Protein phosphorylation is a post-translational modification that is essential to cellular signaling, cellular function, and associated disease progression. Bottom-up proteomics based on enzymatic digestion is the most widely used approach for identifying and quantifying phosphoproteins in complex biological samples. Researchers have largely optimized the experimental conditions for trypsin digestion, and it is now a routine procedure. However, trypsin digestion is impaired by the presence of phosphorylated residues in the protein sequence. This impairment arises from the fact that there are commonly salt bridges between a negatively charged phosphate group and the side chain of protonated arginine or lysine. On average, 55% of all phosphopeptides have their phosphosites located less than three amino acid residues from a cleavage site. Salt bridges reduce the cleavage accessibility for trypsin by masking the basic site chain groups of arginine and lysine. Thus, there are frequent missed cleavages in the vicinity of phosphorylation sites, thereby lessening both the depth of proteome coverage and the quantification accuracy of phosphoproteomics. In this work, we propose a method termed PhosphoShield to mitigate salt bridge formation by adding a digallium complex that exhibits a high binding affinity to the phosphate group. We tested our method using quantitative mass spectrometry analysis of the phosphoproteome of human liver cancer cells (HepG2). PhosphoShield enhances the cleavage frequency of at least 17% of tryptic phosphopeptides having cleavage sites close to the phosphate group.

中文翻译：

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PhosphoShield：通过屏蔽带负电荷的磷酸盐部分来改善磷蛋白的胰蛋白酶消化。

蛋白质磷酸化是一种翻译后修饰，对细胞信号传导、细胞功能和相关疾病进展至关重要。基于酶消化的自下而上蛋白质组学是识别和定量复杂生物样品中磷蛋白的最广泛使用的方法。研究人员在很大程度上优化了胰蛋白酶消化的实验条件，现在已成为常规程序。然而，蛋白质序列中磷酸化残基的存在会削弱胰蛋白酶消化。这种损害源于以下事实：带负电荷的磷酸基团和质子化精氨酸或赖氨酸的侧链之间通常存在盐桥。平均而言，所有磷酸肽中有 55% 的磷酸位点位于距切割位点不到三个氨基酸残基的位置。盐桥通过掩盖精氨酸和赖氨酸的基本位点链基团来降低胰蛋白酶的裂解可及性。因此，在磷酸化位点附近经常会丢失裂解，从而降低了蛋白质组覆盖的深度和磷酸化蛋白质组学的定量准确性。在这项工作中，我们提出了一种称为 PhosphoShield 的方法，通过添加对磷酸基团具有高结合亲和力的二镓复合物来减轻盐桥的形成。我们使用人肝癌细胞 (HepG2) 磷酸化蛋白质组的定量质谱分析测试了我们的方法。PhosphoShield 可提高至少 17% 的裂解位点靠近磷酸基团的胰蛋白酶磷酸肽的裂解频率。