8 research outputs found
Autopilot: An Online Data Acquisition Control System for the Enhanced High-Throughput Characterization of Intact Proteins
The ability to study organisms by
direct analysis of their proteomes
without digestion via mass spectrometry has benefited greatly from
recent advances in separation techniques, instrumentation, and bioinformatics.
However, improvements to data acquisition logic have lagged in comparison.
Past workflows for Top Down Proteomics (TDPs) have focused on high
throughput at the expense of maximal protein coverage and characterization.
This mode of data acquisition has led to enormous overlap in the identification
of highly abundant proteins in subsequent LC-MS injections. Furthermore,
a wealth of data is left underutilized by analyzing each newly targeted
species as unique, rather than as part of a collection of fragmentation
events on a distinct proteoform. Here, we present a major advance
in software for acquisition of TDP data that incorporates a fully
automated workflow able to detect intact masses, guide fragmentation
to achieve maximal identification and characterization of intact protein
species, and perform database search online to yield real-time protein
identifications. On <i>Pseudomonas aeruginosa</i>, the software
combines fragmentation events of the same precursor with previously
obtained fragments to achieve improved characterization of the target
form by an average of 42 orders of magnitude in confidence. When HCD
fragmentation optimization was applied to intact proteins ions, there
was an 18.5 order of magnitude gain in confidence. These improved
metrics set the stage for increased proteome coverage and characterization
of higher order organisms in the future for sharply improved control
over MS instruments in a project- and lab-wide context
Autopilot: An Online Data Acquisition Control System for the Enhanced High-Throughput Characterization of Intact Proteins
The ability to study organisms by
direct analysis of their proteomes
without digestion via mass spectrometry has benefited greatly from
recent advances in separation techniques, instrumentation, and bioinformatics.
However, improvements to data acquisition logic have lagged in comparison.
Past workflows for Top Down Proteomics (TDPs) have focused on high
throughput at the expense of maximal protein coverage and characterization.
This mode of data acquisition has led to enormous overlap in the identification
of highly abundant proteins in subsequent LC-MS injections. Furthermore,
a wealth of data is left underutilized by analyzing each newly targeted
species as unique, rather than as part of a collection of fragmentation
events on a distinct proteoform. Here, we present a major advance
in software for acquisition of TDP data that incorporates a fully
automated workflow able to detect intact masses, guide fragmentation
to achieve maximal identification and characterization of intact protein
species, and perform database search online to yield real-time protein
identifications. On <i>Pseudomonas aeruginosa</i>, the software
combines fragmentation events of the same precursor with previously
obtained fragments to achieve improved characterization of the target
form by an average of 42 orders of magnitude in confidence. When HCD
fragmentation optimization was applied to intact proteins ions, there
was an 18.5 order of magnitude gain in confidence. These improved
metrics set the stage for increased proteome coverage and characterization
of higher order organisms in the future for sharply improved control
over MS instruments in a project- and lab-wide context
Cell-free Protein Synthesis from a Release Factor 1 Deficient <i>Escherichia coli</i> Activates Efficient and Multiple Site-specific Nonstandard Amino Acid Incorporation
Site-specific incorporation of nonstandard
amino acids (NSAAs)
into proteins enables the creation of biopolymers, proteins, and enzymes
with new chemical properties, new structures, and new functions. To
achieve this, amber (TAG codon) suppression has been widely applied.
However, the suppression efficiency is limited due to the competition
with translation termination by release factor 1 (RF1), which leads
to truncated products. Recently, we constructed a genomically recoded <i>Escherichia coli</i> strain lacking RF1 where 13 occurrences
of the amber stop codon have been reassigned to the synonymous TAA
codon (<i>rEc.E13.ĪprfA</i>). Here, we assessed and
characterized cell-free protein synthesis (CFPS) in crude S30 cell
lysates derived from this strain. We observed the synthesis of 190
Ā± 20 Ī¼g/mL of modified soluble superfolder green fluorescent
protein (sfGFP) containing a single <i>p</i>-propargyloxy-l-phenylalanine (pPaF) or <i>p</i>-acetyl-l-phenylalanine. As compared to the parent <i>rEc.E13</i> strain with RF1, this results in a modified sfGFP synthesis improvement
of more than 250%. Beyond introducing a single NSAA, we further demonstrated
benefits of CFPS from the RF1-deficient strains for incorporating
pPaF at two- and five-sites per sfGFP protein. Finally, we compared
our crude S30 extract system to the PURE translation system lacking
RF1. We observed that our S30 extract based approach is more cost-effective
and high yielding than the PURE translation system lacking RF1, ā¼1000
times on a milligram protein produced/$ basis. Looking forward, using
RF1-deficient strains for extract-based CFPS will aid in the synthesis
of proteins and biopolymers with site-specifically incorporated NSAAs
Metabolic Perturbation of an Essential Pathway: Evaluation of a Glycine Precursor of Coenzyme A
Pantetheine and its
corresponding disulfide pantethine play a key
role in metabolism as building blocks of coenzyme A (CoA), an essential
cofactor utilized in ā¼4% of primary metabolism and central
to fatty acid, polyketide, and nonribosomal peptide synthases. Using
a combination of recombinant engineering and chemical synthesis, we
show that the disulfide of <i>N</i>-pantoylglycyl-2-aminoethanethiol
(GlyPan), with one fewer carbon than pantetheine, can rescue a mutant E. coli strain MG1655Ī<i>panC</i> lacking a functional pantothenate synthetase. Using mass spectrometry,
we show that the GlyPan variant is accepted by the downstream CoA
biosynthetic machinery, ultimately being incorporated into essential
acyl carrier proteins. These findings point to further flexibility
in CoA-dependent pathways and offer the opportunity to incorporate
orthogonal analogues
Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the <i>Echinococcus granulosus</i> Preadult Stage
<i>Echinococcus granulosus</i> is the causative agent
of cystic hydatid disease, a neglected zoonosis responsible for high
morbidity and mortality. Several molecular mechanisms underlying parasite
biology remain poorly understood. Here, <i>E. granulosus</i> subcellular fractions were analyzed by top down and bottom up proteomics
for protein identification and characterization of co-translational
and post-translational modifications (CTMs and PTMs, respectively).
Nuclear and cytosolic extracts of <i>E. granulosus</i> protoscoleces
were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed
by LCāMS/MS. By top down analysis, 186 proteins and 207 proteoforms
were identified, of which 122 and 52 proteoforms were exclusively
detected in nuclear and cytosolic fractions, respectively. CTMs were
evident as 71% of the proteoforms had methionine excised and 47% were
N-terminal acetylated. In addition, <i>in silico</i> internal
acetylation prediction coupled with top down MS allowed the characterization
of 9 proteins differentially acetylated, including histones. Bottom
up analysis increased the overall number of identified proteins in
nuclear and cytosolic fractions to 154 and 112, respectively. Overall,
our results provided the first description of the low mass proteome
of <i>E. granulosus</i> subcellular fractions and highlighted
proteoforms with CTMs and PTMS whose characterization may lead to
another level of understanding about molecular mechanisms controlling
parasitic flatworm biology
A Proteomic Survey of Nonribosomal Peptide and Polyketide Biosynthesis in Actinobacteria
Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a āprotein-firstā method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nat. Biotechnol. 2009, 27, 951ā956), for example, improved <i>de novo</i> peptide sequencing, have enabled the discovery of 10 NRPS/PKS gene clusters from 6 strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously āorphanā gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2ā4 Mbp genomes) used previously
Applying Label-Free Quantitation to Top Down Proteomics
With
the prospect of resolving whole protein molecules into their
myriad proteoforms on a proteomic scale, the question of their quantitative
analysis in discovery mode comes to the fore. Here, we demonstrate
a robust pipeline for the identification and stringent scoring of
abundance changes of whole protein forms <30 kDa in a complex system.
The input is ā¼100ā400 Ī¼g of total protein for
each biological replicate, and the outputs are graphical displays
depicting statistical confidence metrics for each proteoform (<i>i.e</i>., a volcano plot and representations of the technical
and biological variation). A key part of the pipeline is the hierarchical
linear model that is tailored to the original design of the study.
Here, we apply this new pipeline to measure the proteoform-level effects
of deleting a histone deacetylase (<i>rpd3</i>) in S. cerevisiae. Over 100 proteoform changes were detected
above a 5% false positive threshold in WT vs the Ī<i>rpd3</i> mutant, including the validating observation of hyperacetylation
of histone H4 and both H2B isoforms. Ultimately, this approach to
label-free top down proteomics in discovery mode is a critical technical
advance for testing the hypothesis that whole proteoforms can link
more tightly to complex phenotypes in cell and disease biology than
do peptides created in shotgun proteomics
Applying Label-Free Quantitation to Top Down Proteomics
With
the prospect of resolving whole protein molecules into their
myriad proteoforms on a proteomic scale, the question of their quantitative
analysis in discovery mode comes to the fore. Here, we demonstrate
a robust pipeline for the identification and stringent scoring of
abundance changes of whole protein forms <30 kDa in a complex system.
The input is ā¼100ā400 Ī¼g of total protein for
each biological replicate, and the outputs are graphical displays
depicting statistical confidence metrics for each proteoform (<i>i.e</i>., a volcano plot and representations of the technical
and biological variation). A key part of the pipeline is the hierarchical
linear model that is tailored to the original design of the study.
Here, we apply this new pipeline to measure the proteoform-level effects
of deleting a histone deacetylase (<i>rpd3</i>) in S. cerevisiae. Over 100 proteoform changes were detected
above a 5% false positive threshold in WT vs the Ī<i>rpd3</i> mutant, including the validating observation of hyperacetylation
of histone H4 and both H2B isoforms. Ultimately, this approach to
label-free top down proteomics in discovery mode is a critical technical
advance for testing the hypothesis that whole proteoforms can link
more tightly to complex phenotypes in cell and disease biology than
do peptides created in shotgun proteomics