A simple MALDI plate functionalization by Vmh2 hydrophobin for serial multi-enzymatic protein digestions

Abstract

The development of efficient and rapid methods for the identification with high sequence coverage of proteins is one of the most important goals of proteomic strategies today. The on-plate digestion of proteins is a very attractive approach, due to the possibility of coupling immobilized-enzymatic digestion with direct matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) analysis. The crucial step in the development of on-plate immobilization is however the functionalization of the solid surface. Fungal self-assembling proteins, the hydrophobins, are able to efficiently functionalize surfaces. We have recently shown that such modified plates are able to absorb either peptides or proteins and are amenable to MALDI-TOF-MS analysis. In this paper, the hydrophobin-coated MALDI sample plates were exploited as a lab-on-plate for noncovalent immobilization of enzymes commonly used in protein identification/characterization, such as trypsin, V8 protease, PNGaseF, and alkaline phosphatase. Rapid and efficient on-plate reactions were performed to achieve high sequence coverage of model proteins, particularly when performing multiple enzyme digestions. The possibility of exploiting this direct on-plate MALDI-TOF/TOF analysis has been investigated on model proteins and, as proof of concept, on entire whey milk proteome.

Recent Advances in Fungal Hydrophobin Towards Using in Industry

“…Immobilization of selfassemble hydrophobins on the steel sample-loading plate of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) has been proposed by this group [137]. The researchers have successfully analysed several proteins such as human serum, alkaline phosphatase, V8 protease, and trypsin using those modified plates [136,137].…”

“…A group from the University of Naples, Italy, has recently studied the use of hydrophobin for enhancing the sensitivity of biosensors [134][135][136][137]. Immobilization of selfassemble hydrophobins on the steel sample-loading plate of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) has been proposed by this group [137].…”

On‐the‐Spot Immobilization of Quantum Dots, Graphene Oxide, and Proteins via Hydrophobins

“…[ 72 ] The Vmh2 monolayer acts as a bioactive substrate to bind other proteins, which show improved stability and activity when bound on the biohybrid chips. [70][71][72][73] In this work, we propose a fast, highly homogeneous, and effi cient glass functionalization method by spontaneous selfassembling of Vmh2 at liquid-solid interface. In addition, we demonstrate for the fi rst time the immobilization of nanomaterials and proteins via hydrophobins in a microarray fashion, which results to be a time-saving process.…”

“…[ 68 ] Vmh2 from the white-rot fungus Pleurotus ostreatus is one of the most hydrophobic HFBs known, soluble in low polar solvents. This Class I HFB has been isolated and studied in our laboratories and used for modifi cation of steel, [ 69,70 ] nanostructured silicon, [ 67 ] and graphene-based materials. [ 71 ] The nanometric layer (≈2.5 nm tick) of Vmh2 has been characterized on different surfaces as well its self-assembled rod-like structures, typically formed by Class I HFBs.…”

Self‐assembly of two hydrophobins from marine fungi affected by interaction with surfaces

“…Only a few proteins belonging to Class I HFBs have been analyzed so far and they show quite different self‐assembling mechanisms (Gandier et al, ; Gravagnuolo et al, ). We have focused our work on the discovery of new Class I HFBs, (Armenante et al, ) and the characterization of their self‐assembling (Gravagnuolo et al, ; Longobardi et al, ) to enable modern biotechnological applications (Gravagnuolo et al, ,; Kaur et al, ; Longobardi et al, , ; Patel et al, ; Piscitelli et al, ,; Politi et al, , ). Additionally, because of their critical role in fungal biology, the knowledge of their mechanism of action may be useful for the discovery of novel antifungals (Pham et al, ).…”