Proteomic Analysis of Chr 18 Proteins Using 2D Fractionation

One of the main goals of the Chromosome-Centric Human Proteome
Project (C-HPP) is detection of “missing proteins” (PE2-PE4).
Using the UPS2 (Universal proteomics standard 2) set as a model to
simulate the range of protein concentrations in the cell, we have
previously shown that 2D fractionation enables the detection of more
than 95% of UPS2 proteins in a complex biological mixture. In this
study, we propose a novel experimental workflow for protein detection
during the analysis of biological samples. This approach is extremely
important in the context of the C-HPP and the neXt-MP50 Challenge,
which can be solved by increasing the sensitivity and the coverage
of the proteome encoded by a particular human chromosome. In this
study, we used 2D fractionation for in-depth analysis of the proteins
encoded by human chromosome 18 (Chr 18) in the HepG2 cell line. Use
of 2D fractionation increased the sensitivity of the SRM SIS method
by 1.3-fold (68 and 88 proteins were identified by 1D fractionation
and 2D fractionation, respectively) and the shotgun MS/MS method by
2.5-fold (21 and 53 proteins encoded by Chr 18 were detected by 1D
fractionation and 2D fractionation, respectively). The results of
all experiments indicate that 111 proteins encoded by human Chr 18
have been identified; this list includes 42% of the Chr 18 protein-coding
genes and 67% of the Chr 18 transcriptome species (Illumina RNaseq)
in the HepG2 cell line obtained using a single sample. Corresponding
mRNAs were not registered for 13 of the detected proteins. The combination
of 2D fractionation technology with SRM SIS and shotgun mass spectrometric
analysis did not achieve full coverage, i.e., identification of at
least one protein product for each of the 265 protein-coding genes
of the selected chromosome. To further increase the sensitivity of
the method, we plan to use 5–10 crude synthetic peptides for
each protein to identify the proteins and select one of the peptides
based on the obtained mass spectra for the synthesis of an isotopically
labeled standard for subsequent quantitative analysis. Data are available
via ProteomeXchange with the identifier PXD019263.

染色体中心人类蛋白质组项目（C-HPP）的主要目标之一是检测“缺失蛋白质”（PE2-PE4）。以通用蛋白质组学标准2（UPS2）集为模型，模拟细胞内蛋白质浓度的范围，我们先前已证明二维分级分离技术能够检测到复杂生物混合物中超过95%的UPS2蛋白质。在本研究中，我们提出了一种新颖的实验工作流程，用于在分析生物样品过程中进行蛋白质检测。这一方法在C-HPP和neXt-MP50挑战赛的大背景下具有重要意义，它可以通过提高特定人类染色体编码的蛋白质组的敏感性和覆盖率来解决。在本研究中，我们利用二维分级分离技术对HepG2细胞系中人类染色体18（Chr 18）编码的蛋白质进行了深入研究。二维分级分离技术的应用使得SRM SIS方法的灵敏度提高了1.3倍（分别通过1D分级分离和2D分级分离识别出68和88种蛋白质）以及质谱/质谱（MS/MS）方法的灵敏度提高了2.5倍（分别通过1D分级分离和2D分级分离检测到21和53种由Chr 18编码的蛋白质）。所有实验结果均表明，已识别出111种由人类染色体18编码的蛋白质；该列表包括42%的Chr 18蛋白质编码基因和67%的Chr 18转录组物种（Illumina RNaseq），这些均是在单个样品中获得的。对于检测到的13种蛋白质，未注册相应的mRNA。将二维分级分离技术与SRM SIS和质谱分析相结合并未实现全面覆盖，即未能对所选染色体上的265个蛋白质编码基因中的每一个至少识别出一个蛋白质产物。为进一步提高方法的灵敏度，我们计划对每种蛋白质使用5-10个粗合成肽进行识别，并根据获得的质谱选择其中一个肽进行同位素标记标准的合成，以进行后续的定量分析。数据可通过ProteomeXchange平台，使用标识符PXD019263获取。