Errol W. Robinson

Mass spectrometry-based proteomics

Mass spectrometry (MS)-based proteomics is emerging as a broadly effective means for identification, characterization, and quantification of proteins that are integral components of the processes essential for life. Characterization of proteins at the proteome and sub-proteome (e.g., the phosphoproteome, proteoglycome, or degradome/peptidome) levels provides a foundation for understanding fundamental aspects of biology. Emerging technologies such as ion mobility separations coupled with MS and microchip-based-proteome measurements combined with MS instrumentation and chromatographic separation techniques, such as nanoscale reversed phase liquid chromatography and capillary electrophoresis, show great promise for both broad undirected and targeted highly sensitive measurements. MS-based proteomics increasingly contribute to our understanding of the dynamics, interactions, and roles that proteins and peptides play, advancing our understanding of biology on a systems wide level for a wide range of applications including investigations of microbial communities, bioremediation, and human health.

Trapped-ion cell with improved DC potential harmonicity for FT-ICR MS.

The trapped-ion cell is a key component critical for optimal performance in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). To extend the performance of FT-ICR MS, we have developed a new cell design that is capable of generating a DC trapping potential which closely approaches that of an ideal Penning trap, i.e., a 3D axial quadrupolar potential distribution. The new cell design was built upon an open cylindrical geometry, supplemented with two pairs of cylindrical compensation segments. Electric potential calculations for trial cell geometries were aimed at minimizing spatial variations of the radial electric field divided by radius. The resulting cell proportions and compensation voltages delivered practically constant effective ion cyclotron frequency that was independent of ion radial and axial positions. Our customized 12 tesla FT-ICR instrument was upgraded with the new cell, and the performance was characterized for a range of ion excitation power and ion populations. Operating the compensated cell at increased postexcitation radii, ∼0.7 of the cell inner radius, resulted in improved mass measurement accuracy together with increased signal intensity. Under these same operating conditions the noncompensated open cell configuration exhibited peak splitting and reduced signal life time. Mass accuracy tests using 11 calibrants covering a wide m/z range reproducibly produced under 0.05 ppm RMS precision of the internal calibration for reduced ion populations and the optimal excitation radius. Conditions of increased ion population resulted in a twofold improvement in mass accuracy compared with the noncompensated cell, due to the larger achievable excitation radii and correspondingly lower space charge related perturbations of the calibration law.

Multidimensional separations of ubiquitin conformers in the gas phase: Relating ion cross sections to H/D exchange measurements

Investigating gas-phase structures of protein ions can lead to an improved understanding of intramolecular forces that play an important role in protein folding. Both hydrogen/deuterium (H/D) exchange and ion mobility spectrometry provide insight into the structures and stabilities of different gas-phase conformers, but how best to relate the results from these two methods has been hotly debated. Here, high-field asymmetric waveform ion mobility spectrometry (FAIMS) is combined with Fourier-transform ion cyclotron resonance mass spectrometry (FT/ICR MS) and is used to directly relate ubiquitin ion cross sections and H/D exchange extents. Multiple conformers can be identified using both methods. For the 9+ charge state of ubiquitin, two conformers (or unresolved populations of conformers) that have cross sections differing by 10% are resolved by FAIMS, but only one conformer is apparent using H/D exchange at short times. For the 12+ charge state, two conformers (or conformer populations) have cross sections differing by <1%, yet H/D exchange of these conformers differ significantly (6 versus 25 exchanges). These and other results show that ubiquitin ion collisional cross sections and H/D exchange distributions are not strongly correlated and that factors other than surface accessibility appear to play a significant role in determining rates and extents of H/D exchange. Conformers that are not resolved by one method could be resolved by the other, indicating that these two methods are highly complementary and that more conformations can be resolved with this combination of methods than by either method alone.

Enhanced top-down characterization of histone post-translational modifications

Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS analysis of 7.5 µg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.

The role of conformation on electron capture dissociation of ubiquitin.

Effects of protein conformation on electron capture dissociation (ECD) were investigated using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and Fourier-transform ion cyclotron resonance mass spectrometry. Under the conditions of these experiments, the electron capture efficiency of ubiquitin 6+ formed from three different solution compositions differs significantly, ranging from 51 ± 7% for ions formed from an acidified water/methanol solution to 88 ± 2% for ions formed from a buffered aqueous solution. This result clearly indicates that these protein ions retain a memory of their solution-phase structure and that conformational differences can be probed in an ECD experiment. Multiple conformers for the 7+ and 8+ charge states of ubiquitin were separated using FAIMS. ECD spectra of conformer selected ions of the same charge states differ both in electron capture efficiency and in the fragment ion intensities. Conformers of a given charge state that have smaller collisional cross sections can have either a larger or smaller electron capture efficiency. A greater electron capture efficiency was observed for ubiquitin 6+ that has the same collisional cross section as one ubiquitin 7+ conformer, despite the lower charge state. These results indicate that the shape of the molecule can have a greater effect on electron capture efficiency than either collisional cross section or charge state alone. The cleavage locations of different conformers of a given charge state were the same indicating that the presence of different conformers in the gas phase is not due to difference in where charges are located, but rather reflect conformational differences most likely originating from solution. Small neutral losses observed from the singly- and doubly-reduced ubiquitin 6+ do not show a temperature dependence to their formation, consistent with these ions being formed by nonergodic processes.

An integrated top-down and bottom-up strategy for broadly characterizing protein isoforms and modifications

We present an integrated top-down and bottom-up approach that is facilitated by concurrent liquid chromatography-mass spectrometry (LC-MS) analysis and fraction collection for comprehensive high-throughput intact protein profiling. The approach employs high-resolution, reversed-phase (RP) LC separations coupled on-line with a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer to profile and tentatively identify modified proteins, using detected intact protein masses in conjunction with bare protein identifications from the bottom-up analysis of the corresponding LC fractions. Selected identifications are incorporated into a target ion list for subsequent off-line gas-phase fragmentation that uses an aliquot of the original fraction used for bottom-up analysis. In a proof-of-principle demonstration, this comprehensive strategy was applied to identify protein isoforms arising from various amino acid modifications (e.g., acetylation, phosphorylation) and genetic variants (e.g., single nucleotide polymorphisms, SNPs). This strategy overcomes major limitations of traditional bottom-up (e.g., inability to characterize multiple unexpected protein isoforms and genetic variants) and top-down (e.g., low throughput) approaches.

Enhanced Sensitivity for Selected Reaction Monitoring Mass Spectrometry-based Targeted Proteomics Using a Dual Stage Electrodynamic Ion Funnel Interface

Selected reaction monitoring mass spectrometry (SRM-MS) is playing an increasing role in quantitative proteomics and biomarker discovery studies as a method for high throughput candidate quantification and verification. Although SRM-MS offers advantages in sensitivity and quantification compared with other MS-based techniques, current SRM technologies are still challenged by detection and quantification of low abundance proteins (e.g. present at ∼10 ng/ml or lower levels in blood plasma). Here we report enhanced detection sensitivity and reproducibility for SRM-based targeted proteomics by coupling a nanospray ionization multicapillary inlet/dual electrodynamic ion funnel interface to a commercial triple quadrupole mass spectrometer. Because of the increased efficiency in ion transmission, significant enhancements in overall signal intensities and improved limits of detection were observed with the new interface compared with the original interface for SRM measurements of tryptic peptides from proteins spiked into non-depleted mouse plasma over a range of concentrations. Overall, average SRM peak intensities were increased by ∼70-fold. The average level of detection for peptides also improved by ∼10-fold with notably improved reproducibility of peptide measurements as indicated by the reduced coefficients of variance. The ability to detect proteins ranging from 40 to 80 ng/ml within mouse plasma was demonstrated for all spiked proteins without the application of front-end immunoaffinity depletion and fractionation. This significant improvement in detection sensitivity for low abundance proteins in complex matrices is expected to enhance a broad range of SRM-MS applications including targeted protein and metabolite validation.

Proteogenomic Analysis of a Thermophilic Bacterial Consortium Adapted to Deconstruct Switchgrass

Thermophilic bacteria are a potential source of enzymes for the deconstruction of lignocellulosic biomass. However, the complement of proteins used to deconstruct biomass and the specific roles of different microbial groups in thermophilic biomass deconstruction are not well-explored. Here we report on the metagenomic and proteogenomic analyses of a compost-derived bacterial consortium adapted to switchgrass at elevated temperature with high levels of glycoside hydrolase activities. Near-complete genomes were reconstructed for the most abundant populations, which included composite genomes for populations closely related to sequenced strains of Thermus thermophilus and Rhodothermus marinus, and for novel populations that are related to thermophilic Paenibacilli and an uncultivated subdivision of the little-studied Gemmatimonadetes phylum. Partial genomes were also reconstructed for a number of lower abundance thermophilic Chloroflexi populations. Identification of genes for lignocellulose processing and metabolic reconstructions suggested Rhodothermus, Paenibacillus and Gemmatimonadetes as key groups for deconstructing biomass, and Thermus as a group that may primarily metabolize low molecular weight compounds. Mass spectrometry-based proteomic analysis of the consortium was used to identify >3000 proteins in fractionated samples from the cultures, and confirmed the importance of Paenibacillus and Gemmatimonadetes to biomass deconstruction. These studies also indicate that there are unexplored proteins with important roles in bacterial lignocellulose deconstruction.

C60 Secondary Ion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Secondary ion mass spectrometry (SIMS) has seen increased application for high spatial resolution chemical imaging of complex biological surfaces. The advent and commercial availability of cluster and polyatomic primary ion sources (e.g., Au and Bi cluster and buckminsterfullerene (C(60))) provide improved secondary ion yield and decreased fragmentation of surface species, thus improving accessibility of intact molecular ions for SIMS analysis. However, full exploitation of the advantages of these new primary ion sources has been limited, due to the use of low mass resolution mass spectrometers without tandem MS to enable enhanced structural identification capabilities. Similarly, high mass resolution and high mass measurement accuracy would greatly improve the chemical specificity of SIMS. Here we combine, for the first time, the advantages of a C(60) primary ion source with the ultrahigh mass resolving power and high mass measurement accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Mass resolving power in excess of 100 000 (m/Δm(50%)) is demonstrated, with a root-mean-square mass measurement accuracy below 1 part-per-million. Imaging of mouse brain tissue at 40 μm pixel size is shown. Tandem mass spectrometry of ions from biological tissue is demonstrated and molecular formulas were assigned for fragment ion identification.

Evidence supporting dissimilatory and assimilatory lignin degradation in Enterobacter lignolyticus SCF1

Lignocellulosic biofuels are promising as sustainable alternative fuels, but lignin inhibits access of enzymes to cellulose, and by-products of lignin degradation can be toxic to cells. The fast growth, high efficiency and specificity of enzymes employed in the anaerobic litter deconstruction carried out by tropical soil bacteria make these organisms useful templates for improving biofuel production. The facultative anaerobe Enterobacter lignolyticus SCF1 was initially cultivated from Cloud Forest soils in the Luquillo Experimental Forest in Puerto Rico, based on anaerobic growth on lignin as sole carbon source. The source of the isolate was tropical forest soils that decompose litter rapidly with low and fluctuating redox potentials, where bacteria using oxygen-independent enzymes likely play an important role in decomposition. We have used transcriptomics and proteomics to examine the observed increased growth of SCF1 grown on media amended with lignin compared to unamended growth. Proteomics suggested accelerated xylose uptake and metabolism under lignin-amended growth, with up-regulation of proteins involved in lignin degradation via the 4-hydroxyphenylacetate degradation pathway, catalase/peroxidase enzymes, and the glutathione biosynthesis and glutathione S-transferase (GST) proteins. We also observed increased production of NADH-quinone oxidoreductase, other electron transport chain proteins, and ATP synthase and ATP-binding cassette (ABC) transporters. This suggested the use of lignin as terminal electron acceptor. We detected significant lignin degradation over time by absorbance, and also used metabolomics to demonstrate moderately significant decreased xylose concentrations as well as increased metabolic products acetate and formate in stationary phase in lignin-amended compared to unamended growth conditions. Our data show the advantages of a multi-omics approach toward providing insights as to how lignin may be used in nature by microorganisms coping with poor carbon availability.