Advancements in clinical approaches, analytical methods, and smart sampling for LC-MS-based protein determination from dried matrix spots.

Determination of proteins from dried matrix spots using MS is an expanding research area. Mainly, the collected dried matrix sample is whole blood from a finger or heal prick, resulting in dried blood spots. However as other matrices such as plasma, serum, urine, and tear fluid also can be collected in this way, the term dried matrix spot is used as an overarching term.

Is this the end of dried blood spots as we know it?

In 2017 integrated sampling and sample preparation for simplified liquid chromatography-mass spectrometry analysis of proteins from dried blood spots were introduced. The concept, called smart samplers or smart sampling, enables proteolysis or affinity clean-up, two common sample preparation steps in liquid chromatography-mass spectrometric bioanalysis of proteins, to start at the moment of sampling. The idea is to utilize the time for sampling and drying to perform these time-consuming and labour-intensive steps.

One-step functionalization of paper and simplified antibody immobilization for on-the-spot immunocapture from dried serum in liquid chromatography-tandem mass spectrometry based targeted protein determination.

This work aimed to simplify and improve the process of binding monoclonal antibodies (mAbs) covalently to filter paper for use in dried blood spot sampling, enabling instant capture of protein biomarkers for targeted protein determination. Incorporating the necessary immunocapture sample preparation step in the initial sampling stage saves time and reduces the workload. The biomarker human chorionic gonadotropin (hCG) was used as the model analyte.

Smart sampling as the "Spot-on" Method for LC-MS protein analysis from dried blood spots.

This perspective explores the feasibility of smart sampling with dried blood spots for the determination of proteins and peptides from human biomatrices using liquid chromatography coupled to mass spectrometry for clinical purposes. The focus is on innovative approaches to transform filter paper from a mere sample carrier to an active element in sample preparation, with the aim of reducing the need for extensive and intensive sample preparation in the conventional sense. Specifically, we discuss the use of modified cellulose to integrate sample preparation at an early stage of sample handling.

Dried blood spot analysis with liquid chromatography and mass spectrometry: Trends in clinical chemistry.

Dried blood spot samples are simple to prepare and transport, enabling safe and accessible diagnostics, both locally and globally. We review dried blood spot samples for clinical analysis, focusing on liquid chromatography-mass spectrometry as a versatile measurement tool for these samples. Dried blood spot samples can provide information for, for example, metabolomics, xenobiotic analysis, and proteomics.

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins.

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.

The utility of molecularly imprinted polymers for mass spectrometric protein and proteomics analysis.

Selective and efficient sample clean-up is important in mass spectrometric protein- and proteomics analyses from biological matrices. Molecularly imprinted polymers (MIPs), polymers prepared to have tailor-made cavities for capture of target analytes may by such represent an interesting alternative for selective clean-up. The present review aims to give an overview of the utility of MIPs for protein capture from biological matrices prior to mass spectrometry (MS) analysis.

On the spot immunocapture in targeted biomarker analysis using paper-bound streptavidin as anchor for biotinylated antibodies.

The modification of an easily available resource like paper to circumvent expensive or intensive sample pretreatment could be the answer to sample analysis in resource-poor regions. Therefore, a novel on-paper device combining sample collection with affinity sample pretreatment is introduced here. Universal smart affinity samplers are produced by a simple KIO-mediated oxidation of cellulose, which functionalizes the paper.

A Novel Porcine Model of Ischemia-Reperfusion Injury After Cross-Clamping the Thoracic Aorta Revealed Substantial Cardiopulmonary, Thromboinflammatory and Biochemical Changes Without Effect of C1-Inhibitor Treatment.

Ischemic injury worsens upon return of blood and innate immunity including the complement system play a central role in ischemia-reperfusion injury (IRI) as in thoracic aortic surgery. Complement component1 inhibitor (C1-INH) has been shown to reduce IRI and is a broad-acting plasma cascade inhibitor. We established a new porcine model of IRI by cross-clamping the thoracic aorta and evaluated the global changes occurring in organ function, systemic inflammatory response and organ damage with or without treatment with C1-INH-concentrate.

Affinity capture in bottom-up protein analysis - Overview of current status of proteolytic peptide capture using antibodies and molecularly imprinted polymers.

Antibody-based affinity capture has become the gold standard in sample preparation for determination of low-abundance protein biomarkers in biological matrices prior to liquid chromatography-mass spectrometry (LC-MS) determination. This comprises both capture of intact proteins prior to the digestion step and capture of proteolytic peptides after digestion of the sample. The latter can be performed both using antibodies specifically developed to capture target proteolytic peptides, as well as by the less explored use of anti-protein antibodies to capture the proteolytic epitope peptide.

On-line duplex molecularly imprinted solid-phase extraction for analysis of low-abundant biomarkers in human serum by liquid chromatography-tandem mass spectrometry.

In the present work, a pair of molecularly imprinted polymers (MIPs) targeting distinct peptide targets were packed into trap columns and combined for automated duplex analysis of two low abundant small cell lung cancer biomarkers (neuron-specific enolase [NSE] and progastrin-releasing peptide [ProGRP]). Optimization of the on-line molecularly imprinted solid-phase extraction (MISPE) protocol ensured that the MIPs had the necessary affinity and selectivity towards their respective signature peptide targets - NLLGLIEAK (ProGRP) and ELPLYR (NSE) - in serum. Two duplex formats were evaluated: a physical mixture of the two MIPs (1:1 w/w ratio) inside a single trap column, and two separate MIP trap columns connected in series.

Facilitating serum determination of neuron specific enolase at clinically relevant levels by coupling on-line molecularly imprinted solid-phase extraction to LC-MS/MS.

The identification and quantification of biomarkers is essential for the diagnosis, treatment, and long-term monitoring of many human diseases. In the present work, macromolecular synthetic receptors with pre-determined affinity and selectivity for the signature peptide of a prognostically significant small cell lung cancer (SCLC) biomarker - neuron-specific enolase (NSE) - were prepared in a porous polymer microsphere format using a template-directed synthesis strategy performed under precipitation polymerization conditions. The polymer microspheres were packed into short trap columns and then exploited as molecularly selective sorbents in a fully automated, on-line molecularly imprinted solid-phase extraction (MISPE) protocol.

Liquid chromatography mass spectrometry based characterization of epitope configurations.

Here we evaluate a quick and easy tool for determination of epitope configuration using immunocapture and liquid chromatography mass spectrometry (LC-MS) subsequent to pre-treatment of the target protein to disrupt its three-dimensional structure. The approach can be a valuable screening tool to identify antibodies that can be used in peptide capture by anti-protein antibodies. The experimental set-up was established using seven monoclonal antibodies (mAbs) with known linear or conformational epitope recognition.

Magnetic Synthetic Receptors for Selective Clean-Up in Protein Biomarker Quantification.

Biomarker analysis by mass spectrometry (MS) can allow for the rapid quantification of low abundant biomarkers. However, the complexity of human serum is a limiting factor in MS-based bioanalysis; therefore, novel biomarker enrichment strategies are of interest, particularly if the enrichment strategies are of low cost and are easy to use. One such strategy involves the use of molecularly imprinted polymers (MIPs) as synthetic receptors for biomarker enrichment.

Immunocapture sample clean-up in determination of low abundant protein biomarkers - a feasibility study of peptide capture by anti-protein antibodies.

Immunocapture in mass spectrometry based targeted protein analysis using a bottom-up workflow is nowadays mainly performed by target protein extraction using anti-protein antibodies followed by tryptic digestion. Already available monoclonal antibodies (mAbs) which were developed against intact target proteins (anti-protein antibodies) can capture proteotypic epitope containing peptides after tryptic digestion of the sample. In the present paper considerations when developing a method for targeted protein quantitation through capture of epitope containing peptides are discussed and a method applying peptide capture by anti-protein antibodies is compared with conventional immunocapture MS.

All-in-one paper-based sampling chip for targeted protein analysis.

A novel all-in-one paper-based sampling concept for mass spectrometric bottom-up protein analysis is here demonstrated in a chip format integrating instant immunocapture, protein reduction, - alkylation and tryptic digestion all in-device. Conventional laboratory grade filter paper was coated with the polymer 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (pHEMA-VDM) with a subsequent covalent immobilization of the monoclonal antibody E27 targeting the biomarker human chorionic gonadotropin (hCG). In-device protein reduction and alkylation was optimized with regards to reagent concentration and reaction pH.

Nano volume fractionation strategy for dilute-and-shoot injections in off-line loss-less proteomic workflows for extensive protein identifications of ultra-low sample amounts.

A proteomic workflow for a simple loss-less manual nano-fractionation (300 nL/fraction) for low µg sample amounts which avoids the need to dry down or transfer fractions to autosampler vials is shown to be feasible. It is demonstrated that the conventional procedure of drying samples down followed by reconstitution negatively affects the number of protein and peptide identifications. Furthermore, these losses seem to disproportionately affect hydrophobic peptides from the drying down and reconstitution step.

Paper-based immunocapture for targeted protein analysis.

A novel sampling concept for mass spectrometric bottom-up targeted protein analysis is here demonstrated with polymeric sampling spots integrated with instant immunocapture for analysis of dried matrix spots. The polymers 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (pHEMA-VDM) and pHEMA-Tosyl for covalent attachment of antibodies where investigated alongside with adsorption on non-treated filter paper. From performance characterization, the pHEMA-VDM had the best performance.

Data-Independent Acquisition for the Orbitrap Q Exactive HF: A Tutorial.

Data-independent acquisition (DIA) is a powerful mass spectrometric technique to perform both protein identification and quantification of complex protein samples. Setting up DIA methods on Orbitrap analyzers requires a thorough overview of the actions the Orbitrap mass spectrometers carry out. This Tutorial is written with the intention to give an overview of the important parameters to consider as well as which measurements to carry out to get the most out of your DIA method when setting it up on an Orbitrap mass analyzer.

Selective Fishing for Peptides with Antibody-Immobilized Acrylate Monoliths, Coupled Online with NanoLC-MS.

An online microfluidics-mass spectrometry platform was developed for determining proteotypic peptides from in-solution digested samples. Accelerated and selective sample cleanup was achieved by integrating proteotypic epitope peptide immunoextraction with nano liquid chromatography-tandem mass spectrometry (online IE-nanoLC-MS/MS). Ten individually prepared 180 μm inner diameter capillaries with ethylene glycol dimethacrylate- co-vinyl azlactone (EDMA- co-VDM) monoliths were immobilized with anti-protein antibodies that are used in routine immunoassays of the intact small cell lung cancer biomarker ProGRP.

Smart blood spots for whole blood protein analysis.

A reactor for whole blood sampling integrated with instant protein digestion in a "lab-on-paper" format is introduced here. The sampling reactor was fabricated on commercially available filter paper using 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (HEMA-VDM) polymerization followed by the immobilization of trypsin. Immobilization conditions were investigated with respect to temperature, enzyme amount and immobilization time.

Correction to: Exploring the peptide retention mechanism in molecularly imprinted polymers.

The authors would like to call the reader's attention to the fact that unfortunately due to the file formatting during the exporting of the data matrix from the program Unscrambler (used for the development of the statistical model) to the Word office file.

Instant on-paper protein digestion during blood spot sampling.

A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step.

Exploring the peptide retention mechanism in molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) have been used as useful sorbents in solid-phase extraction for a wide range of molecules and sample matrices. Their unique selectivity can be fine-tuned in the imprinting process and is crucial for the extraction of macromolecules from complex matrices such as serum. A relevant example of this is the application of MIPs to peptides in diagnostic assays.

To elute or not to elute in immunocapture bottom-up LC-MS.

Immunocapture-based bottom-up LC-MS is a promising technique for the quantification of low abundant proteins. Magnetic immunocapture beads provide efficient enrichment from complex samples through the highly specific interaction between the target protein and its antibody. In this article, we have performed the first thorough comparison between digestion of proteins while bound to antibody coated beads versus after elution from the beads.