A simple MALDI plate functionalization by Vmh2 hydrophobin for serial multi-enzymatic protein digestions.
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.
Department of Chemical Sciences, University of Naples "Federico II", Via Cintia 4, 80126, Naples, Italy, sara.longobardi@unina.it.
Sara
S
Longobardi
Alfredo Maria
AM
Gravagnuolo
Riccardo
R
Funari
Bartolomeo
B
Della Ventura
Francesca
F
Pane
Eugenio
E
Galano
Angela
A
Amoresano
Gennaro
G
Marino
Paola
P
Giardina
D000469:Alkaline Phosphatase / Q000737:chemistry; D000595:Amino Acid Sequence; D002364:Caseins / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry; D008894:Milk Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D058525:Quartz Crystal Microbalance Techniques; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
D002364:Caseins; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D008894:Milk Proteins; D011506:Proteins; D000469:Alkaline Phosphatase; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase