Jiang Jialu, Zhan Lingpeng, Dai Liuyan, Yao Xiaopeng, Qin Yao, Zhu Zhongqin, Zhang Mei, Tong Wenjun, Wang Guanbo
School of Chemistry and Materials Science, Nanjing Normal University, Nanjing, China.
Shenzhen Bay Laboratory, Institute for Cell Analysis, Shenzhen, China.
Rapid Commun Mass Spectrom. 2025 May;39 Suppl 1:e9369. doi: 10.1002/rcm.9369. Epub 2022 Aug 22.
The profiling of natural urinary peptides is a valuable indicator of kidney condition. While front-end separation limits the speed of peptidomic profiling, MS1-based results suffer from limited peptide coverage and specificity. Clinical studies on chronic kidney disease require an effective strategy to balance the trade-off between identification depth and throughput.
CKD273, a urinary proteome classifier associated with chronic kidney disease, in samples from diabetic nephropathy patients was profiled in parallel using capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography with mass spectrometry (LC-MS), and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Through cross-comparison of results from MS1 of unfractionated peptides and elution-time-resolved MS1 as well as MS/MS in LC- and CE-MS approaches, we evaluated the contribution of false-positive identification to MS1-based identification and quantitation, and analyzed the benefit of front-end separation in terms of accuracy and efficiency.
In LC- and CE-MS, although MS1 data resulted in higher number of identifications than MS/MS, elution-time-dependent analysis revealed extensive interference by non-CKD273 peptides, which would contribute up to 50% to quantitation if they are not separated from genuine CKD273 peptides. In the absence of separation, MS1 data resulted in lower numbers of identifications and abundance pattern that significantly deviated from those by liquid chromatography with tandem mass spectrometry (LC-MS/MS) or capillary electrophoresis with tandem mass spectrometry (CE-MS/MS). CE showed higher identification efficiency even when less sample was used or achieved faster separation.
To ensure the reliability of MS1-based urinary peptide profiling, front-end separation should not be omitted, and elution time should be used in addition to intact mass for identification. Including MS/MS in data acquisition does not compromise the speed or identification number, while benefiting data reliability by providing real-time sequence verification.
天然尿肽谱是肾脏状况的重要指标。虽然前端分离限制了肽组学分析的速度，但基于MS1的结果存在肽覆盖范围和特异性有限的问题。慢性肾病的临床研究需要一种有效的策略来平衡鉴定深度和通量之间的权衡。
使用毛细管电泳-质谱（CE-MS）、液相色谱-质谱（LC-MS）和基质辅助激光解吸/电离-质谱（MALDI-MS）对糖尿病肾病患者样本中与慢性肾病相关的尿蛋白质组分类器CKD273进行平行分析。通过对未分级肽的MS1结果、洗脱时间分辨的MS1以及LC-和CE-MS方法中的MS/MS结果进行交叉比较，我们评估了假阳性鉴定对基于MS1的鉴定和定量的贡献，并从准确性和效率方面分析了前端分离的益处。
在LC-和CE-MS中，虽然MS1数据比MS/MS鉴定出的肽数量更多，但洗脱时间依赖性分析显示非CKD273肽存在广泛干扰，如果不与真正的CKD273肽分离，这些干扰肽对定量结果的贡献可达50%。在没有分离的情况下，MS1数据鉴定出的肽数量较少，且丰度模式与液相色谱-串联质谱（LC-MS/MS）或毛细管电泳-串联质谱（CE-MS/MS）显著不同。即使使用较少的样品或实现更快的分离，CE也显示出更高的鉴定效率。
为确保基于MS1的尿肽谱分析的可靠性，不应省略前端分离，除了完整质量外，还应使用洗脱时间进行鉴定。在数据采集中纳入MS/MS不会影响速度或鉴定数量，同时通过提供实时序列验证提高数据可靠性。
