Gold M R, Yungwirth T, Sutherland C L, Ingham R J, Vianzon D, Chiu R, van Oostveen I, Morrison H D, Aebersold R
Biomedical Research Centre, University of British Columbia, Vancouver.
Electrophoresis. 1994 Mar-Apr;15(3-4):441-53. doi: 10.1002/elps.1150150161.
The activation of protein tyrosine kinase (PTKs) and subsequent tyrosine phosphorylation of cellular proteins is a critical initial signal in the response of eukaryotic cells to mitogens, differentiative signals, and other stimuli. A number of PTK substrates have been identified and many of these are components of signal transduction pathways that regulate cell function. However, the majority of proteins that are tyrosine-phosphorylated in response to receptor signaling remain unidentified. As some of these unidentified PTK substrates may also be signal-transducing proteins, their identification and functional characterization is an important objective towards understanding receptor signaling. We describe the development of a comprehensive and general process for the isolation and structural characterization of tyrosine-phosphorylated proteins. The method involves enrichment by anti-phosphotyrosine affinity chromatography, electrophoretic concentration and separation, and proteolytic fragmentation of individual purified phosphoproteins. Resulting peptide fragments are separated by microbore reverse-phase high performance liquid chromatography (RP-HPLC) and a portion of the eluted peptides are subjected to electrospray-mass spectrometry (ES/MS) for accurate determination of peptide masses. Proteolytic fragmentation of a protein produces a characteristic set of peptide masses that can be used to rapidly identify the protein by searching databases containing the peptide mass "fingerprints" for all known proteins. The identity of the protein established by this method can be confirmed by sequence analysis of selected peptides. We have applied this procedure to the analysis of PTK substrates from B lymphocytes that have been stimulated through the B cell antigen receptor (BCR). Signaling by this receptor is involved in the generation of antibodies against foreign molecules (antigens). The BCR activates multiple PTKs which phosphorylate at least 30 different proteins. We have identified several of these tyrosine-phosphorylated proteins, including Syk, a PTK that is known to be tyrosine-phosphorylated in activated B cells. Thus, the procedure described here can be used to identify regulatory proteins of low abundance. The process consists of a logical succession of compatible steps that avoids pitfalls inherent to prior attempts to characterize low abundance phosphoproteins and should find wide use for the identification of tyrosine-phosphorylated proteins in other cell types.
蛋白质酪氨酸激酶（PTKs）的激活以及随后细胞蛋白质的酪氨酸磷酸化是真核细胞对有丝分裂原、分化信号和其他刺激作出反应时的关键初始信号。许多PTK底物已被鉴定出来，其中许多是调节细胞功能的信号转导途径的组成部分。然而，大多数因受体信号传导而发生酪氨酸磷酸化的蛋白质仍未被鉴定。由于这些未鉴定的PTK底物中的一些可能也是信号转导蛋白，因此对它们的鉴定和功能表征是理解受体信号传导的一个重要目标。我们描述了一种用于分离和结构表征酪氨酸磷酸化蛋白质的全面通用方法。该方法包括通过抗磷酸酪氨酸亲和色谱法进行富集、电泳浓缩和分离，以及对单个纯化的磷酸化蛋白质进行蛋白水解片段化。所得肽片段通过微径反相高效液相色谱（RP-HPLC）进行分离，一部分洗脱的肽进行电喷雾质谱（ES/MS）以准确测定肽质量。蛋白质的蛋白水解片段化产生一组特征性的肽质量，可通过搜索包含所有已知蛋白质的肽质量“指纹”的数据库来快速鉴定该蛋白质。通过这种方法确定的蛋白质身份可通过对选定肽段的序列分析来确认。我们已将此程序应用于对经B细胞抗原受体（BCR）刺激的B淋巴细胞中的PTK底物的分析。该受体的信号传导参与针对外来分子（抗原）的抗体的产生。BCR激活多种PTK，这些PTK使至少30种不同的蛋白质磷酸化。我们已经鉴定出其中几种酪氨酸磷酸化蛋白质，包括Syk，一种已知在活化B细胞中发生酪氨酸磷酸化的PTK。因此，这里描述的程序可用于鉴定低丰度的调节蛋白。该过程由一系列兼容步骤组成，避免了先前表征低丰度磷酸化蛋白质尝试中固有的陷阱，并且应该在鉴定其他细胞类型中的酪氨酸磷酸化蛋白质方面得到广泛应用。
