361 research outputs found
Electrostatic and affinity enhancements of protein partitioning in two-phase aqueous micellar systems
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2005.Includes bibliographical references (p. 175-188).This thesis was motivated by the practical need to develop a scalable and cost-effective separation method for low-cost, high-volume protein products. This unmet challenge can potentially be addressed by extraction in two-phase aqueous micellar systems, in which biomolecules can be partitioned in mild, predominantly aqueous environments. The goal of this thesis was to explore various ways of enhancing protein partitioning in two-phase aqueous micellar systems, by the incorporation of electrostatic and affinity interactions, to obtain satisfactory yield and specificity for the purification of industrially relevant hydrophilic proteins. The electrostatically-enhanced partitioning of the enzyme glucose-6-phosphate dehydrogenase (G6PD) in two-phase aqueous mixed (nonionic/cationic) micellar systems was investigated experimentally and theoretically. The successful enhancement, up to 22-fold, of the partitioning of the negatively-charged G6PD was attained by adding the positively- charged surfactant alkyltrimethylammonium bromide (CnTAB) to form charged mixed micelles with the phase-forming nonionic surfactant, decyl tetra(ethylene oxide) (C₁₀E₄).(cont.) The effects of the tail length of the positively-charged surfactant on protein denaturation and protein partitioning behavior were also studied. Furthermore, the experimental results were used to validate a predictive theory for electrostatic enhancement. In the area of affinity enhancement, the affinity-enhanced partitioning of an engineered affinity-tagged protein, CBM9-GFP (Green Fluorescent Protein linked to a carbohydrate- binding module), in two-phase aqueous micellar systems was investigated experimentally and theoretically. The experimental results showed that the partition coefficient of the target protein, CBM9-GFP, can be improved more than 6-fold, by virtue of the affinity interactions, and that the enhancement is specific to the target protein. The system utilized requires only one surfactant, decyl [beta]-D-glucopyranoside (C₁₀G₁), which acts simultaneously as the affinity ligand and as the phase-forming surfactant, and as such, has important practical advantages. A novel theoretical framework to describe affinity- enhanced protein partitioning in two-phase aqueous micellar systems was developed and validated experimentally. In addition, the separation method developed was successfully applied to a real cell lysate.(cont.) It was found that the protein impurities in the cell lysate do not interfere with the partitioning of the target protein (CBM9-GFP) at industrially relevant concentrations, and that the protein impurities were concentrated away from the target protein. Lastly, the theoretical description developed was used to identify various strategies for improving the affinity-enhanced partitioning of the target protein in two-phase aqueous micellar systems. Although more work remains to be done before the separation methods studied in this thesis can reach their full potential and be eventually commercialized, this thesis nevertheless represents an essential starting point for future efforts to improve, extend, and commercialize this promising bioseparation method.by Hei Ning Henry Lam.Ph.D
Robust estimation of bacterial cell count from optical density
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data
Expanding Tandem Mass Spectral Libraries of Phosphorylated Peptides: Advances and Applications
The identification of phosphorylated proteins remains a challenge in proteomics, partially due to the difficulty in assigning tandem mass (MS/MS) spectra to their originating peptide sequences with correct phosphosite localization. Because of its advantages in efficiency and sensitivity, spectral library searching is a promising alternative to conventional sequence database searching. Our work aims to construct the largest collision-induced dissociation (CID) MS/MS spectral libraries of phosphorylated peptides in human (Homo sapiens) and four model organisms (Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus) to date, to facilitate phosphorylated peptide identification by spectral library searching. We employed state-of-the-art search methods to published data and applied two recently published phosphorylation site i localization tools (PhosphoRS and PTMProphet) to ascertain the phosphorylation sites. To further increase the coverage of this library, we predicted "semi-empirical" spectra for peptides containing known phosphorylation sites from the corresponding template unphosphorylated peptide spectra. The performance of the spectral libraries built were evaluated and found to be superior to conventional database searching in terms of sensitivity. Updated spectral libraries of phosphorylated peptides are made freely available for use with the spectral search engine SpectraST. The work flow being developed will be used to continuously update the libraries when new data become available
Expanding spectral libraries of phosphorylated peptides: Progress in coverage and accuracy
A semi-empirical approach to predict unobserved peptide MS/MS spectra from spectral libraries
Utilizing unidentified tandem mass spectral libraries for biological sample fingerprinting
Graph-based time alignment algorithms for LC-MS datasets with large retention-time drifts
Liquid chromatography coupled to mass spectrometry (LC-MS) is the dominant technological platform for proteomics. An LC-MS analysis of a complex biological sample can be visualized as a “map” of which the positional coordinates are the mass-to-charge ratio (m/z) and chromatographic retention time (RT) of the chemical species profiled. Label-free quantitative proteomics requires the alignment and comparison of multiple LC-MS maps to ascertain the reproducibility of experiments or reveal proteome changes under different conditions. The main challenge in this task lies in correcting retention time shifts, which are inevitable even on the same instrument and under the same elution conditions. For large-scale studies, multiple instruments or multi-week experiments are often required, which exacerbates the problem. Similar, but not identical, LC instruments and settings can cause peptides to elute in a different order, violating the key assumption of many state-of-the-art alignment tools. We present a new graph-based time alignment algorithm that can align these less similar LC-MS maps, which cannot be effectively handled by existing methods. We developed WBMatch, which is based on an efficient weighted bipartite matching algorithm from graph theory, to align different LC-MS maps. Instead of finding warping functions to correct the variations in the retention time dimension, WBMatch directly tries to find a peak-to-peak mapping that maximize a similarity function between two LC-MS maps. The similarity function is a combination of m/z, retention time and intensity deviations of all aligned peaks. In order to handle large retention time shifts between LC-MS experiments conducted from different instruments or settings, an additional step of locally weighted scatterplot smoothing is performed prior to WBMatch, forming a new method called LWBMatch. For validation, we defined the ground-truth for alignment success based on MS/MS identifications from sequence searching. We showed that our method outperforms several existing tools in terms of precision and recall, and is capable of aligning maps from different instruments and settings
A semi-empirical approach for predicting unobserved peptide MS/MS spectra from spectral libraries
Spectral library searching is a promising alternative to sequence database searching in peptide identification from MS/MS spectra. The key advantage of spectral library searching is the utilization of more spectral features to improve score discrimination between good and bad matches, and hence sensitivity. However, the coverage of reference spectral library is limited by current experimental and computational methods. We developed a computational approach to expand the coverage of spectral libraries with semi-empirical spectra predicted from perturbing known spectra of similar sequences, such as those with single amino acid substitutions. We hypothesized that the peptide of similar sequences should produce similar fragmentation patterns, at least in most cases. Our results confirm our hypothesis and specify when this approach can be applied. In actual spectral searching of real data sets, the sensitivity advantage of spectral library searching over sequence database searching can be mostly retained even when all real spectra are replaced by semi-empirical ones. We demonstrated the applicability of this approach by detecting several known non-synonymous single-nucleotide polymorphisms in three large human data sets by spectral searching. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Site-specific N-linked glycoproteomics analysis by nano-LC tandem mass spectrometry and spectral library searching
A new N-linked glycopeptide identification tool, for large-scale glycoproteomics analysis by spectral library searching
- …