CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China.
Liaoning Key Laboratory of Molecular Recognition and Imaging School of Bioengineering, Dalian University of Technology, Dalian 116024, China.
Anal Chem. 2022 Mar 15;94(10):4155-4164. doi: 10.1021/acs.analchem.1c03704. Epub 2022 Mar 3.
Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 μg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.
蛋白质酪氨酸磷酸化 (pTyr) 在所有真核细胞的信号转导和调节中起着重要作用。在传统的免疫亲和纯化 (IP) 方法中，磷酸酪氨酸肽是用磷酸酪氨酸特异性抗体从细胞蛋白提取物的消化物中分离出来的，并通过串联质谱鉴定。然而，低灵敏度、重现性差和高成本是 IP 方法的普遍关注点。在这项研究中，我们提出了一种无需抗体即可通过蛋白质酪氨酸磷酸酶 (PTP) 来鉴定磷酸酪氨酸肽的方法。研究发现，包括 PTP1B、TCPTP 和 SHP1 在内的大多数 PTP 可以有效地、选择性地去磷酸化磷酸酪氨酸肽。然后，我们设计了一种工作流程，该流程将基于 Ti-IMAC 的两个磷酸肽富集步骤与 PTP 催化的去磷酸化相结合，用于酪氨酸磷酸蛋白质组学分析。该工作流程首先通过选择性检测半复杂样品中的磷酸酪氨酸肽进行验证，然后应用于分析 Jurkat T 细胞的酪氨酸磷酸蛋白质组。从不到 500μg 的细胞裂解物中鉴定出了约 1000 个假定的前磷酸酪氨酸肽。这些肽中的大多数酪氨酸磷酸化位点可以通过这种方法明确确定，其中超过 70%的肽只含有一个酪氨酸残基。此外，通过这种方法鉴定的酪氨酸位点与基于 SH2 超结合剂的方法鉴定的酪氨酸位点高度互补。因此，Ti-IMAC 富集与 PTP 去磷酸化的结合为酪氨酸磷酸蛋白质组学分析提供了一种替代的、具有成本效益的方法。
