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dc.identifier.citationArdakani, Dorna Misaghian. Porous layer open tubular (PLOT) protein separation and trypsin open tubular (TOT) columns. Masteroppgave, University of Oslo, 2012en_US
dc.description.abstractPorous layer open tubular (PLOT) columns and immobilized trypsin open tubular (TOT) columns of 10 μm inner diameter (i.d.) were made and characterized for fast intact protein analysis and protein digestion, respectively. A 0.1 mg/mL standard protein solution of cytochrome C, myoglobin, and carbonic anhydrase was used to investigate the PLOT column performance and on-column trypsination was investigated using 5 and 0.3 mg/mL cytochrome C solutions. Mobile phase combination of A (H2O/FA/TFA, 100/0.1/0.05, v/v/v) and B (ACN/H2O/FA/TFA, 90/10/0.1/0.05, v/v/v/v) were used for protein separation on PLOT columns and mobile phase combination of A (50 mM NH4OAc pH 8.75) and B (ACN) were used for protein digestion on the TOT columns. The optimal combination of flow rate and gradient time for separation of intact proteins on the poly(styrene-divinylbenzene) (PS-DVB) PLOT column was explored in the range of 20 to 80 nL/min, and 60 nL/min was found as the efficient flow rate regarding peak shape and peak width at a gradient time of 40 min. The mass spectrometric (MS) signal intensity was found to depend on flow rate and the temperature in the investigated range from 25 to 80℃. Gradient steepness on different column lengths, and column i.d.s, was also examined. Long columns provided better performance at longer gradient times. At elevated flow rates the pressure increases, thus 60 ℃ was found as the optimal temperature at 60 nL/min. The optimal gradient time on a 3 m long column at a flow rate of 60 nL/min was 15 min. Due to difficulties with mass spectrometric detection of many intact proteins the possibility of an on-line digestion using a TOT column was explored. Unfortunately, no digestion was observed with continuous flow at 150 nL/min. Different stop flow times from 20 to 120 s, and 15 min at different flow rates from 20 to 340 nL/min at temperatures 25 and 37℃, different amount of ACN within a range of 5 to 90% at 25℃, and 37℃ and column length in the range of 27 to 50 cm was examined to obtain digestion, and a 2 min stop flow time at 37℃ were found as optimal digestion time and temperature, respectively. Shorter columns gave better digestion and a column length of 30 cm column was found to be optimal. Proteins mostly elute from a reversed phase column within a range of 40 to 90% of ACN. No difference in protein digestion on the TOT column was found in the range 40 to 90% of ACN, at both 25 and 37℃.eng
dc.titlePorous layer open tubular (PLOT) protein separation and trypsin open tubular (TOT) columnsen_US
dc.typeMaster thesisen_US
dc.creator.authorArdakani, Dorna Misaghianen_US
dc.identifier.bibliographiccitationinfo:ofi/fmt:kev:mtx:ctx&ctx_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft.au=Ardakani, Dorna Misaghian&rft.title=Porous layer open tubular (PLOT) protein separation and trypsin open tubular (TOT) columns&rft.inst=University of Oslo&rft.date=2012&rft.degree=Masteroppgaveen_US
dc.contributor.supervisorElsa Lundanesen_US

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