Electron monochromator mass spectrometry for the analysis of whole bacteria and bacterial spores

Spores from a variety of Bacillus species were analyzed with direct probe mass spectrometry using an electron monochromator to select electrons of distinct energies for ionization. Electron energies were chosen to match the electron capture energies of taxonomically important compounds such as dipicolinic acid and fatty acids. Previous negative ion interferences were not observed when the monochromator was used, and the signal-to-noise ratio of targeted compounds was significantly enhanced using this approach. To demonstrate the selectivity of the technique, the monochromator was swept over a range of electron energies while monitoring the masses of compounds with known electron capture energies. Scanning the monochromator while the mass spectrometer was operated in single-ion mode enabled dipicolinic acid to be detected in 10(5) spores. The results presented here demonstrate the utility of the electron monochromator for selectively ionizing compounds directly in bacteria and bacterial spores.     

Hollow-fiber flow field-flow fractionation for whole bacteria analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This work proposes for the first time the use of hollow-fiber flow field-flow fractionation (HF FlFFF) for improved matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOFMS) of whole bacteria. HF FlFFF has proved to be able to prepurify or fractionate different species of whole bacteria. Sample preparation by HF FlFFF gives improved spectra quality because noncellular components possibly present in the sample can be separated from the cells. When a mixture of two bacteria (Bacillus subtilis and Escherichia coli) is fractionated through HF FlFFF, MALDI/TOFMS analysis of each separated bacterial species preserves the most characteristic ion signals of the species without the presence of characteristic signals of the other species. The main advantages of HF FlFFF for MALDI/TOFMS analysis of whole bacteria are miniaturization, simplicity, and low cost of the fractionator components. This low cost makes disposable usage of the fractionator possible, thus eliminating the risk of run-to-run contamination of spectra due to sample carryover. The low fractionator volume yields bacterial fractionation on the order of a few minutes, which is comparable to MALDI/TOFMS analysis time. The small fractionation volume makes sample dilution low enough so that additional sample concentration steps are not strictly required to preserve MALDI/TOFMS detection.     

Charge monitoring cell mass spectrometry

An instrument to directly measure the charge carried by a cell or a microparticle as well as mass-to-charge ratio of the cell/microparticle was developed for rapid mass distribution measurement. A successful mass spectrum with a record high mass has been demonstrated. In this article, the details of the construction and operation of the charge monitoring cell mass spectrometer are reported. Examples are also given for demonstration and discussion.     

On the scalability and requirements of whole protein mass spectrometry

Top-down proteomics has improved over the past decade despite the significant challenges presented by the analysis of large protein ions. Here, the detection of these high mass species by electrospray-based mass spectrometry (MS) is examined from a theoretical perspective to understand the mass-dependent increases in the number of charge states, isotopic peaks, and interfering species present in typical protein mass spectra. Integrating these effects into a quantitative model captures the reduced ability to detect species over 25 kDa with the speed and sensitivity characteristic of proteomics based on <3 kDa peptide ions. The model quantifies the challenge that top-down proteomics faces with respect to current MS instrumentation and projects that depletion of (13)C and (15)N isotopes can improve detection at high mass by only <2-fold at 100 kDa whereas the effect is up to 5-fold at 10 kDa. Further, we find that supercharging electrosprayed proteins to the point of producing <5 charge states at high mass would improve detection by more than 20-fold.     

High-speed mass analysis of whole erythrocytes by charge-detection quadrupole ion trap mass spectrometry

Herein, we report an application of charge-detection quadrupole ion trap mass spectrometry to the measurement of total dry masses of mammalian and poultry erythrocytes evaporated/ionized by laser-induced acoustic desorption. The method is rapid and widely applicable. Eight different types of red blood cells (RBCs) have been successfully analyzed, including those of human, goat, cow, mouse, pig, and chicken. The measured mean masses (weights per corpuscle) range from 0.58 x 10(13) Da (9.6 pg) of goat RBCs to 2.80 x 10(13) Da (46.5 pg) of chicken RBCs. The total dry weights determined for human RBCs from a healthy male adult, a patient with iron-deficiency anemia, and a patient with thalassemia are 34.8, 28.8, and 20.6 pg, respectively. These weights, except that of thalassemia, are all approximately 10% higher than their corresponding mean corpuscular hemoglobin values determined by a commercial automated hematology analyzer. The mass distribution profiles of the cells are all near-Gaussian, with a standard deviation of 15% for the normal human RBCs. The deviation increases significantly to 20% for RBCs with thalassemia characteristics and 27% for RBCs with iron-deficiency anemia characteristics. All the observations are in accord with their corresponding mean corpuscular volume measurements, indicating an increase in anisocytosis (variation in RBC size) in the anemic samples. Our results suggest a broad and promising application of this new technology to high-speed mass analysis of RBCs and other biological whole cells as well.     

Explanatory optimization of protein mass spectrometry via genetic search

Optimizing experimental conditions for the effective analysis of intact proteins by mass spectrometry is challenging, as many analytical factors influence the spectral quality, often in very different ways for different proteins and especially with complex protein mixtures. We show that genetic search methods are highly effective in this kind of optimization and that it was possible in 6 generations with a total of <500 experiments out of some 10(14) to find good combinations of experimental variables (electrospray ionization mass spectral settings) that would not have been detected by optimizing each variable alone (i.e., the search space is epistatic). Moreover, by inspecting the evolution of the variables to be optimized using genetic programming, we discovered an important relationship between two of the mass spectrometer settings that accounts for much of this success. Specifically, the conditions that were evolved included very low values of skimmer 1 voltage (the sample cone) and a skimmer 2 voltage (extraction cone) above a threshold that would nevertheless minimize the potential difference between the sample and extraction skimmers. The discovery of this relationship demonstrates the hypothesis-generating ability of genetic search in optimization processes where the size of the search space means that little or no a priori knowledge of the optimal conditions is available.     

Phosphorylation site identification via ion trap tandem mass spectrometry of whole protein and peptide ions: bovine alpha-crystallin A chain

Tandem mass spectrometry was applied both to ions of a tryptic fragment and intact protein of bovine alpha-crystallin A chain to localize the single site of phosphorylation. The [M + 19H](19+) to [M + 11H](11+) charge states of both phosphorylated and unphosphorylated bovine alpha-crystallin A chain whole protein ions were subjected to collisional activation in a quadrupole ion trap. Ion parking was used to increase the number of parent ions over that yielded by electrospray. Ion-ion proton-transfer reactions were used to reduce the product ion charge states largely to +1 to simplify spectral interpretation. In agreement with previous studies on whole protein ion fragmentation, both protein forms showed backbone cleavages C-terminal to aspartic acid residues at lower charge states. The phosphorylated protein showed competitive fragmentation between backbone cleavage and the neutral loss of phosphoric acid. Analysis of which backbone cleavage products did or did not contain the phosphate was used to localize the site of phosphorylation to one of two possible serine residues. A tryptic digest of the bovine alpha-crystallin A chain yielded a phosphopeptide containing one missed cleavage site. The peptide provided information complementary to that obtained from the intact protein and localized the modified serine to residue 122. Fragmentation of the triply charged phosphopeptide yielded five possible serine phosphorylation sites. Fragmentation of the doubly charged phosphopeptide, formed by ion/ion proton-transfer reactions, positively identified the phosphorylation site as serine-122.     

Discrimination of polymers by using their characteristic collision energy in tandem mass spectrometry

The characteristic collision energy to obtain 50% fragmentation, expressed as the characteristic collision voltage (CCV), was used as a tool to discriminate different classes of polymers. The CCV value of different polymers was determined in a quadrupole ion trap mass spectrometer. Good linear correlation (0.980 < R(2) < 0.999) between the CCV values and precursor ion mass was found for all polymers studied. The position of the various linear trend lines varied among the various polymers, which allowed their grouping based on the respective CCV values. The collision energy necessary to drive fragmentation was decreasing in the order of polyethers > polymethacrylates > polyesters > polysaccharides. This suggests that polysaccharides fragment most easily (low CCVs), while polyethers require the highest collision energy among the polymers studied. The effect of end group on the CCV was also studied, showing a minor influence in most cases. In addition, the applicability of CCV as discriminator was studied for a mixture of (1) polylactic acid (PLA), (2) poly(tetramethylene glycol) (PTMEG), and (3) PLA-block-PTMEG-block-PLA block copolymer. Differences between the CCV values of four nominally isobaric polymers (of which two were copolymers and two were homopolymers) were observed. These results demonstrate that the insertion of a "weak" link into a polymer chain significantly affects the energy required for fragmentation.     

Nano-LC FTICR tandem mass spectrometry for top-down proteomics: routine baseline unit mass resolution of whole cell lysate proteins up to 72 kDa

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.     

Elemental imaging via laser ionization orthogonal time-of-flight mass spectrometry

An elemental imaging method using a laser ionization orthogonal time-of-flight mass spectrometer system was developed for the simultaneous detection of all metal and nonmetal elements. The instrument control and data processing were realized by self-developed programs. This system is capable of simultaneous detection of metal and nonmetal elements, with a spatial resolution of 50 μm, the lowest detection limits of 3 × 10(-7) g/g (Li), and a dynamic range of 7 orders of magnitude. Moreover, this technique does not require standards for quantitative analysis and can be a powerful and versatile tool for elemental imaging.     

Identification of biomarkers of whole Coxiella burnetii phase I by MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) specific biomarkers have been shown to be an effective tool for identifying microorganisms. In this study, we demonstrate the feasibility of using this technique to detect the obligate intracellular bacterium Coxiella burnetii, a category B bioterrorism agent. Specific biomarkers were detected in C. burnetii Nine Mile phase I (NMI) strain purified from embryonated egg yolk sac preparations. Whole organisms were applied directly to the MALDI target. MALDI-TOF MS analysis of C. burnetii NMI grown and purified at different times and places revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 1000-25000 Da. Statistical analysis of the averaged centroided masses uncovered at least 24 peptides or biomarkers. Three biomarkers observed in the MALDI-TOF MS spectrum consistently matched proteins that had been previously described in C. burnetii, one of them being the small cell variant protein A. MALDI-TOF MS analysis of whole organisms represents a sensitive and specific option for characterizing C. burnetii isolates, especially when coupled with antigen capture techniques. The method also has potential for several applications in basic microbial research, including regulation of gene expression.     

Imaging mass spectrometry of three-dimensional cell culture systems

Three-dimensional (3D) cell cultures have increased complexity compared to simple monolayer and suspension cultures, recapitulating the cellular architecture and molecular gradients in tissue. As such, they are popular for in vitro models in biological research. Classical imaging methodologies, like immunohistochemistry, are commonly used to examine the distribution of specific species within the spheroids. However, there is a need for an unbiased discovery-based methodology that would allow examination of protein/peptide distributions in 3D culture systems, without a need for prior knowledge of the analytes. We have developed a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS)-based imaging approach to examine protein distributions in 3D cell culture models. Using colon carcinoma cell lines, we detect changes in the spatial distribution of proteins across 3D culture structures. To identify the protein species present, we are combining results from the MS/MS capabilities of MALDI-MS to sequence peptides in a de novo fashion and nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) of homogenized cultures. As a proof-of-principle, we have identified cytochrome C and Histone H4 as two of the predominant protein species in the 3D colon carcinoma cultures.     

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.     

Molecular analysis of native tissue and whole oils by infrared laser mass spectrometry

We have employed infrared laser desorption ionization orthogonal time-of-flight mass spectrometry (IR-LDI-o-TOF-MS) to generate molecular ion profiles directly from native tissue and from whole oils. The method requires little sample preparation besides an eventual dissection of the areas of interest and drying of particularly water-rich samples. The lateral resolution of the analysis is on the order of the laser focal diameter, and in the third dimension, defined by the depth of material ejection, a few to 10 microm per laser pulse. Various types of small molecules are readily detected from minute volumes of sample. Among these are carbohydrates, phospholipids, triglycerides, and flavonoids. Substantially different molecular profiles were recorded from different areas of a single strawberry seed.     

Valved sampling cell for membrane introduction mass spectrometry

Membrane extractors comprising a membrane house inside of a valve have been developed to separate compounds of interest from a sample matrix and introduce these compounds into a mass spectrometer. Experimental control over parameters that affect permeability or that may damage the membrane, such as the membrane temperature, is provided with the valve. The valve was tested for response and response times with the valve separated from the mass spectrometer by various interface tube lengths. Data for steady state response measurements showed no significant change with the valve at different distances from the ion source. Polar compounds show a strong response time dependency on the interface tube length. This adsorption phenomenon is minimized by simply heating the interface tube. Other factors affecting the performance of the device are discussed.     

Qualitative and quantitative analysis of tumor cell metabolism via stable isotope labeling assisted microfluidic chip electrospray ionization mass spectrometry

In this work, a stable isotope labeling assisted microfluidic chip electrospray ionization mass spectrometry (SIL-chip-ESI-MS) platform for qualitative and quantitative analysis of cell metabolism was developed. Microfluidic cell culture, drug-induced cell apoptosis analysis, and cell metabolism measurements were performed simultaneously on the specifically designed device. MCF-7 cells were cultivated in vitro and exposed in anticancer agent (genistein and genistein-d(2)) for cell-based drug assay. A dual-isotopic labeling was presented for effective qualitative analysis of multiplex metabolites. Interestingly, three coeluting pairs of isotopomers appeared with an m/z difference of two. Despite complex biological matrixes, they can be easily recognized and identified by chip-ESI-MS/MS, which significantly facilitates candidate biomarker discovery. The quantitative performance of this system was evaluated using genistein as a model drug by means of stable isotope dilution analysis. The linear equation obtained is y = 0.06x - 3.38 × 10(-3) (R(2) = 0.995) at the dynamic range from 0.5 to 40 μM. The detection limit is 0.2 μM. The method shows an excellent stability of 2.2% relative standard deviation (RSD) and a good repeatability of 5.5% RSD. Our results have successfully demonstrated the capability of selective and quantitative analysis of cell-based drug absorption and metabolites with high stability, sensitivity, and repeatability on the chip-ESI-MS system. Consequently, the present device shows promise as a high-throughput, low-cost, and online platform for cell metabolism studies and drug screening processes.     

Characterization of protein cross-links via mass spectrometry and an open-modification search strategy

Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying cross-linked peptides from single LC/MS/MS data sets, without using specialized cross-linkers or custom-written software. To achieve this, each peptide in the pair of cross-linked peptides was considered to be "post-translationally" modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of cross-linked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich data sets for cross-linked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native cross-links in proteins. The pipeline was validated by characterizing the known interactions in the chemically cross-linked CYP2E1-b5 complex. Utility of this method in identifying native cross-links was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.     

Automated proteomics of E. coli via top-down electron-transfer dissociation mass spectrometry

Electron-transfer dissociation (ETD) has recently been introduced as a fragmentation method for peptide and protein analysis. Unlike collisionally induced dissociation (CID), fragmentation by ETD occurs randomly along the peptide backbone. With the use of the sequences determined from the protein termini and the parent protein mass, intact proteins can be unambiguously identified. Because of the fast kinetics of these reactions, top-down proteomics can be performed using ETD in a linear ion trap mass spectrometer on a chromatographic time scale. Here we demonstrate the utility of ETD in high-throughput top-down proteomics using soluble extracts of E. coli. Development of a multidimensional fractionation platform, as well as a custom algorithm and scoring scheme specifically designed for this type of data, is described. The analysis resulted in the robust identification of 322 different protein forms representing 174 proteins, comprising one of the most comprehensive data sets assembled on intact proteins to date.