13 research outputs found
Culture-Independent Analysis of Fecal Enterobacteria in Environmental Samples by Single-Cell mRNA Profiling
A culture-independent method called mRNA profiling has been developed for the analysis of fecal enterobacteria and their physiological status in environmental samples. This taxon-specific approach determines the single-cell content of selected gene transcripts whose abundance is either directly or inversely proportional to growth state. Fluorescence in situ hybridization using fluorochrome-labeled oligonucleotide probes was used to measure the cellular concentration of fis and dps mRNA. Relative levels of these transcripts provided a measure of cell growth state and the ability to enumerate fecal enterobacterial cell number. Orthologs were cloned by inverse PCR from several major enterobacterial genera, and probes specific for fecal enterobacteria were designed using multiple DNA sequence alignments. Probe specificity was determined experimentally using pure and mixed cultures of the major enterobacterial genera as well as secondary treated wastewater samples seeded with pure culture inocula. Analysis of the fecal enterobacterial community resident in unseeded secondary treated wastewater detected fluctuations in transcript abundance that were commensurate with incubation time and nutrient availability and demonstrated the utility of the method using environmental samples. mRNA profiling provides a new strategy to improve wastewater disinfection efficiency by accelerating water quality analysis
Single-Cell Protein Profiling of Wastewater Enterobacterial Communities Predicts Disinfection Efficiency
The efficiency of enterobacterial disinfection is dependent largely on enterobacterial community physiology. However, the relationship between enterobacterial community physiology and wastewater processing is unclear. The purpose of this study was to investigate this relationship. The influence of wastewater treatment processes on enterobacterial community physiology was examined at the single-cell level by using culture-independent methods. Intracellular concentrations of two conserved proteins, the growth-related protein Fis and the stationary-phase protein Dps, were analyzed by epifluoresence microscopy of uncultivated cells by using enterobacterial group-specific polyclonal fluorochrome-coupled antibodies. Enterobacterial single-cell community protein profiles were distinct for different types of biological treatment. The differences were not apparent when bulk methods of protein analysis were used. Trickling filter wastewater yielded Fis-enriched communities compared to the communities in submerged aeration basin wastewater. Community differences in Fis and Dps contents were used to predict disinfection efficiency. Disinfection of community samples by heat exposure combined with cultivation in selective media confirmed that enterobacterial communities exhibited significant differences in sensitivity to disinfection. These findings provide strategies that can be used to increase treatment plant performance, reduce the enterobacterial content in municipal wastewater, and minimize the release of disinfection by-products into receiving water
Single-cell protein profiling of wastewater enterobacterial communities predicts disinfection efficiency
Characterization of Cysteinylation and Trisulfide Bonds in a Recombinant Monoclonal Antibody
Accurate Determination of Protein Methionine Oxidation by Stable Isotope Labeling and LC-MS Analysis
Methionine
(Met) oxidation is a major modification of proteins,
which converts Met to Met sulfoxide as the common product. It is challenging
to determine the level of Met sulfoxide, because it can be generated
during sample preparation and analysis as an artifact. To determine
the level of Met sulfoxide in proteins accurately, an isotope labeling
and LC-MS peptide mapping method was developed. Met residues in proteins
were fully oxidized using hydrogen peroxide enriched with <sup>18</sup>O atoms before sample preparation. Therefore, it was impossible to
generate Met sulfoxide as an artifact during sample preparation. The
molecular weight difference of 2 Da between Met sulfoxide with the <sup>16</sup>O atom and Met sulfoxide with the <sup>18</sup>O atom was
used to differentiate and calculate the level of Met sulfoxide in
the sample originally. Using a recombinant monoclonal antibody as
a model protein, much lower levels of Met sulfoxide were detected
for the two susceptible Met residues with this new method compared
to a typical peptide mapping procedure. The results demonstrated efficient
elimination of the analytical artifact during LC-MS peptide mapping
for the measurement of Met sulfoxide. This method can thus be used
when accurate determination of the level of Met sulfoxide is critical
Accurate Determination of Protein Methionine Oxidation by Stable Isotope Labeling and LC-MS Analysis
Methionine
(Met) oxidation is a major modification of proteins,
which converts Met to Met sulfoxide as the common product. It is challenging
to determine the level of Met sulfoxide, because it can be generated
during sample preparation and analysis as an artifact. To determine
the level of Met sulfoxide in proteins accurately, an isotope labeling
and LC-MS peptide mapping method was developed. Met residues in proteins
were fully oxidized using hydrogen peroxide enriched with <sup>18</sup>O atoms before sample preparation. Therefore, it was impossible to
generate Met sulfoxide as an artifact during sample preparation. The
molecular weight difference of 2 Da between Met sulfoxide with the <sup>16</sup>O atom and Met sulfoxide with the <sup>18</sup>O atom was
used to differentiate and calculate the level of Met sulfoxide in
the sample originally. Using a recombinant monoclonal antibody as
a model protein, much lower levels of Met sulfoxide were detected
for the two susceptible Met residues with this new method compared
to a typical peptide mapping procedure. The results demonstrated efficient
elimination of the analytical artifact during LC-MS peptide mapping
for the measurement of Met sulfoxide. This method can thus be used
when accurate determination of the level of Met sulfoxide is critical
Detection and Quantitation of Low Abundance Oligosaccharides in Recombinant Monoclonal Antibodies
Oligosaccharides are critical for
structural integrity, stability,
and biological functions of recombinant monoclonal antibodies. It
is relatively easy to characterize, quantify, and determine the impact
of major glycoforms. While challenging to detect and quantify, certain
low abundance oligosaccharides are highly relevant to the stability
and functions of recombinant monoclonal antibodies. Methods were established
in this study based on enzymatic digestion to consolidate peaks of
the same type of oligosaccharides by removing heterogeneity and thus
increase detectability of low abundance peaks. Endo H was used to
collapse high mannose oligosaccharides to a single peak of GlcNAc
for ease of detection and quantitation. β-Galactosidase and
β-<i>N</i>-acetylhexosaminidase were used to convert
complex oligosaccharides into two peaks containing either GlcNAc<sub>2</sub>Man<sub>3</sub>Fuc or GlcNAc<sub>2</sub>Man<sub>3</sub>, which
simplified the chromatograms and data analysis. More importantly,
low abundance hybrid oligosaccharides can only be detected and qualified
after β-galactosidase and β-<i>N</i>-acetylhexosaminidase
digestion. Detection and quantitation of low abundance oligosaccharides
can also be achieved using a combination of all three enzymes. These
methods can be applied to the development of recombinant monoclonal
antibody therapeutics
Characterization of Recombinant Monoclonal Antibody Charge Variants Using OFFGEL Fractionation, Weak Anion Exchange Chromatography, and Mass Spectrometry
Recombinant
monoclonal antibody charge heterogeneity has been commonly
observed as multiple bands or peaks when analyzed by charge-based
analytical methods such as isoelectric focusing electrophoresis and
cation or anion exchange chromatography. Those charge variants have
been separated by some of the above-mentioned methods and used for
detailed characterization. The utility of a combination of OFFGEL
fractionation and weak anion exchange chromatography to separate the
charge variants of a recombinant monoclonal antibody was demonstrated
in the current study. Charge variants were separated into various
fractions of high purity and then analyzed thoroughly by liquid chromatography
mass spectrometry. Analysis of intact molecular weights identified
the presence of heavy chain leader sequence, C-terminal lysine, and
C-terminal amidation. The identified modifications were further localized
into different regions of the antibody from analysis of antibody fragments
obtained from FabRICATOR digestion. Analysis of tryptic peptides from
various fractions further confirmed the previously identified modifications
in the basic variants. Asparagine deamidation and aspartate isomerization
were identified in acidic fractions from analysis of tryptic peptides.
Basic variants have been fully accounted for by the identified modifications.
However, only a portion of the acidic variants can be explained by
deamidation and isomerization, suggesting that additional modifications
are yet to be identified or acidic variants are an ensemble of molecules
with different structures
Characterization of the Acidic Species of a Monoclonal Antibody Using Weak Cation Exchange Chromatography and LC-MS
Charge
variants, especially acidic charge variants, of recombinant
monoclonal antibodies have been challenging to fully characterize
despite the fact that several posttranslational modifications have
already been identified. The acidic species of a recombinant monoclonal
antibody were collected using weak cation exchange (WCX)-10 chromatography
and characterized by LC-MS at multiple levels. In this study, methionine
oxidation and asparagine deamidation are the only two modifications
identified in the acidic species. Incubation of the collected main
chromatographic peak with hydrogen peroxide generated acidic species,
which confirmed that acidic species were enriched in oxidized antibody.
Differences observed between the original acidic species and the oxidization-induced
acidic species indicate that different mechanisms are involved in
the formation of acidic species. Additionally, acidic species were
generated by thermal stress of the collected main peak from the original
sample. Thermal stress of the collected main peak in pH 9 buffer or
ammonium bicarbonate generated chromatograms that are highly similar
to those from the analysis of the original molecule. LC-MS analysis
identified oxidation of the same methionine residue and deamidation
of the same asparagine in the corresponding acidic fractions generated
by thermal stress; however, relatively lower levels of methionine
oxidation and higher levels of asparagine deamdiation were observed.
The results support the use of stressed conditions to generate low
abundance species for detailed characterization of recombinant monoclonal
antibody charge variants, but with caution