A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post‐translational modifications. In top‐down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top‐down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.
Labeling approaches
using isobaric chemical tags (e.g., isobaric
tagging for relative and absolute quantification, iTRAQ and tandem
mass tag, TMT) have been widely applied for the quantification of
peptides and proteins in bottom-up MS. However, until recently, successful
applications of these approaches to top-down proteomics have been
limited because proteins tend to precipitate and “crash”
out of solution during TMT labeling of complex samples making the
quantification of such samples difficult. In this study, we report
a top-down TMT MS platform for confidently identifying and quantifying
low molecular weight intact proteoforms in complex biological samples.
To reduce the sample complexity and remove large proteins from complex
samples, we developed a filter-SEC technique that combines a molecular
weight cutoff filtration step with high-performance size exclusion
chromatography (SEC) separation. No protein precipitation was observed
in filtered samples under the intact protein-level TMT labeling conditions.
The proposed top-down TMT MS platform enables high-throughput analysis
of intact proteoforms, allowing for the identification and quantification
of hundreds of intact proteoforms from Escherichia coli cell lysates. To our knowledge, this represents the first high-throughput
TMT labeling-based, quantitative, top-down MS analysis suitable for
complex biological samples.
The development of novel high-resolution
separation techniques is crucial for advancing the complex sample
analysis necessary for high-throughput top-down proteomics. Recently,
our group developed an offline 2D high-pH RPLC/low-pH RPLC separation
method and demonstrated good orthogonality between these two RPLC
formats. Specifically, ultrahigh-pressure long capillary column RPLC
separation has been applied as the second dimensional low-pH RPLC
separation for the improvement of separation resolution. To further
improve the throughput and sensitivity of the offline approach, we
developed an online 2D ultrahigh-pressure nano-LC system for high-pH
and low-pH RPLC separations in top-down proteomics. An online microtrap
column with a dilution setup was used to collect eluted proteins from
the first dimension high-pH separation and inject the fractions for
ultrahigh-pressure long capillary column low-pH RPLC separation in
the second dimension. This automatic platform enables the characterization
of 1000+ intact proteoforms from 5 μg of intact E. coli cell lysate in 10 online-collected fractions. Here, we have demonstrated
that our online 2D pH RP/RPLC system coupled with top-down proteomics
holds the potential for deep proteome characterization of mass-limited
samples because it allows the identification of hundreds of intact
proteoforms from complex biological samples at low microgram sample
amounts.
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