High-Throughput Determination of Endogenous Peptides in Urine Using Electromembrane Extraction Combined With Liquid Chromatography-Tandem Mass Spectrometry
Abstract
Objective
To develop a precise method for analyzing urinary peptides based on electromembrane extraction (EME) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to evaluate its potential applicability in tumor biomarker screening.
Methods
A total of 15 disease-associated peptides were selected as the target analytes. A supported liquid membrane (SLM) composed of n-octanol containing 5% di (2-ethylhexyl) phosphate was employed, with the donor phase being a 1∶1 mixture of urine and 100 mmol/L formic acid and urine, and the acceptor phase being 20 mmol/L formic acid containing 50% dimethyl sulfoxide (DMSO). After EME at 40 V for 15 min, the acceptor phase solution was analyzed by LC-MS/MS. Subsequently, the method, EME combined with LC-MS/MS (EME-LC-MS/MS), was preliminarily validated utilizing urine samples from 12 healthy controls and 7 patients with urinary system tumors.
Results
All 15 peptides exhibited excellent linearity in the range of 0.1-100.0 ng/mL (r ≥ 0.995), with the limits of detection (LODs) being 0.01-0.50 ng/mL and the limits of quantification (LOQs) being 0.03-1.50 ng/mL. The spiked recoveries ranged from 21.0% to 71.2%, with relative standard deviations (RSDs) of 0.8%-20.0% (n = 3). Small-sample analysis of clinical specimens revealed that the concentration of bradykinin 1-5 in the urine were significantly higher in tumor patients (median: 0.65 ng/mL) than that in healthy controls (median: 0.37 ng/mL) (P < 0.05), suggesting its potential as a specific biomarker for urinary system tumors.
Conclusion
The EME-LC-MS/MS method established in the study features simplicity, high efficiency, and high sensitivity, enabling precise determination of trace-level peptides in urine samples. Moreover, this approach provides a reliable methodological basis for disease biomarker screening and promotes the clinical application of electromembrane extraction.
Keywords: Electromembrane extraction, Microextraction, Tumor biomarkers, Urine, Peptides
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BRAY F, LAVERSANNE M, SUNG H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2024, 74(3): 229-263. doi: 10. 3322/caac.21834.
HAN B, ZHENG R, ZENG H, et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent, 2024, 4(1): 47-53. doi: 10.1016/j.jncc. 2024.01.006.
CAO W, QIN K, LI F, et al. Socioeconomic inequalities in cancer incidence and mortality: an analysis of GLOBOCAN 2022. Chin Med J (Engl), 2024, 137(12): 1407-1413. doi: 10.1097/cm9.0000000000003140.
MOHAMMADI K, SHAFIE D, GHOMASHI N, et al. Kinin-kallikrein system: new perspectives in heart failure. Heart Fail Rev, 2024, 29(3): 729-737. doi: 10.1007/s10741-024-10393-y.
LATOSINSKA A, SIWY J, MISCHAK H, et al. Peptidomics and proteomics based on CE-MS as a robust tool in clinical application: the past, the present, and the future. Electrophoresis, 2019, 40(18/19): 2294-2308. doi: 10.1002/elps.201900091.
CATANESE L, SIWY J, MISCHAK H, et al. Recent advances in urinary peptide and proteomic biomarkers in chronic kidney disease: a systematic review. Int J Mol Sci, 2023, 24(11): 9156. doi: 10.3390/ijms24119156.
JIA L, LI X, SHEN J, et al. Ang-1, Ang-2, and Tie2 are diagnostic biomarkers for Henoch-Schönlein purpura and pediatric-onset systemic lupus erythematous. Open Life Sci, 2024, 19(1): 20220812. doi: 10.1515/biol-2022-0812.
ONO D, MATSUSAKI T, MATSUOKA Y, et al. A pilot study of urine oxytocin as an objective biomarker for chronic pain. Ann Neurosci, 2024, 32(4): 242-247. doi: 10.1177/09727531231224141.
FARKONA S, KOTLYAR M, BURNS K, et al. Urine measurements of the renin-angiotensin system-regulated proteins predict death and graft loss in kidney transplant recipients enrolled in a ramipril versus placebo randomized controlled trial. J Proteome Res, 2025, 24(4): 2040-2052. doi: 10.1021/acs.jproteome.4c01100.
DARYANAVARD S M, ZOLFAGHARI H, ABDEL-REHIM A, et al. Recent applications of microextraction sample preparation techniques in biological samples analysis. Biomed Chromatogr, 2021, 35(7): e5105. doi: 10.1002/bmc.5105.
DEMEUSE J, HUYGHEBAERT L, DETERME W, et al. Development and validation of an LC-MS/MS method for the simultaneous quantitation of angiotensin (1-7), (1-8), (1-9) and (1-10) in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci, 2024, 1232: 123943. doi: 10.1016/j. jchromb.2023.123943.
SOARES Da SILVA BURATO J, VARGAS MEDINA D A, De TOFFOLI A L, et al. Recent advances and trends in miniaturized sample preparation techniques. J Sep Sci, 2020, 43(1): 202-225. doi: 10.1002/jssc.201900776.
PEDERSEN-BJERGAARD S, RASMUSSEN K E. Electrokinetic migration across artificial liquid membranes. New concept for rapid sample preparation of biological fluids. J Chromatogr A, 2006, 1109(2): 183-190. doi: 10.1016/j.chroma.2006.01.025.
GAO T F, DONG Z Z, SHEN X T, et al. Advances in electromembrane extraction of drugs-of-abuse from biological samples. J Instrumen Anal, 2025, 44(1): 25-33. doi: 10.12452/j.fxcsxb.241024482.
EIE L V, RYE T K, HANSEN F, et al. Electromembrane extraction of peptides and amino acids-status and perspectives. Bioanalysis, 2021, 13(4): 277-289. doi: 10.4155/bio-2020-0285.
EIE L V, PEDERSEN-BJERGAARD S, HANSEN F A. Electromembrane extraction of polar substances-status and perspectives. J Pharm Biomed Anal, 2022, 207: 114407. doi: 10.1016/j.jpba.2021.114407.
VÅRDAL L, ØIESTAD E L, GJELSTAD A, et al. Electromembrane extraction with solvent modification of the acceptor solution: improved mass transfer of drugs of abuse from human plasma. Bioanalysis, 2019, 11(8): 755-771. doi: 10.4155/bio-2018-0308.
HANSEN F A, SANTIGOSA-MURILLO E, RAMOS-PAYÁN M, et al. Electromembrane extraction using deep eutectic solvents as the liquid membrane. Anal Chim Acta, 2021, 1143: 109-116. doi: 10.1016/j.aca.2020.11.044.
NOJAVAN S, BIDARMANESH T, MOHAMMADI A, et al. Electromembrane extraction of gonadotropin-releasing hormone agonists from plasma and wastewater samples. Electrophoresis, 2016, 37(5/6): 826-833. doi: 10.1002/elps.201500555.
BUSSY U, WANG H, CHUNG-DAVIDSON Y W, et al. Simultaneous determination of gonadotropin-inhibitory and gonadotropin-releasing hormones using ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry. Anal Bioanal Chem, 2015, 407(2): 497-507. doi: 10.1007/s00216-014-8214-9.
GANGNUS T, BURCKHARDT B B. Targeted LC-MS/MS platform for the comprehensive determination of peptides in the kallikrein-kinin system. Anal Bioanal Chem, 2021, 413(11): 2971-2984. doi: 10.1007/s00216-021-03231-9.
LIU Y, LI L, WANG Z, et al. A comprehensive profiling of renin-angiotensin system in mouse and human plasma by a rapid quantitative analysis of 14 angiotensin peptides using ultrahigh-performance liquid chromatography with tandem mass spectrometry. Rapid Commun Mass SP, 2023, 37(24): e9637. doi: 10.1002/rcm.9637.
CHEN F, CHENG Z, PENG Y, et al. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay for simultaneous quantification of aldosterone, renin activity, and angiotensin Ⅱ in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci, 2021, 1179: 122740. doi: 10.1016/j.jchromb.2021.122740.
MURPHEY L J, MALAVE H A, PETRO J, et al. Bradykinin and Its metabolite bradykinin 1-5 inhibit thrombin-induced platelet aggregation in Humans. J Pharmacol Exp Ther, 2006, 318(3): 1287-1292. doi: 10.1124/jpet.106.104026.
NIEMAN M T, PAGAN-RAMOS E, WARNOCK M, et al. Mapping the interaction of bradykinin 1-5 with the exodomain of human protease activated receptor 4. FEBS Lett, 2005, 579(1): 25-29. doi: 10.1016/j.febslet. 2004.11.041.
HISADA Y, MACKMAN N. Cancer-associated pathways and biomarkers of venous thrombosis. Blood, 2017, 130(13): 1499-1506. doi: 10.1182/blood-2017-03-743211.
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