Microwave-assisted high-throughput acid hydrolysis in silicon carbide microtiter platforms--a rapid and low volume sample preparation technique for total amino acid analysis in proteins and peptides.
An efficient microwave-assisted high-throughput protein hydrolysis protocol was developed utilizing strongly microwave absorbing silicon carbide-based microtiter platforms. The plates are equipped with 20 bore holes having the proper dimensions for holding standard screw-capped HPLC/GC vials. Due to the possibility of heating up to four heating platforms simultaneously (80 vials), parallel microwave-assisted acid hydrolyses can be performed under carefully controlled conditions significantly reducing the overall time required for protein hydrolysis and the subsequent evaporation step required for larger volumes of acid. An extensive optimization of the hydrolysis conditions has demonstrated that 5min irradiation at 160°C with 6N HCl leads to comparable results in terms of total and individual amino acid recovery as the traditional method requiring 24h heating at 110°C. Complete hydrolysis of several proteins and synthetic peptides was performed using 25μg of sample material and 100μL of 6N HCl in a dedicated low-volume HPLC/GC vial. Since the hydrolysis and subsequent analysis can be performed from the same vial, errors caused by sample transfer can be minimized. Control experiments have demonstrated that the observed rate enhancements are the result of a purely thermal/kinetic effect as a consequence of the considerable higher reaction temperatures.
Christian Doppler Laboratory for Microwave Chemistry (CDLMC) and Institute of Chemistry, Karl-Franzens-University Graz, Heinrichstraße 28, A-8010 Graz, Austria.
Markus
M
Damm
Michael
M
Holzer
Günther
G
Radspieler
Gunther
G
Marsche
C Oliver
CO
Kappe
D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D017554:Carbon Compounds, Inorganic / Q000737:chemistry*; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000295:instrumentation* / Q000379:methods*; D006358:Hot Temperature; D006851:Hydrochloric Acid; D006868:Hydrolysis; D008872:Microwaves; D008904:Miniaturization; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D017655:Silicon Compounds / Q000737:chemistry*
D000596:Amino Acids; D017554:Carbon Compounds, Inorganic; D010455:Peptides; D011506:Proteins; D017655:Silicon Compounds; D012710:Serum Albumin, Bovine; D006851:Hydrochloric Acid; C022088:silicon carbide