蛋白质磷酸化在细胞信号传导和疾病发展中起重要作用。基于质谱的蛋白质组学的进展使得定性和定量磷酸化研究以及深入的生物标志物发现和信号通路分析的生物学探索成为可能。然而,磷酸化过程中发生的动态变化和目标分析物的低丰度使直接分析变得困难,因为质谱检测没有选择性,不同于免疫分析,如蛋白质印迹和酶联免疫吸附测定(ELISA)。本研究旨在解决磷酸化肽的特异性和高效分离的关键问题之一。开发了一种基于磁性氮化碳复合材料与基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)耦合的方法,用于富集和分析复杂样品中丰度低的磷酸肽。合成了磁性氮化碳复合材料,并通过电子显微镜对其进行了表征,红外光谱,和X射线衍射。复合材料表现出分布良好的二维层状结构和具有优异顺磁性能的官能团。两种经典的磷蛋白,即,α-和β-酪蛋白,被选择为模型磷酸化样品,以评估所提出的富集技术的性能。磁性氮化碳复合材料对磷酸肽富集具有高选择性和灵敏度。检测限通过MALDI-TOF-MS分析确定为0.1fmol。使用α-酪蛋白的消化混合物研究了该方法的选择性,β-酪蛋白,和牛血清白蛋白(BSA)的质量比(1∶1∶1000,1∶1∶2000和1∶1∶5000)。样品的直接分析揭示了来自BSA中丰富肽的光谱信号的优势。用磁性氮化碳复合材料富集后,高浓度的背景蛋白被洗掉,只有磷酸肽的信号被捕获。酪蛋白的信号被清晰地观察到,背景噪音很小,表明复合材料的高选择性。通过评估同一批次的磁性氮化碳材料在20个富集循环中的可重用性来测试该方法的稳健性。即使经过多次重复使用,该复合材料也显示出几乎相同的富集能力,证明其对大量临床样本的潜在适用性。最后,该方法用于分析几种常用的含磷蛋白样品中的磷酸肽,包括脱脂乳消化物,人血清,和人类唾液;这些样本在食品质量分析中具有重要意义,疾病生物标志物,和液体活检癌症。没有浓缩,没有检测到磷酸肽,因为大量的非磷酸肽材料主导了获得的光谱信号。用开发的磁性氮化碳复合材料预处理后,通过MALDI-TOF-MS以高选择性和灵敏度鉴定了大多数磷酸位点。这些结果揭示了所开发的方法用于临床应用的实用性。此外,我们的方法有可能用于实际复杂生物样品的磷酸蛋白质组学.
Protein phosphorylation plays an important role in cellular signaling and disease development. Advances in mass spectrometry-based proteomics have enabled qualitative and quantitative phosphorylation studies as well as in-depth biological explorations for biomarker discovery and signaling pathway analysis. However, the dynamic changes that occur during phosphorylation and the low abundance of target analytes render direct analysis difficult because mass spectral detection offers no selectivity, unlike immunoassays such as Western blot and enzyme-linked immunosorbent assay (ELISA). The present study aimed to solve one of the key problems in the specific and efficient isolation of phosphorylated peptides. A method based on a magnetic carbon nitride composite coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed for the enrichment and analysis of phosphopeptides with low abundance in complex samples. Magnetic carbon nitride composite was synthesized and characterized by electron microscopy, infrared spectroscopy, and X-ray diffractometry. The composite showed a well-distributed two-dimensional layered structure and functional groups with excellent paramagnetic performance. Two classical phosphoproteins, namely, α- and β-caseins, were selected as model phosphorylated samples to assess the performance of the proposed enrichment technique. The magnetic carbon nitride composite exhibited high selectivity and sensitivity for phosphopeptide enrichment. The limit of detection was determined by MALDI-TOF-MS analysis to be 0.1 fmol. The selectivity of the method was investigated using the digest mixtures of α-casein, β-casein, and bovine serum albumin (BSA) with different mass ratios (1∶1∶1000, 1∶1∶2000, and 1∶1∶5000). Direct analysis of the samples revealed the dominance of spectral signals from the abundant peptides in BSA. After enrichment with the magnetic carbon nitride composite, the high concentration of background proteins was washed away and only the signals of the phosphopeptides were captured. The signals from the casein proteins were clearly observed with little background noise, indicating the high selectivity of the composite material. The robustness of the method was tested by assessing the reusability of the same batch of magnetic carbon nitride materials over 20 cycles of enrichment. The composite showed nearly the same enrichment ability even after several cycles of reuse, demonstrating its potential applicability for a large number of clinical samples. Finally, the method was applied to the analysis of phosphopeptides from several commonly used phosphoprotein-containing samples, including skimmed milk digest, human serum, and human saliva; these samples are significant in the analysis of food quality, disease biomarkers, and liquid biopsies for cancer. Without enrichment, no phosphopeptide was detected because of the high abundance of nonphosphopeptide materials dominating the spectral signals obtained. After pretreatment with the developed magnetic carbon nitride composite, most of the phosphosites were identified with high selectivity and sensitivity via MALDI-TOF-MS. These results revealed the practicality of the developed approach for clinical applications. In addition, our method may potentially be employed for phosphoproteomics with real complex biological samples.