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.

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.

Paper-based sampling has been implemented in the determination of small molecules in biological samples for many decades. However, the use of paper-based sampling in combination with mass spectrometric analysis of proteins has been limited. Promising efforts have previously been demonstrated with blood and serum, sampled on paper materials. However, common for the

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