Influence of the Digestion Technique, Protease, and Missed Cleavage Peptides in Protein Quantitation

Quantitative determination
of absolute and relative protein amounts
is an essential requirement for most current bottom-up proteomics
applications, but protein quantitation estimates are affected by several
sources of variability such as sample preparation, mass spectrometric
acquisition, and data analysis. Among them, sample digestion has attracted
much attention from the proteomics community, as protein quantitation
by bottom-up proteomics relies on the efficiency and reproducibility
of protein enzymatic digestion, with the presence of missed cleavages,
nonspecific cleavages, or even the use of different proteases having
been postulated as important sources of variation in protein quantitation.
Here we evaluated both in-solution and filter-aided digestion protocols
and assessed their influence in the estimation of protein abundances
using five E. coli mixtures with known amounts of
spiked proteins. We observed that replicates of trypsin specificity
digestion protocols are highly reproducible in terms of peptide quantitation,
with digestion technique and the chosen proteolytic enzyme being the
major sources of variability in peptide quantitation. Finally, we
also evaluated the result of including peptides with missed cleavages
in protein quantitation and observed no significant differences in
precision, accuracy, specificity, and sensitivity compared with the
use of fully tryptic peptides.

对蛋白质绝对与相对含量进行定量测定，是当前绝大多数自下而上蛋白质组学（bottom-up proteomics）应用的核心必要条件，但蛋白质定量结果往往受到多种变量来源的干扰，例如样品前处理、质谱采集与数据分析环节。其中，样品酶解过程一直受到蛋白质组学界的广泛关注：自下而上蛋白质组学的蛋白质定量结果，依赖于蛋白质酶解的效率与重现性，而漏切位点、非特异性酶切，乃至不同蛋白酶的使用，均被认为是蛋白质定量的重要变异来源。本研究针对溶液内酶解与滤膜辅助酶解两种方案开展评估，并通过5组含有已知浓度加标蛋白的大肠杆菌（E. coli）混合物，分析二者对蛋白质丰度估算的影响。结果显示，胰蛋白酶特异性酶解方案的重复实验在肽段定量层面具有极高的重现性，而酶解技术与所选用的蛋白水解酶，是影响肽段定量的主要变量来源。最后，本研究还评估了在蛋白质定量中纳入含漏切位点肽段的效果，结果表明，相较于仅使用完全胰酶酶切肽段的方案，纳入漏切肽段并不会对定量的精密度、准确度、特异性与灵敏度产生显著影响。