色谱2024，Vol.42Issue(6)：564-571,8.DOI:10.3724/SP.J.1123.2023.11007
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
摘要
Abstract
Protein phosphorylation plays an important role in cellular signaling and disease de-velopment.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 be-cause 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 phos-phoproteins,namely,α-and β-caseins,were selected as model phosphorylated samples to as-sess the performance of the proposed enrichment technique.The magnetic carbon nitride com-posite exhibited high selectivity and sensitivity for phosphopeptide enrichment.The limit of de-tection was determined by MALDI-TOF-MS analysis to be 0.1 fmol.The selectivity of the meth-od was investigated using the digest mixtures of α-casein,β-casein,and bovine serum albumin(BSA)with different mass ratios(1∶1∶1 000,1∶1∶2 000,and 1∶1∶5 000).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 sig-nals 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 assess-ing the reusability of the same batch of magnetic carbon nitride materials over 20 cycles of en-richment.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 phos-phoprotein-containing samples,including skimmed milk digest,human serum,and human sali-va;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 pre-treatment 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.关键词
氮化碳/磁性固相萃取/基质辅助激光解吸电离飞行时间质谱/磷酸化肽/富集Key words
carbon nitride/magnetic-solid phase extraction(MSPE)/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS)/phosphopeptide/enrichment分类
化学化工引用本文复制引用
姜丽艳,张玮璐,赵璐,胡良海..磁性氮化碳复合材料的制备及其对磷酸化肽的富集[J].色谱,2024,42(6):564-571,8.基金项目
吉林省自然科学基金(20230101155JC).Natural Science Foundation of Jilin Province,China(No.20230101155JC). （20230101155JC）
