4 research outputs found
An Improved Stable Isotope N‑Terminal Labeling Approach with Light/Heavy TMPP To Automate Proteogenomics Data Validation: dN-TOP
<i>In silico</i> gene prediction has proven to be prone
to errors, especially regarding precise localization of start codons
that spread in subsequent biological studies. Therefore, the high
throughput characterization of protein N-termini is becoming an emerging
challenge in the proteomics and especially proteogenomics fields.
The trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP)
is an efficient N-terminomic approach that allows the characterization
of both N-terminal and internal peptides in a single experiment. Due
to its permanent positive charge, TMPP labeling strongly affects MS/MS
fragmentation resulting in unadapted scoring of TMPP-derivatized peptide
spectra by classical search engines. This behavior has led to difficulties
in validating TMPP-derivatized peptide identifications with usual
score filtering and thus to low/underestimated numbers of identified
N-termini. We present herein a new strategy (dN-TOP) that overwhelmed
the previous limitation allowing a confident and automated N-terminal
peptide validation thanks to a combined labeling with light and heavy
TMPP reagents. We show how this double labeling allows increasing
the number of validated N-terminal peptides. This strategy represents
a considerable improvement to the well-established N-TOP method with
an enhanced and accelerated data processing making it now fully compatible
with high-throughput proteogenomics studies
An Improved Stable Isotope N‑Terminal Labeling Approach with Light/Heavy TMPP To Automate Proteogenomics Data Validation: dN-TOP
<i>In silico</i> gene prediction has proven to be prone
to errors, especially regarding precise localization of start codons
that spread in subsequent biological studies. Therefore, the high
throughput characterization of protein N-termini is becoming an emerging
challenge in the proteomics and especially proteogenomics fields.
The trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP)
is an efficient N-terminomic approach that allows the characterization
of both N-terminal and internal peptides in a single experiment. Due
to its permanent positive charge, TMPP labeling strongly affects MS/MS
fragmentation resulting in unadapted scoring of TMPP-derivatized peptide
spectra by classical search engines. This behavior has led to difficulties
in validating TMPP-derivatized peptide identifications with usual
score filtering and thus to low/underestimated numbers of identified
N-termini. We present herein a new strategy (dN-TOP) that overwhelmed
the previous limitation allowing a confident and automated N-terminal
peptide validation thanks to a combined labeling with light and heavy
TMPP reagents. We show how this double labeling allows increasing
the number of validated N-terminal peptides. This strategy represents
a considerable improvement to the well-established N-TOP method with
an enhanced and accelerated data processing making it now fully compatible
with high-throughput proteogenomics studies