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

Abstract

Here the relation between digestion of proteins by trypsin covalently bound to paper and trypsin in‐solution is investigated. The trypsin acting on paper is covalently bound. A trypsin concentration of 0.5% (w/v) results in the highest digestion activity of all concentrations tested. Additionally, it can be seen that trypsin on‐paper has retained approx. 50% of its activity. Unlike trypsin in‐solution, the stability of the smart proteolysis samplers was regarded to be stable for at least four months when kept refrigerated. Autolysis was very small for covalently bound trypsin: less than 2% compared to in‐solution trypsin. Proteomic analysis of diluted human serum showed more protein identifications (214) in‐solution digestions than on‐paper digestions (76). Also, higher coverage for the in‐solution digestion was obtained. Those proteins identified after on‐paper digestion with no or few disulfide bonds seem to have more similar sequence coverages compared to those identified after in‐solution digestion. Smart samplers allow the determination of at least 70–75 proteins without performing the overnight digestion. All in all, trypsin covalently bound to paper shows to retain high proteolytic activity and is a stable alternative for conventional digestions. In this way, smart proteolytic samplers show their feasibility in pre‐lab sample preparation.

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

“…In all cases, the integrated proteolysis step allowed to digest even complex samples as serum and whole blood identifying up to 200 proteins per spot. Additionally, it appeared that paper with covalently bound trypsin was surprisingly stable: it did not lose its activity when kept dry for 4 months [70].…”

“…Although successful, a major drawback with covalent binding via 2hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone chemistry was that the paper lost much of its absorbing properties [68]. This was overcome by covalent binding of trypsin via periodate oxidation [69,70]. In all cases, the integrated proteolysis step allowed to digest even complex samples as serum and whole blood identifying up to 200 proteins per spot.…”

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

“…Additionally, the trypsin sampler did not show a significant decrease in activity over a period of 4 months (stored dry at 4 • C). 18 Trypsin samplers have also briefly been investigated for use in targeted protein determination. Serum spiked with the biomarker progastrin-releasing peptide (ProGRP) was added to trypsin samplers and subsequently, the ProGRP-specific peptide ALGNQQP-SWDSEDSSNFK was isolated using peptide affinity capture.…”

An efficient in‐gel digestion method on small amounts of protein sample from large intact gel pieces

“…There are various methods for protein digestion in bottom‐up proteomics, but in‐gel digestion [1] is labor‐intensive and time‐consuming, and its reproducibility has been a concern due to the large number of steps, the tendency for keratin and other substances to be mixed in, and loss of peptides [2, 3]. For this reason, recently gel‐free methods have been developed, such as filter‐assisted sample preparation [4], single‐pot solid‐phase enhanced sample preparation (SP3) [5] using carboxylated beads or HILIC paramagnetic beads, and on‐paper digestion using trypsin covalently bound to paper [6].…”

“…For this reason, recently gel-Article Related Abbreviations: ABC, ammonium bicarbonate; FDR, false discovery rate; PPM, positive pressure manifold. free methods have been developed, such as filter-assisted sample preparation [4], single-pot solid-phase enhanced sample preparation (SP3) [5] using carboxylated beads or HILIC paramagnetic beads, and on-paper digestion using trypsin covalently bound to paper [6].…”