磁性氮化碳复合材料的制备及其对磷酸化肽的富集

Preparation of magnetic carbon nitride composite toward phosphopeptide enrichment

蛋白质的磷酸化在细胞信号传导和疾病发生发展中起着重要作用,但磷酸化的动态变化和低丰度的特点使得对其直接分析有着较大的困难。为了解决磷酸化肽难离子化、检测丰度低的瓶颈问题,本研究制备了一种磁性氮化碳复合材料,结合基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),建立了一种对复杂样品中低丰度磷酸化肽富集与分析的方法。采用电子显微镜、红外光谱分析及X射线衍射分析等手段对合成的磁性氮化碳材料进行表征。以酪蛋白酶解产物为实验模型,发现磁性氮化碳材料能够实现对磷酸化肽的高选择性富集和高灵敏度检测,检出限为0.1 fmol。选择脱脂牛奶、人唾液和人血清为实际分析样品,发现磁性氮化碳材料对微量蛋白生物样品中磷酸化肽的分析具有较高的应用潜力。

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-andβ-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 ofα-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.