Instant on-paper protein digestion during blood spot sampling

Abstract

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.

Smart proteolysis samplers for pre‐lab bottom‐up protein analysis – Performance of on‐paper digestion compared to conventional digestion

“…The latter does not out rule the possibility of good accessibility when few disulfide bonds are present, however, on the other hand, it is not given that the absence of disulfide bonds results in good protein coverages for on-paper digests. In accordance with previously published results [45]. This is probably dependent on additional protein properties.…”

“…Our group has been working with the concept of smart sampling in combination with MS determination since 2017 [45]. This concept is based on the covalent binding of trypsin to cellulose allowing a DBS sampler to start with the digestion step at the moment of sampling [46][47][48][49].…”

Next-Generation Dried Blood Spot Samplers for Protein Analysis: Describing Trypsin-Modified Smart Sampling Paper

“…This is also seen in the number of high-confidence peptides qualified. As in earlier studies [35], it is seen that carboxymethylated tryptic peptides are found in expected amounts [40]. The latter is of interest since the reduction and alkylation take place after the digestion.…”

“…The long-term goal of our research is to simplify MS-based protein analysis from dried matrix samples (such as DBS), and we aim to integrate time-consuming procedures within the sampling device for a prompt start to the sample preparation upon sample collection [29,[35][36][37][38].…”

Matrix‐assisted ionization mass spectrometry in targeted protein analysis – An initial evaluation

“…One reason for a too low digestion output may be the lack of a reduction and alkylation step prior to digestion. 33 As a simple precautionary measure, initially this step was left out to avoid introduction of the DTT and IAA into the mass spectrometer during direct injection MAI-MS using the syringe method. However, to…”