As a part of an on going project aimed to the development of convenient and sensitive procedures for proteomics applications [1-3], our group has recently rationally designed and developed a new enzymatic mesoreactor for ultrafast protein digestion [4]. The high surface areas, the ordered periodicity of the pores with controllable dimensions and morphology, render mesoporous silicate (MPS) optimal supports for a great variety of catalysts, from small molecules catalysts (such as metals, metal complexes, metal oxides) to large molecule catalysts such as enzymes. MPS SBA-15 together with N-(2-aminoethyl)-3-aminopropyl and aminopropyl (indicated as AAPTES and APTES, respectively) functionalized derivatives were prepared with pore dimensions of about 4 nm, slightly larger than the diameter of trypsin (3.8 nm), to obtain a well-fitting physical entrapment. Myoglobin was added to trypsin meso-reactor, with a molar enzyme/substrate ratio of 1:3. Within 1 min of digestion, a rich pattern of proteolytic fragments was obtained for SBA-15- AAPTES and SBA-15-APTES, which allowed unambiguous myoglobin identification. The best performance was achieved for trypsin adsorbed in SBA-15-AAPTES with 100% sequence coverage obtained in just 1 min. The effect of organic functionalities such as AAPTES and APTES grafted on SBA-15 on in situproteolysis are examined. In addition we address the suitability of these bionanocatalysts, for a convenient “proteomic scale” procedure consisting of few sample-handling steps, with increased proteolytic efficiency (1000 times faster and an improved performance compared to the conventional in solution procedure), making them promising for high-speed and high-throughput protein identification. [1] R. Terracciano, M. Gaspari, F. Testa, L. Pasqua, G. Cuda, P. Tagliaferri, M. C. Cheng, A. J. Nijdam, E. F. Petricoin, L. A. Liotta, M. Ferrari and S. Venuta, Proteomics, 6, 2006, 3243. [2] R. Terracciano, L. Pasqua, F. Casadonte, S. Frascà, M. Preianò, D. Falcone and R. Savino, Bioconjugate Chem., 20, 2009, 913. [3] R. Terracciano, F. Casadonte, L. Pasqua, P. Candeloro, E. Di Fabrizio, A. Urbani and R. Savino, Talanta, 80, 2010, 1532. [4] F Casadonte, L. Pasqua, R. Savino and R. Terracciano, Chem. Eur. J., 16, 2010, 8998.
In mesopore protein digestion a new strategy for mass spectrometry-based proteomics
Terracciano R;Savino R
2011-01-01
Abstract
As a part of an on going project aimed to the development of convenient and sensitive procedures for proteomics applications [1-3], our group has recently rationally designed and developed a new enzymatic mesoreactor for ultrafast protein digestion [4]. The high surface areas, the ordered periodicity of the pores with controllable dimensions and morphology, render mesoporous silicate (MPS) optimal supports for a great variety of catalysts, from small molecules catalysts (such as metals, metal complexes, metal oxides) to large molecule catalysts such as enzymes. MPS SBA-15 together with N-(2-aminoethyl)-3-aminopropyl and aminopropyl (indicated as AAPTES and APTES, respectively) functionalized derivatives were prepared with pore dimensions of about 4 nm, slightly larger than the diameter of trypsin (3.8 nm), to obtain a well-fitting physical entrapment. Myoglobin was added to trypsin meso-reactor, with a molar enzyme/substrate ratio of 1:3. Within 1 min of digestion, a rich pattern of proteolytic fragments was obtained for SBA-15- AAPTES and SBA-15-APTES, which allowed unambiguous myoglobin identification. The best performance was achieved for trypsin adsorbed in SBA-15-AAPTES with 100% sequence coverage obtained in just 1 min. The effect of organic functionalities such as AAPTES and APTES grafted on SBA-15 on in situproteolysis are examined. In addition we address the suitability of these bionanocatalysts, for a convenient “proteomic scale” procedure consisting of few sample-handling steps, with increased proteolytic efficiency (1000 times faster and an improved performance compared to the conventional in solution procedure), making them promising for high-speed and high-throughput protein identification. [1] R. Terracciano, M. Gaspari, F. Testa, L. Pasqua, G. Cuda, P. Tagliaferri, M. C. Cheng, A. J. Nijdam, E. F. Petricoin, L. A. Liotta, M. Ferrari and S. Venuta, Proteomics, 6, 2006, 3243. [2] R. Terracciano, L. Pasqua, F. Casadonte, S. Frascà, M. Preianò, D. Falcone and R. Savino, Bioconjugate Chem., 20, 2009, 913. [3] R. Terracciano, F. Casadonte, L. Pasqua, P. Candeloro, E. Di Fabrizio, A. Urbani and R. Savino, Talanta, 80, 2010, 1532. [4] F Casadonte, L. Pasqua, R. Savino and R. Terracciano, Chem. Eur. J., 16, 2010, 8998.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
