Flow injection analysis electrospray ionization mass spectrometry for high-throughput screening of a gene library for amidase activity.
In high-throughput screening of gene and mutant libraries, high analysis speeds and short method development times are important factors. Mass spectrometry (MS) is considered to be a generic analytical technique with a relatively short development time. Furthermore, when applying flow injection analysis (FIA) for sample introduction, the requirements for high throughput are met. In this work, the use of a single quadrupole electrospray MS instrument for assaying amidase activity in a gene library is demonstrated. The desired selectivity for measuring the amino acid, the reaction product of the amidase reaction, in the presence of high concentrations of the corresponding amino acid amide substrate was obtained by selective ionization of the amino acid in negative ion mode electrospray. The only sample preparation required was a 200-fold dilution of the reaction mixture. For obtaining quantitative results, a complementary calibration procedure was set up to correct for the change in ionization suppression as a function of conversion. This approach was used to screen a Mycobacterium neoaurum gene library consisting of 11,520 clones with alpha-methylleucine amide as substrate within 24h. Conversion was measured on the [M-H]- species of the corresponding alpha-methylleucine (m/z 144). Five positive clones were detected with a conversion ranging from 0.2% to 3.4%.
Henderickx
Huub J W
HJ
DSM Resolve, 6160 MD Geleen, The Netherlands. huub.henderickx@dsm.com
Raemakers-Franken
Petronella C
PC
van der Wal
Sjoerd
S
de Koster
Chris G
CG
Duchateau
Alexander L L
AL
Sonke
Theo
T
D000581:Amidohydrolases / Q000032:analysis* / Q000737:chemistry; D002138:Calibration; D017022:Flow Injection Analysis / Q000379:methods; D015723:Gene Library; D009161:Mycobacterium / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D013237:Stereoisomerism; D013379:Substrate Specificity
D000581:Amidohydrolases; C019665:amidase