An Improved Stable Isotope N‑Terminal Labeling Approach with Light/Heavy TMPP To Automate Proteogenomics Data Validation: dN-TOP

In silico 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.

计算机模拟基因预测（in silico gene prediction）已被证实易出现错误，尤其在后续生物学研究中广泛涉及的起始密码子精确定位环节。因此，蛋白质组学，尤其是蛋白质基因组学领域内，蛋白质N端的高通量表征正成为一项新兴挑战。三甲氧基苯基鏻（trimethoxyphenyl phosphonium, TMPP）标记法（N-TOP）是一种高效的N端蛋白质组学分析方法，可在单次实验中同时实现N端肽段与内部肽段的表征。由于其带有永久正电荷，TMPP标记会显著改变串联质谱（MS/MS）的碎裂模式，导致经典质谱搜索引擎无法对TMPP衍生肽段的质谱谱图进行适配打分。这一特性使得借助常规打分过滤验证TMPP衍生肽段的鉴定结果面临重重困难，进而造成已鉴定N端肽段的数量偏低且被低估。本文提出一种全新的dN-TOP策略，该策略通过联用轻、重两种TMPP试剂进行标记，攻克了此前的技术局限，可实现可靠且自动化的N端肽段验证。我们阐明了该双标记策略如何提升已验证N端肽段的数量。相较于业已成熟的N-TOP方法，该策略实现了显著改进：数据处理流程得到优化与加速，如今可完全适配高通量蛋白质基因组学研究。