocol for diagnosis of bloodstream infections: a turnaround time approach

Bloodstream infections (BSIs) are serious infections associated with high rates of morbidity and mortality. Every hour delay in initiation of an effective antibiotic increases mortality due to sepsis by 7%. Turnaround time (TAT) for conventional blood cultures takes 48h, forcing physicians to streamline therapy by exposing patients to broad-spectrum antimicrobials. Our objective was (1) to evaluate the accuracy and TAT of an optimized workflow combining direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and in-house real-time polymerase chain reaction (PCR) for bacterial identification and antimicrobial resistance profiling directly from positive blood bottles for diagnosing bloodstream infections and (2) to verify the effect of reporting results to medical staff. A total of 103 BSI episodes from 91 patients admitted to three hospitals in São Paulo, Brazil were included. TAT from molecular versus conventional methods was measured and compared. Our protocol showed an overall agreement of 93.5% for genus and 78.5% for species identification; 74.2% for methicillin resistance detection, 89.2% for extended-spectrum β-lactamase profiling, 77.8% for metallo-β-lactamase profiling, and 100% for carbapenemase profile and vancomycin-resistance detection when compared with conventional testing. TAT of molecular sample processing according to our protocol was 38h shorter than conventional methods. Antimicrobial interventions were possible in 27 BSI episodes. Antimicrobial discontinuation was achieved in 12 BSI episodes while escalation of therapy occurred in 15 episodes. Antimicrobial therapy was inadequate in three (12%) BSI episodes diagnosed using results of molecular testing. Our in-house rapid protocol for identifying both bacteria and antimicrobial resistance provided rapid and accurate results, having good agreement with conventional testing results. These results could contribute to faster antimicrobial therapy interventions in BSI episodes.

Fluorescent-magnetic Janus nanorods for selective capture and rapid identification of foodborne bacteria

A facile method for foodborne bacterial enrichment and direct identification from milk samples using Janus magnetic mesoporous silica nanoparticles (Janus M-MSNs) is reported. The fluorescent-magnetic nanorods are fabricated using the one-pot sol-gel process, followed by modification with Escherichia coli antibodies to form fluorescent Janus M-MSNs (Janus M-MSNs-Ab). The Janus M-MSNs-Ab combine the fast magnetic response capacity of the magnetic heads with the strong fluorescence and bacterial-targeting properties of the silica bodies, which results in selective capture and fluorescence identification of E. coli from milk samples containing mixed bacteria. After magnetic separation, complexes of the bacteria and nanoparticles are detected through direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which has a strong, anti-interference performance. Thus, the Janus M-MSNs-Ab synthesized in this study show great sensitivity and effectivity in foodborne bacterial capture and is a promising tool for rapid bacterial detection and investigation when combined with MALDI-TOF-MS.

Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of lysozyme contained in hen egg white.

As a natural antibacterial peptide, lysozyme (LZ) is widely used in medicine and the food industry. Despite many years of research on this compound, its new antibacterial properties are still to be determined. The primary aim of this work is to demonstrate the application of the matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometric analysis of LZ directly in hen egg white samples without extraction thereof. The egg white samples were kept over 10 weeks at room temperature and measured every week. The resulting positive and negative ion mass spectra were then compared to determine the intensity of the LZ mass peak. Storage of the egg white for over 10 weeks did not influence the LZ mass peak intensity (both positive and negative). It can be concluded that the LZ concentration in the egg white samples did not vary with time. The effect of the matrix/sample ratio on LZ detection was also examined, and it was found to be different in the case of positive and negative ionization. The mass peaks of LZ oligomeric forms were observed in all mass spectra, so the MALDI method could be used in subsequent studies.

Performance of Two Resin-Containing Blood Culture Media in Detection of Bloodstream Infections and in Direct Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) Broth Assays for Isolate Identification: Clinical Comparison of the BacT/Alert Plus and Bactec Plus Systems

We compared the clinical performances of the BacT/Alert Plus (bioMérieux) and Bactec Plus (Becton Dickinson) aerobic and anaerobic blood culture (BC) media with adsorbent polymeric beads. Patients ≥ 16 years old with suspected bloodstream infections (BSIs) were enrolled in intensive care units and infectious disease wards. A single 40-ml blood sample was collected from each and used to inoculate (10 ml/bottle) one set of BacT/Alert Plus cultures and one set of Bactec Plus cultures, each set consisting of one aerobic and one anaerobic bottle. Cultures were incubated ≤ 5 days in the BacT/Alert 3D and Bactec FX instruments, respectively. A total of 128 unique BSI episodes were identified based on the recovery of clinically significant growth in 212 aerobic cultures (106 BacT/Alert and 106 Bactec) and 151 anaerobic cultures (82 BacT/Alert and 69 Bactec). The BacT/Alert aerobic medium had higher recovery rates for Gram-positive cocci (P = 0.024), whereas the Bactec aerobic medium was superior for recovery of Gram-negative bacilli (P = 0.006). BacT/Alert anaerobic medium recovery rates exceeded those of the Bactec anaerobic medium for total organisms (P = 0.003), Gram-positive cocci (P = 0.013), and Escherichia coli (P = 0.030). In terms of capacity for diagnosing the 128 septic episodes, the BacT/Alert and Bactec sets were comparable, although the former sets diagnosed more BSIs caused by Gram-positive cocci (P = 0.008). They also allowed earlier identification of coagulase-negative staphylococcal growth (mean, 2.8 h; P = 0.003) and growth in samples from patients not on antimicrobial therapy that yielded positive results (mean, 1.3 h; P < 0.001). Similarly high percentages of microorganisms in BacT/Alert and Bactec cultures (93.8% and 93.3%, respectively) were identified by direct matrix-assisted laser desorption ionization-time of flight mass spectrometry assay of BC broths. The BacT/Alert Plus media line appears to be a reliable, timesaving tool for routine detection of BSIs in the population we studied, although further studies are needed to evaluate their performance in other settings.

Orthogonal organic and aqueous‐based washes of tissue sections to enhance protein sensitivity by MALDI imaging mass spectrometry

Imaging mass spectrometry (IMS) is an emergent and innovative approach for measuring the composition, abundance and regioselectivity of molecules within an investigated area of fixed dimension. Although providing unprecedented molecular information compared with conventional MS techniques, enhancement of protein signature by IMS is still necessary and challenging. This paper demonstrates the combination of conventional organic washes with an optimized aqueous-based buffer for tissue section preparation before matrix-assisted laser desorption/ionization (MALDI) IMS of proteins. Based on a 500 mM ammonium formate in water-acetonitrile (9:1; v/v, 0.1% trifluororacetic acid, 0.1% Triton) solution, this buffer wash has shown to significantly enhance protein signature by profiling and IMS (~fourfold) when used after organic washes (70% EtOH followed by 90% EtOH), improving the quality and number of ion images obtained from mouse kidney and a 14-day mouse fetus whole-body tissue sections, while maintaining a similar reproducibility with conventional tissue rinsing. Even if some protein losses were observed, the data mining has demonstrated that it was primarily low abundant signals and that the number of new peaks found is greater with the described procedure. The proposed buffer has thus demonstrated to be of high efficiency for tissue section preparation providing novel and complementary information for direct on-tissue MALDI analysis compared with solely conventional organic rinsing.

Direct Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Improves Appropriateness of Antibiotic Treatment of Bacteremia

Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows the identification of microorganisms directly from positive blood culture broths. Use of the MALDI-TOF MS for rapid identification of microorganisms from blood culture broths can reduce the turnaround time to identification and may lead to earlier appropriate treatment of bacteremia. During February and April 2010, direct MALDI-TOF MS was routinely performed on all positive blood cultures. During December 2009 and March 2010 no direct MALDI-TOF MS was used. Information on antibiotic therapy was collected from the hospital and intensive care units' information systems from all positive blood cultures during the study period. In total, 253 episodes of bacteremia were included of which 89 during the intervention period and 164 during the control period. Direct performance of MALDI-TOF MS on positive blood culture broths reduced the time till species identification by 28.8-h and was associated with an 11.3% increase in the proportion of patients receiving appropriate antibiotic treatment 24 hours after blood culture positivity (64.0% in the control period versus 75.3% in the intervention period (p0.01)). Routine implementation of this technique increased the proportion of patients on adequate antimicrobial treatment within 24 hours.

Rapid characterization of lipids by MALDI MS. Part 2: Artifacts, ion suppression, and TLC MALDI imaging

AbstractSeveral new methods have been developed recently that allow the direct detection of lipids without resorting to derivatization or chromatographic separation. The simplest of these is direct MALDI (matrix‐assisted laser desorption/ionization) mass spectrometry. This approach is most useful for mixtures that contain minimal amounts of ion‐suppressing interfering components. However, when such components are present, their effects can often be minimized by using simple separation techniques beforehand, such as solid phase extraction or thin layer chromatography. For example, direct MALDI has been used for rapid screening of lipids and taxonomic identification of the source organisms with no sample pretreatment. Collecting fractions from solid phase extraction cartridges have also been used to avoid the most extreme effects of ion suppression from more complex lipid mixtures. More recently, direct MALDI has been applied to the analysis of TLC plates allowing the detection of TLC‐separated lipids from the complex lipidome. Herein, we briefly describe the application of rapid MALDI MS to some typical research problems involving the characterization of lipids. In Part 1 [1] we covered bacterial taxonomy by direct analysis of intact lipids and the analysis of food oil triacylglycerols . Part 2 will address ion suppression, spontaneous fragmentation, and coupling MALDI with chromatography. The spontaneous fragmentation of protonated lipids in oils by direct MALDI produces artifactual diacylglycerol‐like ions. An understanding of this process and its minimization facilitates monitoring the decomposition of lipids by direct analysis. Suppression also has an impact on direct analysis of lipids, especially when mixtures contain both polar and non‐polar lipids. We demonstrate the use of solid phase extraction and thin layer chromatography to produce fractions or substrates from complex biological samples in which lipids can be detected by direct and rapid MALDI MS analysis.

Rapid characterization of lipids by MALDI MS. Part 1: Bacterial taxonomy and analysis of food oils

AbstractSeveral new methods have been developed recently that allow the direct detection of lipids without resorting to derivatization or chromatographic separation. The simplest of these is direct MALDI (matrix‐assisted laser desorption/ionization) mass spectrometry. This approach is most useful for mixtures that contain minimal amounts of ion‐suppressing interfering components. However, even when such components are present, their effects can often be minimized by using simple separation techniques beforehand, such as solid phase extraction or thin layer chromatography. For example, direct MALDI has been used for rapid screening of lipids and for taxonomic identification of the source organisms with no sample pretreatment. Fractions collected from solid phase extraction cartridges have also been used to avoid the most extreme effects of ion suppression from more complex lipid mixtures. More recently, direct MALDI has been applied to the analysis of TLC plates allowing the detection of TLC‐separated lipids from the complex lipidome. Herein, we briefly describe the application of rapid MALDI MS to some typical research problems involving the characterization of lipids. In Part 1 these include bacterial taxonomy by direct analysis of intact lipids in simple extracts rather than by conversion to fatty acid methyl esters. Food oils such as triacylglycerols can be characterized simply and easily by direct MALDI MS without resort to any sort of separation. Part 2 (in the next issue of Lipid Technology) will cover the spontaneous fragmentation of protonated lipids, ion suppression and the use of solid phase extraction and thin layer chromatography with MALDI MS to characterize complex biological samples.

Importance of Sialic Acid Residues Illuminated by Live Animal Imaging Using Phosphorylcholine Self-Assembled Monolayer-Coated Quantum Dots

Glycans are expected to be one of the potential signal molecules for controlling drug targeting/delivery or long-term circulation of biopharmaceuticals. However, the effect of the carbohydrates of artificially glycosylated derivatives on in vivo dynamic distribution profiles after intravenous injection of model animals remains unclear due to the lack of standardized methodology and a suitable platform. We report herein an efficient and versatile method for the preparation of multifunctional quantum dots (QDs) displaying common synthetic glycosides with excellent solubility and long-term stability in aqueous solution without loss of quantum yields. Combined use of an aminooxy-terminated thiol derivative, 11,11'-dithio bis[undec-11-yl 12-(aminooxyacetyl)amino hexa(ethyleneglycol)], and a phosphorylcholine derivative, 11-mercaptoundecylphosphorylcholine, provided QDs with novel functions for the chemical ligation of ketone-functionalized compounds and the prevention of nonspecific protein adsorption concurrently. In vivo near-infrared (NIR) fluorescence imaging of phosphorylcholine self-assembled monolayer (SAM)-coated QDs displaying various simple sugars (glyco-PC-QDs) after administration into the tail vein of the mouse revealed that distinct long-term delocalization over 2 h can be achieved in cases of QDs modified with α-sialic acid residue (Neu5Ac-PC-QDs) and control PC-QDs, while QDs bearing other common sugars, such as α-glucose (Glc-PC-QDs), α-mannose (Man-PC-QDs), α-fucose (Fuc-PC-QDs), lactose (Lac-PC-QDs), β-glucuronic acid (GlcA-PC-QDs), N-acetyl-β-D-glucosamine (GlcNAc-PC-QDs), and N-acetyl-β-D-galactosamine (GalNAc-PC-QDs) residues, accumulated rapidly (5-10 min) in the liver. Sequential enzymatic modifications of GlcNAc-PC-QDs gave Galβ1,4GlcNAc-PC-QDs (LacNAc-PC-QDs), Galβ1,4(Fucα1,3)GlcNAc-PC-QDs (Le(x)-PC-QDs), Neu5Acα2,3Galβ1,4GlcNAc-PC-QDs (sialyl LacNAc-PC-QDs), and Neu5Acα2,3Galβ1,4(Fucα1,3)GlcNAc-PC-QDs (sialyl Le(x)-PC-QDs) in quantitative yield as monitored by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses. Live animal imaging uncovered for the first time that Le(x)-PC-QDs also distributed rapidly in the liver after intravenous injection and almost quenched over 1 h in similar profiles to those of LacNAc-PC-QDs and Lac-PC-QDs. On the other hand, sialyl LacNAc-PC-QDs and sialyl Le(x)-PC-QDs were still retained stably in the whole body after 2 h, while they showed significantly different in vivo dynamics in the tissue distribution, suggesting that structure/sequence of the neighboring sugar residues in the individual sialyl oligosaccharides might influence the final organ-specific distribution. The present results clearly visualize the evidence of an essential role of the terminal sialic acid residue(s) for achieving prolonged in vivo lifetime and biodistribution of various glyco-PC-QDs as a novel class of functional platforms for nanomaterial-based drug targeting/delivery. A standardized protocol using multifunctional PC-QDs should facilitate live animal imaging of ligand-displayed QDs using versatile NIR fluorescence photometry without influence of size-dependent accumulation/excretion pathway for nanoparticles (e.g., viruses) >10 nm in hydrodynamic diameter by the liver.

Molecular MALDI imaging: An emerging technology for neuroscience studies

Mass spectrometry (MS) has become an essential tool for the detection, identification, and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of the nervous system. Generally, the application of these powerful techniques requires the destruction of the specimen under study, but recent technological advances have made it possible to apply the matrix-assisted laser desorption/ionization (MALDI) MS technique directly to tissue sections. The major advantage of direct MALDI analysis is that it enables the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification, and separation steps, which have the potential for introducing artifacts. With automation and the ability to display complex spectral data using imaging software, it is now possible to create multiple 2D maps of selected biomolecules in register with tissue sections, a method now known as MALDI Imaging, or MSI (for Mass Spectrometry Imaging). This creates, for example, an opportunity to correlate functional states, determined a priori with live recording or imaging, with the corresponding molecular maps obtained at the time the tissue is frozen and analyzed with MSI. We review the increasing application of MALDI Imaging to the analysis of molecular distributions of proteins and peptides in nervous tissues of both vertebrates and invertebrates, focusing in particular on recent studies of neurodegenerative diseases and early efforts to implement assays of neuronal development.

New Developments in MALDI Imaging for Pathology Proteomic Studies

With new emerging mass spectrometry technologies, it can now be demonstrated that direct tissue analysis is feasible using matrix-assisted laser desorption/ionization (MALDI) sources. A major advantage of direct MALDI analysis is to avoid time-consuming extraction, purification or separation steps, which have the potential for producing artifacts. Direct MALDI analysis of tissue sections enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. With automation and the ability to reconstruct complex spectral data using imaging software, it is now possible to produce multiplex imaging maps of selected bio-molecules within tissue sections. Thus, direct MALDI spectral data obtained from tissue sections can be converted into imaging maps, a method now known as MALDI-imaging. MALDI-imaging combines the power of mass spectrometry, namely exquisite sensitivity and unequivocal structural information, within an intact and unaltered morphological context. Critical improvements to increase image resolution are presented in this manuscript e.g., solvent treatment, new solid ionic matrices, gold sputtering, nickel support or laser focalization. One of the most important developments is the ability to carry out either direct MALDI analysis or MALDI imaging on paraffin tissue sections, thus opening the path to an archival "gold-mine" of existing pathology samples to proteomic analysis. These developments provide new avenues for biomarker hunting and diagnostic follow-up in the clinical setting. Further developments in MALDI-imaging of specific targets provide an added dimension, as validated disease-marker-gene RNA transcripts can be analyzed along with their translation by targeting their specific protein products or metabolites. Disease/health states will thus be closely molecularly monitored at protein and nucleic acids levels, with a single technique. Taken together, MALDI imaging will become a key tool for pathology proteomic studies.

Comparison of MALDI to ESI on a Triple Quadrupole Platform for Pharmacokinetic Analyses

This present work describes the systematic experimental comparison of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) for pharmacokinetic (PK) analysis of two drug candidates from rat plasma using single reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The electrospray assay is an established method using a fast liquid chromatography (LC) separation of the sample extracts prior to mass spectrometry analysis. The novel MALDI assays measured the concentration levels of the drug candidates directly from the spotted sample extracts. Importantly, for both LC-ESI and MALDI the same solid-phase sample extraction protocol, internal standards, triple quadrupole mass analyzer platform, and SRM conditions were used, thus effectively standardizing all experimental parameters of the two assays. Initially, analytical figures of merit such as linearity, limit of quantitation, precision, and accuracy were determined from the calibration curves, indicating very similar performance for both LC-ESI and MALDI. Moreover, the LC-ESI rat plasma concentration time profiles of the drug candidates after orally dosing the animals were accurately reproduced by the MALDI assay, giving virtually identical PK results. The direct MALDI assay, however, was able to generate the data at least 50 x faster than the LC-ESI assay. It is shown in this study that analyzing the entire PK curve for one animal took less than 2 min using MALDI (with five replicate analyses per sample), whereas the corresponding LC-ESI assay required 80 min, however, allowing only two replicate measurements in that time frame.

Identification of protein-binding peptides by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of peptide beads selected from the screening of one bead–one peptide combinatorial libraries

A fast and inexpensive strategy for the identification of peptide ligands by direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of peptide beads screened from one bead –one peptide combinatorial libraries is herein described. Streptavidin was used as the model protein. A combinatorial library of 6561 peptides was synthesized on ChemMatrix resin by the divide –couple –recombine method. 4-Hydroxymethylbenzoic acid was used as the linker and five residues of Gly were incorporated at the C termini to increase the final peptide molecular weight. Positive control peptides with the HPQ motif and negative control peptides without the HPQ motif evidenced that the linker and the five residues of Gly have neither impaired the specific binding nor facilitated unspecific binding. After screening the library, positive beads were isolated and washed with 8 M guanidine hydrochloride. The beads were sliced into two or four pieces, deposited onto the stainless steel MALDI sample plate, and treated with ammonia vapor to release the peptides. In addition, 26 beads picked at random from the library were subjected to the same treatment. All samples were analyzed by MALDI-TOF-MS and the peptides were unambiguously identified with very good reproducibility between the bead pieces, thus evidencing the good homogeneity of the bead. All sequences obtained from the screening contained HPQ.

Small protein biomarkers of culture in Bacillus spores detected using capillary liquid chromatography coupled with matrix assisted laser desorption/ionization mass spectrometry

Capillary liquid chromatography (cLC) coupled with matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) was used to compare small proteins and peptides extracted from Bacillus subtilis spores grown on four different media. A single, efficient protein separation, compatible with MALDI-MS analysis, was employed to reduce competitive ionization between proteins, and thus interrogate more proteins than possible using direct MALDI-MS. The MALDI-MS data files for each fraction are assembled as two-dimensional data sets of retention time and mass information. This method of visualizing small protein data required careful attention to background correction as well as mass and retention time variability. The resulting data sets were used to create comparative displays of differences in protein profiles between different spore preparations. Protein differences were found between two different solid media in both phase bright and phase dark spore phenotype. The protein differences between two different liquid media were also examined. As an extension of this method, we have demonstrated that candidate protein biomarkers can be trypsin digested to provide identifying peptide fragment information following the cLC-MALDI experiment. We have demonstrated this method on two markers and utilized acid breakdown information to identify one additional marker for this organism. The resulting method can be used to identify discriminating proteins as potential biomarkers of growth media, which might ultimately be used for source attribution.

Immunomagnetic Isolation of Enterohemorrhagic Escherichia coli O157:H7 from Ground Beef and Identification by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Database Searches

A rapid (25 min) and facile method was developed for the isolation and identification of the enterohemorrhagic Escherichia coli (serotype O157:H7) in ground beef. The isolation method employed microscopic magnetic beads coated with antibodies covalently bonded to the surface that were specific to antigens of serotype O157. This selective preconcentration step was necessary because direct matrix-assisted laser desorption/ionization (MALDI) MS analysis of bacteria was not amenable, serving to isolate the bacteria from meat components and other nontarget bacteria. The immunomagnetic separation increased the sensitivity of the method and permitted the detection of bacteria in meat. MALDI time-of-flight MS furnished bacterial mass spectra that were useful for organism identification. Molecular weight database searches using the Expert Protein Analysis System proved useful for confirmation of the organism's identity. Bacterial biomarkers from direct MALDI analysis of pure bacterial suspensions were consistently present in bacterial suspensions of buffer/tryptic soy broth (positive controls) and meat extract samples. The detection limits were 2 x 10(6) cells/mL for the experimental approach used herein. Cross-reactivity studies performed on three nontarget bacterial strains revealed that the immunomagnetic beads are specific only to E. coli strain serotype O157:H7, and there is no cross-reactivity with the other relatively innocuous strains studied.

Bacterial Identification by Protein Mass Mapping Combined with an Experimentally Derived Protein Mass Database

A protein mass mapping approach using mass spectrometry (MS) combined with an experimentally derived protein mass database is presented for rapid and effective identification of bacterial species. A prototype mass database from the protein extracts of nine bacterial species has been created by off-line high-performance liquid chromatography (HPLC) matrix-assisted laser desorption/ionization (MALDI) MS, in which the microbiological parameter of bacterial growth time is considered. A numerical method using a statistical weight factor algorithm is devised for matching the protein masses of an unknown bacterial sample against the database. The sum of these weight factors produces a corresponding summed weight factor score for each bacterial species listed in the database, and the database species producing the highest score represents the identity of the respective unknown bacterium. The applicability and reliability of this protein mass mapping approach has been tested with seven bacterial species in a single-blind study by both direct MALDI MS and HPLC electrospray ionization MS methods, and identification results with 100% accuracy are obtained. Our studies have demonstrated that the protein mass database can be rapidly established and readily adopted with relatively less dependency on experimental factors. Furthermore, it is shown that a number of proteins can be detected using a protein sample amount equivalent to an extract of less than 1000 cells, demonstrating that this protein mass mapping approach can potentially be highly sensitive for rapid bacterial identification.