High-throughput Sample Preparation Method Research Articles

Miniaturized on-spot protein denaturation/microwave-assisted extraction for spectrofluorimetric determination of favipiravir in dried plasma: Application to real human sample and inclusive incurred sample reanalysis study

The development of high-throughput sample preparation methods is a key step for upgrading the throughput of classical analytical techniques. In this work, a miniaturized on-spot protein denaturation coupled with microwave-assisted extraction sample treatment method was developed for extraction and subsequent spectrofluorimetric determination of favipiravir (FAV) in human plasma spots. Dried plasma spotting was performed using only 10 µL of plasma on filter paper discs followed by on-spot protein denaturation using 10 µL methanol. Maximum extraction efficiency was achieved using water: methanol (1:1) as extraction solvent with 50 % microwave power for 60 sec. The fluorescence intensity of extracted FAV was measured at 432 nm after excitation at 361 nm. According to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH -M10) guidelines for bioanalytical method validation, spiked plasma samples showed very good linearity between 100 and 1000 ng/mL. The accuracy and precision of the developed method were indicated by mean recovery (%) within 100 ± 15 % for all tested concentrations levels. The obtained results were further compared with a reported protein precipitation method using both F-test and t-test revealing no significant difference between either method. Moreover, the method was successfully applied for FAV determination in real human samples for an incurred sample reanalysis study revealing no metabolite back conversion. The developed method showed superior green performance using analytical Eco-scale, Green Analytical Procedure Index (GAPI), and Analytical Greenness Metric Approach (AGREE) tools compared to a classical protein precipitation method. According to Blue Applicability Grade Index (BAGI), and whiteness tools, the developed method outperformed all reported methods for FAV determination in plasma in terms of practicality and analytical functionality. Compared to reported sample treatment methods, this method exhibited excellent analytical performance, increased throughput, hundred-fold reduction of sample volume (10 μL), significant reduction of organic solvent consumption (260 μL methanol) and produced negligible wastes. Thus, it can be adopted for routine work in pharmacokinetics and clinical trial studies.

Nail analysis for the assessment of caffeine abuse in Northwest China: A population-based study.

Caffeine is the most widely consumed psychoactive agent worldwide and has the potential for abuse, but studies monitoring caffeine abuse in China are scarce. This study aims to estimate the prevalence of caffeine abuse in northwest China and investigate the correlation between caffeine and other drugs in hair and nails using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Fingernail clippings were collected from 376 participants in northwest China to detect caffeine and 13 other illicit psychoactive drugs and their metabolites. Paired hair and nail samples were collected from 39 participants to investigate the correlation between caffeine and other drugs in hair and nails. The samples were decontaminated, pulverized, and extracted by a high-throughput nail sample preparation method and analyzed by UPLC-MS/MS. The results showed a risk of caffeine abuse in northwest China, with concentrations ranging from 0.43 to 10.6 ng/mg for healthy volunteers, 0.49-246 ng/mg for caffeine abusers, and 0.25-363 ng/mg for drug addicts in community rehabilitation centers. Caffeine was detected together with other illicit psychoactive drugs and their metabolites. Furthermore, positive detection correlations were found between hair and nail samples. This study provides a current perspective on caffeine abuse in northwest China and demonstrates the practical use of UPLC-MS/MS for the simultaneous detection of caffeine and 13 illicit psychoactive drugs and their metabolites in hair and nails. The results highlight the potential of nails as a supplementary matrix when hair samples are unavailable and emphasize the need for handling caffeine carefully given its potential for abuse.

An effective and high-throughput sample preparation method involving demalonylation followed by an ultrahigh-performance liquid chromatography–charged aerosol detector for analyzing gypenoside XLIX and gypenoside A in Gynostemma longipes

Gypenosides (Gps) are the major bioactive components in Gynostemma species. They include neutral Gps and acidic malonylgypenosides (MGps). MGps are abundant in Gynostemma species and can be transformed into corresponding Gps via extraction, concentration, and drying. If only the Gps were quantified and MGps were ignored, the quality of Gynostemma species would be underestimated. This study aimed to develop a sample preparation method involving demalonylation and ultrahigh-performance liquid chromatography–charged aerosol detector (UHPLC-CAD) analysis to determine the contents of gypenoside XLIX (Gp XLIX) and gypenoside A (Gp A). First, the optimized ultrasonic extraction method was established to extract G. longipes powder ultrasonically. Then, the extracted solution was put into a closed container (centrifuge tube) and heated in a water bath at 95 °C. Then, MGps were converted into corresponding Gps. The proposed preparation method was compared with the other three methods, including water bath reflux heating, alkali hydrolysis, and extraction of heated powder, and was shown to exhibit higher conversion and better convenience. Subsequently, an UHPLC-CAD method was established and validated. Gp XLIX and Gp A showed excellent linear correlations between 15.55 and 248.8 μg/mL and 24.10–385.5 μg/mL, respectively (R2 > 0.999). The limit of detection was 1.40 ng (Gp XLIX) and 2.41 ng (Gp A), and the limit of quantification was 7.77 ng and 14.46 ng, respectively. The relative standard deviation for precision, stability, and repeatability was 0.63–3.15%. The average recovery of Gp XLIX and Gp A was 98.97% and 98.23%, respectively. The established method was applied for determining Gp XLIX and Gp A contents in wild or cultivated G. longipes samples collected from the Qinba Mountains area. The contents of Gp XLIX and Gp A were 5.16–23.02 mg/g and 15.78–54.55 mg/g, respectively. Conclusively, the proposed sample preparation and analysis method could be used for the quality control and evaluation of G. longipes.

Label-Free Quantification from Direct Infusion Shotgun Proteome Analysis (DISPA-LFQ) with CsoDIAq Software.

Large-scale proteome analysis requires rapid and high-throughput analytical methods. We recently reported a new paradigm in proteome analysis where direct infusion and ion mobility are used instead of liquid chromatography (LC) to achieve rapid and high-throughput proteome analysis. Here, we introduce an improved direct infusion shotgun proteome analysis protocol including label-free quantification (DISPA-LFQ) using CsoDIAq software. With CsoDIAq analysis of DISPA data, we can now identify up to ∼2000 proteins from the HeLa and 293T proteomes, and with DISPA-LFQ, we can quantify ∼1000 proteins from no more than 1 μg of sample within minutes. The identified proteins are involved in numerous valuable pathways including central carbon metabolism, nucleic acid replication and transport, protein synthesis, and endocytosis. Together with a high-throughput sample preparation method in a 96-well plate, we further demonstrate the utility of this technology for performing high-throughput drug analysis in human 293T cells. The total time for data collection from a whole 96-well plate is approximately 8 h. We conclude that the DISPA-LFQ strategy presents a valuable tool for fast identification and quantification of proteins in complex mixtures, which will power a high-throughput proteomic era of drug screening, biomarker discovery, and clinical analysis.

A Simple High-Throughput Field Sample Preparation Method Based on Matrix-Induced Sugaring-Out for the Simultaneous Determination of 5-Hydroxymethylfurfural and Phenolic Compounds in Honey.

In the present work, a high-throughput field sample preparation method was reported for the simultaneous determination of 5-hydroxymethylfurfural and phenolic compounds in honey. Combining a simple and green homogenous liquid−liquid extraction, matrix-induced sugaring-out, with the use of a 96-deepwell plate and multichannel pipette, the proposed method showed its merits in instrument-free and high-throughput preparation. Due to the high-throughput property, the parameters of the method were rapidly and systematically studied using a constructed 4 × 2 × 4 × 3 array (sample amount × ratio of ACN:H2O × standing time × replicates) in a 96-deepwell plate. Analytical performance was fully validated, and the limits of detection and limits of quantification were in the range of 0.17−1.35 μg/g and 0.51−4.14 μg/g, respectively. Recoveries were between 83.98 and 117.11%, and all the precisions were <5%. Furthermore, the developed method was successfully applied in the outdoor preparation of commercial honey samples and the in-field preparation of raw honey samples in apiary. The current work presented a simple, rapid, and high-throughput method for the field sample preparation of honey and provides a valuable strategy for the design of field and on-site sample preparation.

High-throughput interspecies profiling of acidic plant hormones using miniaturised sample processing

BackgroundAcidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants.ResultsA high-throughput sample preparation method for liquid chromatography–tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports.ConclusionThe method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

Simultaneous detection of salivary cortisol and cortisone using an automated high-throughput sample preparation method for LC-MS/MS.

High-Performance Liquid Chromatography-Tandem Mass Spectrometry has emerged triumphant over the years as a reliable and high throughput clinical instrument to assess different metabolic irregularities. One of the latest applications of LC-MS/MS has been in the field of quantification of glucocorticoids, such as cortisol and cortisone from saliva, that can be an indicator of abnormalities such as Congenital Adrenal Hyperplasia (CAH), Cushing's Syndrome, and Addison's disease. We have developed and validated an LC-MS/MS-based assay for simultaneous detection of cortisol and cortisone in human saliva, which requires only 20 µL of sample, to measure cortisol across a 0.5 - 70 ng/mL range, and cortisone across a 1.2- 100 ng/mL range, respectively. The developed method exhibits linearity of R2>0.99, for both analytes, inclusive of both MRMs, and a percent coefficient of variation that is less than or equal to 20%. Using dilute-and-shoot for sample preparation, we have exhibited sample accuracy of 100±20 for the assay calibrators, and integrated Needle Wash Solvent Chemistry for minimal sample carryover, making it adaptable for crucial for potential diagnostic use. We have exhibited a method that is simplistic, specific, and highly automatable on liquid handling platforms, such as the JANUS® G3 Workstation. With our innovation, we have introduced a potential to test 81-unknown samples in singlicate within an on-deck plating time of fewer than four minutes. We performed additional verification studies, including an accelerated stability study and a freeze-thaw study showcasing the potential long-term usability of our proposed prototype kit. Overall, this work presents an optimized LC-MS/MS method with automated sample preparation that is ready to be utilized for cortisol-cortisone detection for clinical diagnostic contexts related to Cushing's Disease, among other adrenal and endocrine disorders.

Simultaneous quantification of 106 drugs or their metabolites in nail samples by UPLC-MS/MS with high-throughput sample preparation: Application to 294 real cases

The nail is an alternative matrix to complement hair analysis in proving drug intake over several months in forensic toxicology investigations. However, because of the high hardness and toughness of nails, the existing pretreatment procedures for nails have the disadvantages of either a high degree of time consumption (from hours to days), or low extraction recoveries. This study aims to propose a high-throughput nail sample preparation method and provide a quantitative analytical method for 106 drugs and their metabolites present in nail. We developed cryogenic grinding, coupled with high-speed grinding in the extraction solvent method, which could improve the extraction recovery by thoroughly destroying the nail keratin for approximately 18 min. Subsequently, an ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the identification and quantification of 34 synthetic cannabinoids, 26 fentanyls, 18 synthetic cathinones, 10 phenylethylamines, eight opioids, three phencyclidine, two tryptamines, two piperazine, cocaine, benzoylecgonine, and tetrahydrocannabinol (THC). Nail samples were collected from people with a history of drug abuse from five different regions of China. The analysis of 294 authentic samples resulted in 213 detected samples, and showed a broad concentration range including 5.04–67.26 pg/mg for nine synthetic cannabinoids, 109.29–250.29 pg/mg for a synthetic cathinone, 5.06–434291 pg/mg for four phenylethylamines, 5.06–464278 pg/mg for three phencyclidine, 5.50–192195 pg/mg for six opioids, 19.44–36.11 pg/mg for cocaine, and 50.53 pg/mg for THC in nail. Furthermore, up to 10 different compounds were detected in a single nail sample. This nail analysis method serves as a useful tool for the large-scale surveillance of illicit drugs abuse.

96DRA-Urine: A high throughput sample preparation method for urinary proteome analysis

Mass spectrometry (MS)-based urinary proteomics is increasingly used for clinical research. A critical step in urinary proteomic analysis comprises the implementation of a reliable sample preparation method with high yields of peptides and proteins. In this study, we developed a urinary sample preparation method, DRA-Urine (Direct reduction/alkylation in urine), which urinary proteins were directly reduced/alkylated in urine, and then precipitated by acetone, washed and digestion on an ultrafilter unit. The qualitative and quantitative comparison of different urinary sample preparation methods (in-solution methods and ultrafilter-assisted methods) showed that DRA-Urine could achieve better results. Adapting DRA-Urine protocol to a 96-well format, namely 96DRA-Urine, shortened the time for buffer change and improved sample preparation throughput. The results showed that 96DRA-Urine displayed similar proteomic performance to DRA-Urine. Finally, the 96DRA-Urine method was used in a label-free, small pilot biomarker discovery analysis for differential urinary proteome analysisof bladder cancer urine. The results showed that urinary proteins could differentiate bladder cancer (BCa) patients from healthy controls and distinguish high-grade BCa from low-grade BCa with area under the curve (AUC) values of 0.972 and 0.847, respectively. Consequently, the 96DRA-Urine method might be a high-throughput method for preparing body fluid samples used in clinical research but needs to be further verified.

An Effective and High-Throughput Sample Preparation Method of Demalonylation Followed by Ultrahigh Performance Liquid Chromatography-Charged Aerosol Detector for the Analysis of Gypenoside XLIX and Gypenoside a in Gynostemma Longipes

An Effective and High-Throughput Sample Preparation Method of Demalonylation Followed by Ultrahigh Performance Liquid Chromatography-Charged Aerosol Detector for the Analysis of Gypenoside XLIX and Gypenoside a in Gynostemma Longipes

Technique Development of High-Throughput and High-Sensitivity Sample Preparation and Separation for Proteomics

Sample preparation and separation methods determine the sensitivity and the quantification accuracy of the proteomics analysis. This article covers a comprehensive review of the recent technique development of high-throughput and high-sensitivity sample preparation and separation methods in proteomics research.

Development and validation of a micro-QuEChERS method with high-throughput enhanced matrix removal followed with UHPLC-QqQ-MS/MS for analysis of raspberry ketone-related phenolic compounds in adipose tissues

Raspberry ketone (RK) is a major flavor compound in red raspberries, and it has been marketed as a popular weight-loss dietary supplement with high potential in accumulating in fatty tissues. However, challenges in extracting and characterizing RK and its associated phenolic compounds in fatty tissues persist due to the complex matrix effect. In this work, we reported a high-throughput sample preparation method for RK and 25 related phenolic compounds in white adipose tissues using an improved micro-scale QuEChERS (quick, efficient, cheap, easy, rugged and safe) approach with enhanced matrix removal (EMR)-lipid cleanup in 96-well plates, followed by UHPLC-QqQ-MS/MS analysis. The absolute recovery was 73–105% at the extraction step, and achieved 71–96% at the EMR cleanup step. The EMR cleanup removed around 66% of total lipids in the acetonitrile extract as profiled by UHPLC-QTOF-MS/MS. The innovative introduction of a reversed-phase C18 sorbent into the extract significantly improved the analytes’ recovery during SpeedVac drying. The final accuracy achieved 80–120% for most analytes. Overall, this newly developed and validated method could serve as a powerful tool for analyzing RK and related phenolic compounds in fatty tissues.

New Advanced Materials and Sorbent-Based Microextraction Techniques as Strategies in Sample Preparation to Improve the Determination of Natural Toxins in Food Samples.

Natural toxins are chemical substances that are not toxic to the organisms that produce them, but which can be a potential risk to human health when ingested through food. Thus, it is of high interest to develop advanced analytical methodologies to control the occurrence of these compounds in food products. However, the analysis of food samples is a challenging task because of the high complexity of these matrices, which hinders the extraction and detection of the analytes. Therefore, sample preparation is a crucial step in food analysis to achieve adequate isolation and/or preconcentration of analytes and provide suitable clean-up of matrix interferences prior to instrumental analysis. Current trends in sample preparation involve moving towards “greener” approaches by scaling down analytical operations, miniaturizing the instruments and integrating new advanced materials as sorbents. The combination of these new materials with sorbent-based microextraction technologies enables the development of high-throughput sample preparation methods, which improve conventional extraction and clean-up procedures. This review gives an overview of the most relevant analytical strategies employed for sorbent-based microextraction of natural toxins of exogenous origin from food, as well as the improvements achieved in food sample preparation by the integration of new advanced materials as sorbents in these microextraction techniques, giving some relevant examples from the last ten years. Challenges and expected future trends are also discussed.

High-throughput quantification of carboxymethyl lysine in serum and plasma using high-resolution accurate mass Orbitrap mass spectrometry.

BackgroundCarboxymethyl lysine is an advanced glycation end product of interest as a potential biomarker of cardiovascular and other diseases. Available methods involve ELISA, with potential interference, or isotope dilution mass spectrometry (IDMS), with low-throughput sample preparation.MethodsA high-throughput sample preparation method based on 96-well plates was developed. Protein-bound carboxymethyl lysine and lysine were quantified by IDMS using reversed phase chromatography coupled to a high-resolution accurate mass Orbitrap Exactive mass spectrometer. The carboxymethyl lysine concentration (normalized to lysine concentration) was measured in 1714 plasma samples from the British Regional Heart Study (BRHS).ResultsFor carboxymethyl lysine, the lower limit of quantification (LLOQ) was estimated at 0.16 μM and the assay was linear between 0.25 and 10 μM. For lysine, the LLOQ was estimated at 3.79 mM, and the assay was linear between 2.5 and 100 mM. The intra-assay coefficient of variation was 17.2% for carboxymethyl lysine, 9.3% for lysine and 10.5% for normalized carboxymethyl lysine. The inter-assay coefficient of variation was 18.1% for carboxymethyl lysine, 14.8 for lysine and 16.2% for normalized carboxymethyl lysine. The median and inter-quartile range of all study samples in each batch were monitored. A mean carboxymethyl lysine concentration of 2.7 μM (IQR 2.0–3.2 μM, range 0.2–17.4 μM) and a mean normalized carboxymethyl lysine concentration of 69 μM/M lysine (IQR 54–76 μM/M, range 19–453 μM/M) were measured in the BRHS.ConclusionThis high-throughput sample preparation method makes it possible to analyse large cohorts required to determine the potential of carboxymethyl lysine as a biomarker.

Development and evaluation of matrix application techniques for high throughput mass spectrometry imaging of tissues in the clinic

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a sensitive label-free technique that can be used to study a wide variety of clinical phenotypes. In this context, MSI offers huge diagnostic potential by supporting decision making in the determination of personalized treatment strategies. However, improvements in throughput and robustness are still needed before it finds a place in routine application. While the field has seen tremendous improvements in the throughput of data acquisition, robust and high-throughput sample preparation methods compatible with these acquisition methods need to be developed. To address this challenge, we have developed several methods to reduce the matrix application time to less than 5 min, while maintaining sensitivity and reproducibility. Workflows incorporating these methods provide a pipeline analysis time for MSI sample preparation and acquisition of less than 30 min. The reduced time for these analyses will contribute towards the integration of MSI into routine molecular pathology for clinical diagnostics.

An automated high-throughput sample preparation method using double-filtration for serum metabolite LC-MS analysis

A novel, automated sample preparation method for serum metabolite LC-MS analysis was developed using a double-filtration system.

High-throughput 96-well solid-phase extraction for preparation of tetracycline followed by liquid chromatography and mass spectrometry analysis.

Tetracyclines abuse has frequently occurred in aquaculture against bacteria, rickettsiae, spirochetes, and mycoplasmas. In this study, a high-throughput sample preparation method was developed using 96-well plate solid-phase extraction (p-SPE) and the extract was analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The experimental conditions were optimized such that the pH is 4, the eluting solvent is methanol (2 mL), and the sorbent is hydrophilic-lipophilic balance (HLB) microsphere. The whole protocol was validated, and it showed that the tetracyclines were linear with correlation coefficients ≥ 0.9990, precision and accuracy (RSD%) in 3.9-6.1%, and mean recoveries of 88.6-103.6%. To exhibit the potential of 96-well p-SPE as a routine tool for inspection and quarantine, fresh aquatic samples were tested, and among which positive samples were observed. This method was demonstrated to be promising for the purification and enrichment of tetracyclines with reduced time and labor, and indeed practically and particularly suitable for widespread tetracyclines analysis.

ICP-MS measurement of toxic and essential elements in human breast milk. A comparison of alkali dilution and acid digestion sample preparation methods

BackgroundFor trace element measurement in human breast milk, an accurate high-throughput sample preparation method would be desirable. In this study, a quick alkali sample dilution method was systematically compared to the traditional acid digestion pretreatment for measuring 10 trace elements in 237 human breast milk samples by inductively coupled plasma mass spectrometry (ICP-MS). MethodsThe alkali method involved dilution of the breast milk samples with a solution consisting of 2% butanol, 0.05% EDTA, 0.05% Triton X-100 and 1% NH4OH. The mixtures were sonicated, centrifuged and analyzed by ICP-MS. Acid digestion was performed in HNO3 at 250°C using a Milestone Ultra CLAVE II microwave digestion system. ResultsOverall, the alkali dilution method gave higher analytical precision compared to acid digestion while LODs and recoveries were similar between the two methods. Spearman's correlations between measured element concentrations by the two sample preparation methods were strong (rS>0.70) for all elements, except for Ca and Cd (rS<0.50). Passing-Bablok regression analyses showed best agreement between the two methods for Li, Mg, Fe, Zn, Se and Cd (slopes 0.8–1.2). Evaluation of Bland-Altman difference plots showed best method agreement (lowest bias) for Na, Mg, Ca, Zn and Cd.Highest difference in element concentration between the methods was observed for iodine (96ng/g by alkali dilution and 33ng/g by acid digestion), probably due to formation of volatile species such as HI and I2 in the acid matrix. ConclusionsThe results indicate that the alkali dilution high-throughput sample preparation method is comparable to the more commonly used acid digestion method for ICP-MS measurements of some toxic and essential elements in human breast milk.

Quick 96FASP for high throughput quantitative proteome analysis

Filter aided sample preparation (FASP) is becoming a central method for proteomic sample cleanup and peptide generation prior to LC-MS analysis. We previously adapted this method to a 96-well filter plate, and applied to prepare protein digests from cell lysate and body fluid samples in a high throughput quantitative manner. While the 96FASP approach is scalable and can handle multiple samples simultaneously, two key advantages compared to single FASP, it is also time-consuming. The centrifugation-based liquid transfer on the filter plate takes 3–5 times longer than single filter. To address this limitation, we now present a quick 96FASP (named q96FASP) approach that, relying on the use of filter membranes with a large MWCO size (~30kDa), significantly reduces centrifugal times. We show that q96FASP allows the generation of protein digests derived from whole cell lysates and body fluids in a quality similar to that of the single FASP method. Processing a sample in multiple wells in parallel, we observed excellent experimental repeatability by label-free quantitation approach. We conclude that the q96FASP approach promises to be a promising cost- and time-effective method for shotgun proteomics and will be particularly useful in large scale biomarker discovery studies. SignificanceHigh throughput sample processing is of particular interests for quantitative proteomics. The previously developed 96FASP is high throughput and appealing, however it is time-consuming in the context of centrifugation-based liquid transfer (~1.5h per spin). This study presents a truly high throughput sample preparation method based on large cut-off 96-well filter plate, which shortens the spin time to ~20min. To our knowledge, this is the first multi-well method that is entirely comparable with conventional FASP. This study thoroughly examined two types of filter plates and performed side-by-side comparisons with single FASP. Two types of samples, whole cell lysate of a UTI (urinary tract infection)-associated Klebsiella pneumoniae cell and human urine, were tested which demonstrated its capability for quantitative proteomics. The q96FSAP approach makes the filter plate-based approach more appealing for protein biomarker discovery projects, and could be broadly applied to large scale proteomics analysis.

Application of volumetric absorptive microsampling for robust, high-throughput mass spectrometric quantification of circulating protein biomarkers

Volumetric absorptive micro sampling (VAMS™) allows accurate sampling of 10µL of blood from a minimally invasive finger prick and could enable remote personalized health monitoring. Moreover, VAMS overcomes effects from hematocrit and sample heterogeneity associated with dried blood spots (DBS). We describe the first application of VAMS with the Mitra® microsampling device for the quantification of protein biomarkers using an automated, high-throughput sample preparation method coupled with mass spectrometric (MS) detection.The analytical performance of the developed workflow was evaluated for 10 peptides from six clinically relevant proteins: apolipoproteins A-I, B, C-I, C-III, E, and human serum albumin (HSA). Extraction recovery from blood with three different levels of hematocrit varied between 100% and 111% for all proteins. Within-day and total assay reproducibility (i.e., 5 replicates on 5days) ranged between 3.2–10.4% and 3.4–12.6%, respectively. In addition, after 22weeks of storage of the Mitra microsampling devices at −80°C, all peptide responses were within ±15% deviation from the initial response. Application to data-independent acquisition (DIA) MS further demonstrated the potential for broad applicability and the general robustness of the automated workflow by reproducible detection of 1661 peptides from 423 proteins (average 15.7%CV (n=3) in peptide abundance), correlating to peptide abundances in corresponding plasma (R=0.8383).In conclusion, we have developed an automated workflow for efficient extraction, digestion, and MS analysis of a variety of proteins in a fixed small volume of dried blood (i.e., 10µL). This robust and high-throughput workflow will create manifold opportunities for the application of remote, personalized disease biomarker monitoring.