Profiling of intact blood proteins by matrix-assisted laser desorption/ionization mass spectrometry without the need for freezing - Dried serum spots as future clinical tools for patient screening.

RATIONALE: To open up new ways for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based patient screening, blood serum is the most preferred specimen because of its richness in patho-physiological information and due to ease of collection. To overcome deleterious freeze/thaw cycles and to reduce high costs for shipping and storage, we sought to develop a procedure which enables MALDI-MS protein profiling of blood serum proteins without the need for serum freezing. METHODS: Blood sera from patients/donors were divided into portions which after pre-incubation were fast frozen. Thawed aliquots were deposited on filter paper discs and air-dried at room temperature. Intact serum proteins were eluted with acid-labile detergent-containing solutions and were desalted by employing a reversed-phase bead system. Purified protein solutions were screened by MALDI-MS using standardized instrument settings. RESULTS: MALDI mass spectra from protein solutions which were eluted from filter paper discs and desalted showed on average 25 strong ion signals (mass range m/z 6000 to 10,000) from intact serum proteins (apolipoproteins, complement proteins, transthyretin and hemoglobin) and from proteolytic processing products. Semi-quantitative analysis of three ion pairs: m/z 6433 and 6631, m/z 8205 and 8916, as well as m/z 9275 and 9422, indicated that the mass spectra from either pre-incubated fast-frozen serum or pre-incubated dried serum spot eluted serum contained the same information on protein composition. CONCLUSIONS: A workflow that avoids the conventional cold-chain and yet enables the investigation of intact serum proteins and/or serum proteolysis products by MALDI-MS profiling was developed. The presented protocol tremendously broadens the clinical application of MALDI-MS and simultaneously allows a reduction in the costs for storage and shipping of serum samples. This will pave the way for clinical screening of patients also in areas with limited access to health care systems, and/or specialized laboratories.

Dried serum spots on pre-punched filter paper discs are ready-to-use storage and shipping devices for blood-borne antigens and antibodies.

Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 µl of serum, named the "Submerge and Dry" protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 µl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests.

High performance liquid chromatography: A versatile tool for assaying antiepileptic drugs in biological matrices.

Epilepsy is a recurrent long-term illness occurring in approximately 1.0% of the world's population. There are currently about 29 approved antiepileptic drugs for the management of epilepsy. Due to narrow therapeutic indices of most antiepileptic drugs, clinical pharmacokinetic characteristics and therapeutic drug monitoring of these drugs are imperative. The objectives of this review were to identify common chromatographic principles, requirements and/or conditions for high-performance liquid chromatography as applied to assay of antiepileptic drugs in biological matrices. The review was conducted using 66 peer reviewed articles (1990 to 2020) from 29 journals that were sought via PubMed, Science Direct and Google Scholar. In all, 29 antiepileptic drugs were assayed from 6 different biological matrices. Forty-three of the reviewed articles estimated the concentration of only one antiepileptic drug, whilst 23 articles focused on simultaneous determination of two or more antiepileptic drugs. Thirty-four, 20, and 14 articles reported using liquid-liquid extraction, protein precipitation, or solid phase extraction for sample clean up, respectively. The ratio of reversed-phase to normal phase, LC-UV to LC-MS and isocratic elution to gradient elution were 61:3, 43:7 and 55:11, respectively. With the exception of one article the reported recoveries ranged from 60.3% to 109.6%. It is noteworthy, that, the performance metrics of high-performance liquid chromatography are better compared to other assays of antiepileptic drugs in biological matrices. This review describes the relevant liquid chromatographic method conditions over the past 30 years for the analysis of this class of drugs, which provides a basis for further method development and optimization.

Precision Diagnostics by Affinity-Mass Spectrometry: A Novel Approach for Fetal Growth Restriction Screening During Pregnancy.

Fetal growth restriction (FGR) affects about 3% to 8% of pregnancies, leading to higher perinatal mortality and morbidity. Current strategies for detecting fetal growth impairment are based on ultrasound inspections. However, antenatal detection rates are insufficient and critical in countries with substandard care. To overcome difficulties with detection and to better discriminate between high risk FGR and low risk small for gestational age (SGA) fetuses, we investigated the suitability of risk assessment based on the analysis of a recently developed proteome profile derived from maternal serum in different study groups. Maternal serum, collected at around 31 weeks of gestation, was analyzed in 30 FGR, 15 SGA, and 30 control (CTRL) pregnant women who delivered between 31 and 40 weeks of gestation. From the 75 pregnant women of this study, 2 were excluded because of deficient raw data and 2 patients could not be grouped due to indeterminate results. Consistency between proteome profile and sonography results was obtained for 59 patients (26 true positive and 33 true negative). Of the proteome profiling 12 contrarious grouped individuals, 3 were false negative and 9 were false positive cases with respect to ultrasound data. Both true positive and false positive grouping transfer the respective patients to closer surveillance and thorough pregnancy management. Accuracy of the test is considered high with an area-under-curve value of 0.88 in receiver-operator-characteristics analysis. Proteome profiling by affinity-mass spectrometry during pregnancy provides a reliable method for risk assessment of impaired development in fetuses and consumes just minute volumes of maternal peripheral blood. In addition to clinical testing proteome profiling by affinitymass spectrometry may improve risk assessment, referring pregnant women to specialists early, thereby improving perinatal outcomes.

Comparison of blood serum protein analysis by MALDI-MS from either conventional frozen samples or storage disc-deposited samples: A study with human serum from pregnant donors and from patients with intrauterine growth restriction.

Mass spectrometric profiling of intact serum proteins, i.e. determination of relative protein abundance differences, was performed using two different serum sample preparation methods: one with frozen and thawed serum, the other with at room temperature deposited and dried serum. Since in a typical clinical setting freezing of serum is difficult to achieve, sampling at room temperature is preferred and can be met when using the Noviplex™ card system. Once deposited and dried, serum proteins can be stored and shipped at room temperature. After resolubilization of serum proteins from "dried serum spots", mass spectra of high quality have been recorded comparable to those that were obtained using fresh-frozen and subsequently thawed serum samples. Differentiation between patients with intrauterine growth restriction and control individuals was achievable, independent from the sample work-up procedure. Having at hand a reliable and robust method for serum storage and shipment which works at room temperature bridges the gap between the clinics and the protein analysis laboratory. Our novel serum handling protocol reduces costs for both, storage and shipping, and ultimately enables clinical risk assessment based on mass spectrometric determination of intact protein abundance profiles.

Maternal Apolipoprotein B100 Serum Levels are Diminished in Pregnancies with Intrauterine Growth Restriction and Differentiate from Controls.

PURPOSE: Intrauterine growth restriction, a major cause of fetal morbidity and mortality, is defined as a condition in which the fetus does not reach its genetically given growth potential. Screening for intrauterine growth restriction biomarkers in the mother's blood would be of great help for optimal pregnancy management and timing of delivery as well as for identifying fetuses requiring further surveillance during their infancies. EXPERIMENTAL DESIGN: A multiplexing serological assay based on liquid chromatography-multiple-reaction-monitoring mass spectrometry is applied for distinguishing serum samples of pregnant women. RESULTS: Assessment of concentrations of apolipoproteins and of proteins that belong to the lipid transport system is performed with maternal serum samples, consuming only 10 µL of serum per multiplex assay from each patient. Of all investigated proteins the serum concentrations of apolipoprotein B100 shows the greatest power for discriminating intrauterine growth restriction from control samples, reaching areas under curves above 0.85 in receiver-operator-characteristics analyses. CONCLUSIONS: These results indicate the potential of liquid chromatography-multiple-reaction-monitoring mass spectrometry to become of clinical importance in the future for intrauterine growth restriction risk assessment based on maternal apolipoprotein B100 serum levels.