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Precision-Engineered Nano-Electrospray Emitters Enhance Ionization Efficiency for Trap-Assisted Direct Infusion Mass Spectrometry: Application in Plasma Alzheimer’s Disease Proteomics

ABSTRACT: Shotgun direct infusion mass spectrometry (DI-MS) enables high-throughput proteomics. Yet, its performance critically depends on nanospray emitter quality. We present here precision-engineered nanospray emitters with an optimized 4-degree emitter tip angle, designed for sensitive, reproducible, user friendly, and compatible across mass spectrometry platforms that demonstrated enhanced ionization efficiency and spray stability, showing a 21-fold improvement in protein identification rates compared to commercial designs. Using both digested HeLa cell protein extracts and plasma samples from Alzheimer's disease (AD) patients and controls, where the 14 most high-abundant proteins were depleted, we performed a comprehensive DI-MS protein identification and precise quantification using both Orbitrap Exploris 480 (OE480) and Orbitrap Astral mass spectrometers. We consistently identified and quantified over 160 proteins in AD plasma samples in 4 min DI-MS runs, with superior reproducibility (CV < 15%) and sensitivity on the Orbitrap Astral. Our analysis revealed several unreported disease-associated protein signatures, while confirming known AD biomarkers, with C5, C4BPA, and FAM135B as the protein combination with the highest potential as AD plasma biomarkers for the detection of the disease. The enhanced performance of these standardized engineered nanospray emitters over the established DI-MS workflow establishes a robust and scalable platform for high-throughput clinical proteomics.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

DISEASE(S): Alzheimer's Disease

SUBMITTER: Ana Montero Calle

LAB HEAD: Rodrigo Barderas

PROVIDER: PXD061620 | Pride | 2025-07-31

REPOSITORIES: Pride

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Project description:Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide, underscoring the need to improve diagnostic strategies. Platelets play a major role, not only in the process of acute thrombosis during plaque rupture, but also in the formation of atherosclerosis itself. MicroRNAs are endogenous small non-coding RNAs that control gene expression and are expressed in a tissue and disease-specific manner. Therefore they have been proposed to be useful biomarkers. The aim of this study was to investigate whether differences in miRNA expression levels in platelets can be found (i) between patients with premature CAD and healthy controls and (ii) within healthy controls after and before aspirin and statin administration. In this case-control study we measured expression levels of platelet miRNAs using microarrays from 40 male patients with premature CAD and 40 age- and sex-matched healthy controls. Premature CAD was defined as a cardiac event before the age of 51 years. The patients were selected from the outpatient clinic of the Academic Medical Center (AMC) of Amsterdam, which is specialised in premature CAD. The control cohort was composed of 40 healthy Caucasian male volunteers, who were recruited by advertisement and who were matched with the cases for age and smoking habits. Individuals of this control cohort did not have a history of CVD, nor did they have a positive family history of CVD and they were not allowed to use any medication. Patients and controls were excluded when they suffered from diabetes. Most CAD patients use aspirin and statins as secondary prevention. The influence of these drugs on miRNA profiles is unknown. To assess the possible influence of medication on miRNA expression and to control for medication as confounding factor, we asked 27 volunteers in our control cohort to also use these drugs. We administered simvastatin 40 mg, once daily, for 6 weeks and during the last two weeks we added 100mg of acetyl salicylic acid, once daily. Blood samples including isolated platelets were collected at baseline in the absence of aspirin and statins and after six weeks of medication use. We also assessed platelet function using the Multiplate® Analyzer (Roche) in the absence of aspirin and statin use. In short, 300 µl whole blood was diluted with 300 µl 0.9% saline and stirred for 3 minutes at 37 ºC. Adenosine diphosphate (ADP) was added in a final concentration of 2.5 ?mol/L to initiate platelet aggregation. Aggregation was measured for 6 minutes and was reported in arbitrary aggregation units plotted against time. Also, the area under the aggregation curve (AUC) was measured. All samples were measured in the absence and presence of 200 ?mol/L indomethacin (20 min incubation with blood) to mimic the effect of aspirin use. We calculated the percentage reduction in AUC after incubation with indomethacin as an in vitro measure of the effect of aspirin use on whole blood platelet aggregation.

			Action	DRS
	20240802_130659_DI-MS_Plasma_AD_Astral.sne	Other
	AD_005.raw	Raw
	AD_007.raw	Raw
	AD_008.raw	Raw
	AD_010.raw	Raw

Dataset Information

Precision-Engineered Nano-Electrospray Emitters Enhance Ionization Efficiency for Trap-Assisted Direct Infusion Mass Spectrometry: Application in Plasma Alzheimer’s Disease Proteomics

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