17th International Mass Spectrometry Conference :: Prague, 2006
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|Session:||Proteomics: New Methods|
|Presentation date:||Tue, Aug 29, 2006|
|Presentation time:||09:50 – 11:20|
Jennie R. Lill1, Wendy N. Sandoval1, Victoria Pham1, Peter S. Liu1, Helga Raab1, Richard Vandlen1, David Arnott11 Genentech Inc, South San Francisco, United States
Correspondence address: Jennie Rebecca Lill, Genentech Inc, Microchemistry, 1 DNA Way, South San Francisco, CA, 94080 United States.
Keywords: Enzymes; Glycoproteins; Mass Spectrometry; Proteomic.
Novel aspect: Microwave assisted proteolysis and deglycosylation is described.
To evaluate microwave irradiation for increased bio-catalysis on a range of both proteolytic and non-proteolytic enzymes.
For evaluating microwave assisted proteolytic cleavage, Asp-N, Trypsin and Lys-C were employed at a concentration of 0.1 mg of enzyme per pmol of protein in 50 mM Tris pH 7.5. Samples were first reduced and alkylated, and either incubated in the CEM microwave at 37°C or 60°C (1-5 W). Various time points were taken from 5 min to 2 h. Alternatively, samples were incubated under identical conditions in a conventional water-bath. Reactions were quenched by addition of 3% TFA (2 µL). Samples were analyzed by micro-capillary reverse phase chromatography LC/MS/MS in data dependent mode, with MS/MS being performed on the 5 most abundant species, on either an LTQ linear ion trap mass spectrometer, or an LTQ-FTMS hybrid mass spectrometer (Thermo, San Jose, CA). Data was analyzed by de novo sequencing or using the search algorithm Mascot (Matrix Science, London, UK). Samples were also analyzed by SDS-PAGE to gauge the level of proteolysis.
For evaluation of non-proteolytic enzymes, the deglycosylating enzyme PNGase F was employed. N-linked glycoproteins were diluted to 0.5 mg/mL concentration and an enzyme to substrate ratio of 1:20 was added in the presence of 0.1 M Tris pH 7.5. Again, samples were incubated either in the presence of microwave irradiation (50 W) at either 37°C, 45°C or 60°C or, at the corresponding temperatures in a water-bath. Time points were taken and reactions were quenched by addition of 3% TFA (5 µL). Samples were separated and desalted on a reverse-phase column, and were introduced into a triple quadrupole TSQ mass spectrometer (Thermo) using electrospray ionization. The Promass software was employed to deconvoluted raw data to determine the intact molecular weight.
Preliminary results for microwave assisted proteolytic cleavage showed that for tryptic in-solution digestions, the reaction proceeded more rapidly in the presence of microwave irradiation, when compared to a traditional convection heating system. For Asp-N and Lys-C proteolysis, results were comparable between microwave irradiated samples and control samples, therefore at this point, no advantage was observed in using microwave assistance for these particular enzymes.
For the PNGase F mediated deglycosylation reactions, a marked overall decrease in incubation times was observed when employing microwave irradiation as compared to using a water-bath catalyzed reaction. For antibodies and other large glyco-proteins, complete deglycosylation could be observed in between 10 min to 1 h using microwave assisted deglycosylation, as compared to water-bath incubation which on average took 2 h to overnight to achieve equivalent results.
Microwave assisted proteomics can mediate increased catalysis for select enzymatic reactions.