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Development of non-targeted proteome analysis of dried blood spots using an automated protein extraction system

ABSTRACT: In this study, we developed a simple protein extraction method of dried blood spots (DBS) that potentially meets the throughput required for newborn screening (NBS) and optimized non-targeted proteomic analysis using this method in combination with liquid chromatography coupled mass spectrometry in data-independent-acquisition mode (DIA-LC-MS/MS).

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yusuke Kawashima

PROVIDER: PXD058804 | JPOST Repository | Fri Dec 12 00:00:00 GMT 2025

REPOSITORIES: jPOST

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			Action	DRS
	DBS_Auto-NANDA_InstB_Days01_11.mzML	Mzml
	DBS_Auto-NANDA_InstB_Days01_12.mzML	Mzml
	DBS_Auto-NANDA_InstB_Days32_01.mzML	Mzml
	DBS_Auto-NANDA_InstB_Days32_02.mzML	Mzml
	DBS_Auto-NANDA_InstB_Days32_03.mzML	Mzml

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Similar Datasets

Development of non-targeted proteome analysis of dried blood spots using an automated protein extraction system (

Project description:In this study, we developed a simple protein extraction method of dried blood spots (DBS) that potentially meets the throughput required for newborn screening (NBS) and optimized non-targeted proteomic analysis using this method in combination with liquid chromatography coupled mass spectrometry in data-independent-acquisition mode (DIA-LC-MS/MS).

2025-08-14 | PXD064701 | JPOST Repository

Dried blood spots analysis for targeted and non-targeted exposomics

Project description:<p>Dried blood spots (DBS) are an established sample type, widely used in newborn screening programs for monitoring metabolic diseases. Their minimally invasive nature offers great promise for assessing chemical exposures, particularly during early life stages and in large-scale epidemiological studies. However, comprehensive evaluations of key analytical parameters such as extraction efficiency and matrix effects across multiple chemical classes remain limited. Moreover, the promising approach of broadly combining targeted and non-targeted mass spectrometric data evaluation remains unexplored in DBS small-molecule omics. Here, we present an optimized LC-HRMS workflow for combined exposomic and metabolomic analysis in DBS samples. Four extraction protocols were systematically compared, with analytical performance evaluated for >200 structurally diverse toxicants, pollutants, and other key biomarkers. The optimized protocol demonstrated acceptable recoveries (60–140%) and reproducibility (median RSD: 18%) for a majority of compounds. Matrix effects showed a median value of 76% (median RSD: 14%). In a proof-of-principle study, twelve exposure compounds of the target panel with varied physicochemical properties were identified in real-life samples, with several reported for the first time in DBS biomonitoring. Complementary non-targeted analysis further expanded the detectable chemical space, enabling reliable annotation of additional exposures. Moreover, high-confidence identification of endogenous metabolites, including amino acids, biogenic amines, fatty acids and acylcarnitines demonstrated the capacity to capture a broad snapshot of the human metabolome. These findings support the use of DBS for integrated exposomic and metabolomic applications, providing toxicological and biological insights from low-volume samples in both, prospective and retrospective studies.</p>

2025-10-09 | MTBLS13065 | MetaboLights

High Field Asymmetric Waveform Ion Mobility Spectrometry in Non-targeted Bottom-up Proteomics of Dried Blood Spot

Project description:Dried Blood Spots samples are a rich source of proteins and have therefore great potential for proteomics biomarker discovery studies. Only a few articles have used DBS samples for non-targeted bottom-up protein analysis, probably due to the complexity of the DBS samples. Gas-phase separation by ion mobility spectrometry such as Field Asymmetric Waveform Ion Mobility (FAIMS) could be useful in analysis of DBS samples as FAIMS has previously shown to be advantageous for bottom-up protein analysis of complex samples. The aim of this project was therefore to evaluate FAIMS in non-targeted analysis of Dried Blood Spots.

2018-05-03 | PXD007926 | Pride

Supporting data for manuscript: A modified fully automated dried blood spot sample preparation system and its application in steroid esters detection

Project description:This project focuses on developing an efficient and sensitive method for detecting steroid esters in dried blood spots (DBS) to support anti-doping efforts. By optimizing a fully automated DBS processing system with an independent desorption pump, we established a high-throughput screening method for 30 steroid esters and a confirmatory method for 26 targets using HPLC-HRMS. The method demonstrates low detection limits (as low as 0.05 ng/mL) and good reproducibility (RSD < 15%), and its performance was validated through blind samples, administration studies, and comparison with traditional IRMS. This approach provides a practical solution for rapid and accurate screening and confirmation of steroid esters, enhancing anti-doping capabilities.

2025-08-20 | PXD067516 |

Visual automated fluorescence electrophoresis provides simultaneous quality, quantity, and molecular weight spectra for genomic DNA from archived neonatal blood spots

2013-07-01 | GSE44249 | GEO

A Mass Spectrometry-based Multiplexed Targeted Assay for Detection of Hemoglobinopathies from Dried Blood Spots

Project description:Hemoglobinopathies are the most common inherited disorders worldwide. Accurate analysis of hemoglobin variants is critical for diagnosis of hemoglobinopathies. Although high-performance liquid chromatography and capillary zone electrophoresis are widely used as screening tools, they possess inherent ambiguities that often preclude accurate detection of hemoglobin variants. Our goal was to develop and optimize a sensitive and specific mass spectrometry-based assay for screening and diagnosis of hemoglobinopathies. A catalog of canonical globin-chain specific peptides as well as mutant peptides corresponding to common hemoglobin variants was generated and their corresponding “heavy” synthetic peptide versions were used as internal standards for quantification and calculation of globin chain ratios. Targeted mass spectrometry analysis was performed by coupling liquid chromatography to a triple quadrupole mass spectrometer, which is the commonest mass spectrometer employed in clinical diagnostics. Dried blood spots from a cohort of 716 individuals (including 211 patients with hemoglobinopathy) were analyzed. The α:β-globin ratios showed a significant difference between normal and β-thalassemia patients, particularly when the disease was homozygous or admixed with structural variants (compound heterozygous). The method presented here permits identification of variants in their homozygous, heterozygous or compound heterozygous states. The intra- and inter-assay precision CV were both <20%. We envision that such mass spectrometry-based assays could be employed as first-line screening assay for hemoglobin variants including sickle cell disease as well as thalassemias.

2025-08-01 | PXD061996 | Pride

Visual automated fluorescence electrophoresis provides simultaneous quality, quantity, and molecular weight spectra for genomic DNA from archived neonatal blood spots

Project description:The Guthrie 903 card archived dried blood spots (DBS) are a unique but terminal resource amenable for individual and population wide genomic profiling. The limited amounts of DBS-derived genomic DNA (gDNA) can be whole-genome amplified (WGA) producing sufficient gDNA for genomic applications, albeit with variable success, and optimizing the isolation of high-quality DNA from these finite, low-yield specimens is essential. Visual automated fluorescence electrophoresis (VAFE) is a novel QC technology affording precise quality, quantity and molecular weight of double-stranded DNA from a single microliter of sample. The VAFE QC data were correlated with subsequent sample performance in PCR, sequencing, and high-density comparative genome hybridization array. The Swedish Repository of DBS samples collected on Guthrie 903 cards for neonatal screening of inborn diseases provided deidentified 3mm blood spot punches. There were total of eight (8) samples representing two (2) decades; 1970s: 1975, 1976, 1977, 1978; 1980s: 1980, 1982, 1984, 1986. gDNA was extracted and whole genome amplified prior to aCGH experiments using control female reference genomic DNA. The objective was to show that large rearrangements (e.g. loss of chrX in male samples) can be detected in WGA gDNA from blood spots >30 years old.

2013-07-01 | E-GEOD-44249 | biostudies-arrayexpress

Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling.

Project description:BackgroundNeonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome wide profiling. Degradation of DNA in DBS often occurs during storage and extraction. Moreover, amplifying small quantities of DNA often leads to a bias in subsequent data, particularly in methylome profiles. Thus it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses.ResultsUsing combinations of in-house-derived and modified commercial extraction kits, we developed a robust and efficient protocol, compatible with methylome studies, many of which require stringent bisulfite conversion steps. Several parameters were tested in a step-wise manner, including blood extraction, cell lysis, protein digestion, and DNA precipitation, purification and elution. DNA quality was assessed based on spectrophotometric measurements, DNA detectability by PCR, and DNA integrity by gel electrophoresis and bioanalyzer analyses. Genome scale Infinium HumanMethylation450 and locus-specific pyrosequencing data generated using the refined DBS extraction protocol were of high quality, reproducible and consistent.ConclusionsThis study may prove useful to meet the increased demand for research on prenatal, particularly epigenetic, origins of human diseases and for newborn screening programs, all of which are often based on DNA extracted from DBS.

| S-EPMC4086704 | biostudies-literature

Multi-mycotoxin analysis using dried blood spots and dried serum spots.

Project description:In this study, a rapid multi-mycotoxin approach was developed for biomonitoring and quantification of 27 important mycotoxins and mycotoxin metabolites in human blood samples. HPLC-MS/MS detection was used for the analysis of dried serum spots (DSS) and dried blood spots (DBS). Detection of aflatoxins (AFB1, AFB2, AFG1, AFG2, AFM1), trichothecenes (deoxynivalenol, DON; DON-3-glucoronic acid, DON-3-GlcA; T-2; HT-2; and HT-2-4-GlcA), fumonisin B1 (FB1), ochratoxins (OTA and its thermal degradation product 2'R-OTA; OTα; 10-hydroxychratoxin A, 10-OH-OTA), citrinin (CIT and its urinary metabolite dihydrocitrinone, DH-CIT), zearalenone and zearalanone (ZEN, ZAN), altenuene (ALT), alternariols (AOH; alternariol monomethyl ether, AME), enniatins (EnA, EnA1, EnB, EnB1) and beauvericin (Bea) was validated for two matrices, serum (DSS), and whole blood (DBS). HPLC-MS/MS analysis showed signal suppression as well as signal enhancement due to matrix effects. However, for most analytes LOQs in the lower pg/mL range and excellent recovery rate were achieved using matrix-matched calibration. Besides validation of the method, the analyte stability in DBS and DSS was also investigated. Stability is a main issue for some analytes when the dried samples are stored under common conditions at room temperature. Nevertheless, the developed method was applied to DBS samples of a German cohort (n = 50). Besides positive findings of OTA and 2'R-OTA, all samples were positive for EnB. This methodical study establishes a validated multi-mycotoxin approach for the detection of 27 mycotoxins and metabolites in dried blood/serum spots based on a fast sample preparation followed by sensitive HPLC-MS/MS analysis. Graphical Abstract ᅟ.

| S-EPMC5395583 | biostudies-literature

Transcriptome profiles derived from parallel venipuncture, dried blood spot (DBS), and saliva samples

Project description:Comparison of transcript abundance estimates derived from DBS vs saliva vs gold standard peripheral blood mononuclear cell (PBMC) samples.

2016-07-27 | GSE79269 | GEO

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