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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. The sampling concept showed a high degree of performance between 10 and 1000 ng/mL (R2 > 0.99) by a five-point calibration curve sampled with hCG spiked to human serum samples and freshly collected whole blood samples, respectively. LOD (experimentally obtained at 100 pg/mL (2.64 pM/0.9 IU/L)) was demonstrated to be up to ten times lower with more than six times faster sample preparation than what has previously been reported for on-paper analysis of hCG in human serum samples.

Electromembrane extraction (EME) in small, stagnant and chip-like devices has the potential for future in-field operation. Literature briefly discuss such systems, but performance suffered from evaporative losses of sample and acceptor. To address this, the current paper reports electromembrane extraction (EME) of five basic drugs (model analytes) from aqueous buffer solutions and whole blood samples under stagnant conditions in a completely closed system. A laboratory-made polyoxymethylene (POM) well plate served as compartment for the sample solution, while a commercially available well filter plate was used to immobilize 2-nitrophenyl octyl ether (NPOE) as supported liquid membrane (SLM) and as closed compartment for the acceptor solution. Major design parameters (sample compartment and electrode geometry) and operational parameters (sample volume, voltage and extraction time) were investigated and optimized. Electrode geometry was not very critical, but extraction efficiency increased with decreasing sample volume. Extraction from 50 μL aqueous buffer solution for 60 min and with a voltage of 75 V was considered exhaustive (sample was depleted), with recoveries ranging between 75% and 87% for loperamide, haloperidol, methadone, nortriptyline, and pethidine (RSD: 2–12%). Extraction from whole blood samples under optimized conditions yielded slightly lower recoveries, ranging between 57 and 96% (RSD: 3–12%). Stagnant EME was evaluated in combination with liquid chromatography-mass spectrometry (LC-MS) as a highly specific instrumental method, and provided evaluation data on methadone from blood samples in accordance with regulatory requirements (LOD: 0.4 ng/mL, LOQ: 1.4 ng/mL, RSD: 6–20%). This work has improved upon the design of stagnant EME, moving it further towards a viable in-field operation device.

Open tubular liquid chromatography columns with organic polymer layers can be powerful tools for high sensitivity measurements in e.g. proteomics. However, these narrow columns are challenging to characterize. A two-electrode system, often used for bioimpendance measurements, was used to study poly(styrene-co-divinylbenzene = PS-DVB) polymer layered open tubular (PLOT) liquid chromatography columns with 10 μm inner diameters. The system performed electrical resistance measurements (ERM) for assessing layer thickness and porosity. Layer determination results were comparable (but more precise) to that obtained with scanning electron microscopy (SEM). Porosity examinations with ERM casted doubt on the presence/availability of pores in the layers investigated.

Rationale Matrix-assisted ionization (MAI) is a relatively new ionization technique for analysis by mass spectrometry (MS). The technique is simple and has been shown to be less influenced by matrix effects than e.g. electrospray ionization (ESI). These features are of interest in the targeted analysis of proteins from biological samples. Methods Targeted protein determination by MAI-MS was evaluated using a triple quadrupole mass analyzer equipped with a stripped nanoESI source in selected reaction monitoring (SRM) mode. The proteins were analyzed using the bottom-up approach with stable isotopic labeled peptides as internal standards (IS). The MAI matrix was 3-nitrobenzonitrile dissolved in acetonitrile. Aqueous sample and matrix solution were mixed in a 1:3 volume ratio. One microlitre of the dried matrix/analyte sample was introduced into the inlet of the mass spectrometer where ionization commences. Results SRM settings established for ESI-SRM-MS of the peptides here investigated were applicable in MAI-SRM-MS for all evaluated peptides except one that is poorly soluble in water. Addition of IS provided efficient correction at most levels (relative standard deviation (RSD) ≤28% (except lowest digest level), r2 ≥ 0.995). This was also true for the more complex biological matrices, diluted urine (1:1; RSD = 20% a synthetic peptide, NLLGLIEAK) and diluted digested serum (1:100; RSD = 7% digested cytochrome C). Biological matrix influenced the signal intensity unless sufficiently diluted. Conclusions The results demonstrate that MAI-SRM-MS has promising potential in targeted protein determination by the bottom-up approach because of its simplicity, ease of use, and speed. However, more data is needed to confirm the results prior to application in a clinical setting.

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. The sampling spots demonstrated fast and easy sampling and preparation of human serum spiked with the biomarker human chorionic gonadotropin. The sampling spots enabled a detection limit of 1 ng/mL (26.4 pM) within a five point concentration curve from 1 ng/mL to 20 ng/mL (R2 = 0.97). The detection limit was demonstrated to be two times lower than previously demonstrated with standard DMPK-C sampling cards. A five point concentration curve from 100 ng/mL to 2000 ng/mL was also investigated (R2 = 0.998). Intra day precision was within 16% and 23% for concentration range 1 ng/mL to 20 ng/mL and 100 ng/mL to 2000 ng/mL, respectively. Inter day precision was within 20%. Accuracy was determined to 10% and 11% for 2.5 ng/mL and 20 ng/mL, respectively. The sampling spots were also demonstrated in a realistic setting where serum samples from two confirmed patients with testicular germ cell cancer were analyzed. These analyses confirmed an elevated hCG content in the sera of 418.5 ± 4.2 ng/mL and 21 ± 0.02 ng/mL hCG for patient one and two respectively.

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. The highest reactor efficiency with respect to protein digestion was obtained with 1.25 mg trypsin per reactor immobilized at room temperature for 3 hours. Commercially available cellulose filter papers and DMPK-C cards were modified and immobilized with trypsin prior to whole blood sampling. Filter paper specifications including thickness (180–220 μm), weight (77–92 g m−2) and porosity (11–25 μm) were investigated with respect to performance (digestion efficiency and extraction recovery). From this study, it was found that a medium thickness paper with higher weight and porosity is optimal for reactor efficiency. The reactors were tested and compared with respect to a standard dried blood spot procedure for protein digestion. The most efficient reactors obtained 134 ± 14 and 124 ± 7 high confidence protein groups for freeze thawed and fresh whole blood samples, respectively.

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. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.