定量研究方法包括平行反应监测（PRM）、数据独立采集（DIA）和数据依赖采集（DDA），这些方法常用于探究蛋白质表达谱。然而，利用PRM、DIA和基于串联质谱标记（TMT）的DDA开发的分析测定法在定量蛋白质组学方面的性能尚未探究。在这里，我们开发了针对六种糖基化蛋白质的测定法，包括富含亮氨酸的α-2糖蛋白（LRG1）、前列腺素-H2 D异构酶（PTGDS）、氨肽酶N（ANPEP）、CD63抗原（CD63）、囊泡膜蛋白（CLU）和前列腺酸性磷酸酶（ACPP），使用PRM、DDA和DIA，并评估了不同采集模式下每个测定法的分析性能。我们还比较了三种轨道阱质谱仪中每种采集模式下的测定法：瑞典泰福飞世尔Q Exactive、Exploris 480和Lumos。我们发现DIA具有最大的线性范围、最高的灵敏度和最高的可再现性。随后，我们将我们开发的DIA测定法应用于非侵袭性（n=48）和侵袭性（n=35）前列腺癌患者的尿液样本中。总之，我们开发了六种糖基化蛋白质的测定法，评估了DIA、PRM和PRM采集模式下不同质谱仪的分析性能，并选择了DIA测定法对侵袭性和非侵袭性前列腺癌患者的尿液样本进行定量分析。©2023 The Authors. Proteomics由Wiley-VCH GmbH出版。

Quantitative approaches encompassing parallel reaction monitoring (PRM), data-independent acquisition (DIA), and data-dependent acquisition (DDA) are commonly used to investigate protein expression profiles. However, analytical performances of assays developed using PRM, DIA, and Tandem Mass Tag (TMT)-based DDA for quantitative proteomics have yet not been investigated. Here, we developed assays for glycopeptides identified from six glycoproteins, including Leucine-rich alpha-2-glycoprotein (LRG1), Prostaglandin-H2 D-isomerase (PTGDS), Aminopeptidase N (ANPEP), CD63 antigen (CD63), Clusterin (CLU), and Prostatic acid phosphatase (ACPP), using PRM, DDA, and DIA and evaluated the analytical performances of each assay using the different acquisition modes. We also compared assays in each acquisition mode on three different orbitrap instruments: Thermo Fisher Q Exactive, Exploris 480, and Lumos. We found that DIA showed the largest linear range, highest sensitivity, and most reproducibility. We then applied our developed DIA assays to urine samples from non-aggressive (n = 48) and aggressive (n = 35) prostate cancer patients. In conclusion, we developed assays for the six glycoproteins, evaluated the analytical performances of each assay in DIA, PRM, and PRM acquisition modes on three types of mass spectrometry instruments, and chose the DIA assays for the quantitative analysis of urine samples from patients with aggressive and non-aggressive prostate cancer.© 2023 The Authors. Proteomics published by Wiley-VCH GmbH.